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CA 02389722 2002-04-30
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15 Human Secreted Proteins
Field of the Invention
This invention relates to newly identified polynucleotides, polypeptides
encoded by these polynucleotides, antibodies that bind these polypeptides,
uses of
such polynucleotides, polypeptides, and antibodies, and their production.
Background of the Invention
Unlike bacterium, which exist as a single compartment surrounded by a
membrane, human cells and other eucaryotes are subdivided by membranes into
many
functionally distinct compartments. Each membrane-bounded compartment, or
organelle, contains different proteins essential for the function of the
organelle. The
cell uses "sorting signals," which are amino acid motifs located within the
protein, to
target proteins to particular cellular organelles.
One type of sorting signal, called a signal sequence, a signal peptide, or a
leader sequence, directs a class of proteins to an organelle called the
endoplasmic
reticulum (ER). The ER separates the membrane-bounded proteins from all other
types of proteins. Once localized to the ER, both groups of proteins can be
further
directed to another organelle called the Golgi apparatus. Here, the Golgi
distributes
the proteins to vesicles, including secretory vesicles, the cell membrane,
lysosomes,
and the other organelles.
Proteins targeted to the ER by a signal sequence can be released into the
extracellular space as a secreted protein. For example, vesicles containing
secreted
proteins can fuse with the cell membrane and release their contents into the
extracellular space - a process called exocytosis. Exocytosis can occur
constitutively
or after receipt of a triggering signal. In the latter case, the proteins are
stored in
secretory vesicles (or secretory granules) until exocytosis is triggered.
Similarly,
proteins residing on the cell membrane can also be secreted into the
extracellular
space by proteolytic cleavage of a "linker" holding the protein to the
membrane.
Despite the great progress made in recent years, only a small number of genes
encoding human secreted proteins have been identified. These secreted proteins
include the commercially valuable human insulin, interferon, Factor VIII,
human
growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of
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the pervasive role of secreted proteins in human physiology, a need exists for
identifying and characterizing novel human secreted proteins and the genes
that
encode them. This knowledge will allow one to detect, to treat, and to prevent
medical diseases, disorders, and/or conditions by using secreted proteins or
the genes
that encode them.
Summary of the Invention
The present invention relates to novel polynucleotides and the encoded
polypeptides. Moreover, the present invention relates to vectors, host cells,
antibodies, and recombinant and synthetic methods for producing the
polypeptides
and polynucleotides. Also provided are diagnostic methods for detecting
diseases,
disorders, and/or conditions related to the polypeptides and polynucleotides,
and
therapeutic methods for treating such diseases, disorders, and/or conditions.
The
invention further relates to screening methods for identifying binding
partners of the
polypeptides.
Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide. The
term "isolated" does not refer to genomic or cDNA libraries, whole cell total
or
mRNA preparations, genomic DNA preparations (including those separated by
electrophoresis and transferred onto blots), sheared whole cell genomic. DNA
preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
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In the present invention, a "secreted" protein refers to those proteins
capable
of being directed to the ER, secretory vesicles, or the extracellular space as
a result of
a signal sequence, as well as those proteins released into the extracellular
space
without necessarily containing a signal sequence. If the secreted protein is
released
into the extracellular space, the secreted protein can undergo extracellular
processing
to produce a "mature" protein. Release into the extracellular space can occur
by many
mechanisms, including exocytosis and proteolytic cleavage.
In specific embodiments, the polynucleotides of the invention are at least 15,
at least 30, at least 50, at least 100, at least 125, at least 500, or at
least 1000
continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb,
50 kb, 15
kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further
embodiment,
polynucleotides of the invention comprise a portion of the coding sequences,
as
disclosed herein, but do not comprise all or a portion of any intron. In
another
embodiment, the polynucleotides comprising coding sequences do not contain
coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest
in the
genome). In other embodiments, the polynucleotides of the invention do not
contain
the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,
4, 3, 2, or
1 genomic flanking gene(s).
As used herein, a "polynucleotide" refers to a molecule having a nucleic acid
sequence contained in SEQ ID NO:X or the cDNA contained within the clone
deposited with the ATCC. For example, the polynucleotide can contain the
nucleotide sequence of the full length cDNA sequence, including the 5' and 3'
untranslated sequences, the coding region, with or without the signal
sequence, the
secreted protein coding region, as well as fragments, epitopes, domains, and
variants
of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers
to a
molecule having the translated amino acid sequence generated from the
polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:X
was often generated by overlapping sequences contained in multiple clones
(contig
analysis). A representative clone containing all or most of the sequence for
SEQ ID
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
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shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and
the
ATCC Deposit Number. The ATCC is located at 10801 University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of the deposit
of
microorganisms for purposes of patent procedure.
A "polynucleotide" of the present invention also includes those
polynucleotides capable of hybridizing, under stringent hybridization
conditions, to
sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within
the clone deposited with the ATCC. "Stringent hybridization conditions" refers
to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x
SSC
(750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared
salmon
sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt conditions, or
temperature. For example, lower stringency conditions include an overnight
incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M
NaCI;
0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with 1 XSSPE,
0.1% SDS. In addition, to achieve even lower stringency, washes performed
following stringent hybridization can be done at higher salt concentrations
(e.g. 5X
SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
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Of course, a polynucleotide which hybridizes only to polyA+ sequences (such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
complementary stretch of T (or U) residues, would not be included in the
definition of
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically
any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA or modified RNA or DNA. For example, polynucleotides can be composed of
single- and double-stranded DNA, DNA that is a mixture of single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or a mixture
of single-
and double-stranded regions. In addition, the polynucleotide can be composed
of
triple-stranded regions comprising RNA or DNA or both RNA and DNA. A
polynucleotide may also contain one or more modified bases or DNA or RNA
backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications can be made to DNA and RNA; thus, "polynucleotide" embraces
chemically, enzymatically, or metabolically modified forms.
The polypeptide of the present invention can be composed of amino acids
joined to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and may contain amino acids other than the 20 gene-encoded amino
acids.
The polypeptides may be modified by either natural processes, such as
posttranslational processing, or by chemical modification techniques which are
well
known in the art. Such modifications are well described in basic texts and in
more
detailed monographs, as well as in a voluminous research literature.
Modifications
can occur anywhere in a polypeptide, including the peptide backbone, the amino
acid
side-chains and the amino or carboxyl termini. It will be appreciated that the
same
type of modification may be present in the same or varying degrees at several
sites in
a given polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched , for example, as a result of
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ubiquitination, and they may be cyclic, with or without branching. Cyclic,
branched,
and branched cyclic polypeptides may result from posttranslation natural
processes or
may be made by synthetic methods. Modifications include acetylation,
acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation, formation
of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation,
gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, pegylation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES,
2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth
Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
"SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ ID NO:Y"
refers to a polypeptide sequence, both sequences identified by an integer
specified in
Table 1.
"A polypeptide having biological activity" refers to polypeptides exhibiting
activity similar, but not necessarily identical to, an activity of a
polypeptide of the
present invention, including mature forms, as measured in a particular
biological
assay, with or without dose dependency. In the case where dose dependency does
exist, it need not be identical to that of the polypeptide, but rather
substantially similar
to the dose-dependence in a given activity as compared to the polypeptide of
the
present invention (i.e., the candidate polypeptide will exhibit greater
activity or not
more than about 25-fold less and, preferably, not more than about tenfold less
activity, and most preferably, not more than about three-fold less activity
relative to
the polypeptide of the present invention.)
Polynucleotides and Polypeptides of the Invention
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FEATURES OF PROTEIN ENCODED BY GENE NO: 1
For purposes of this application, this gene and its corresponding translation
product are known as the B7-H6 gene and B7-H6 protein.
This protein is believed to reside as a cell-surface molecule, and the
transmembrane domain of this protein is believed to approximately embody the
following preferred amino acid residues: NERTHLVILGAILLCLGVALTFIFRLR
(SEQ ID NO: 119). Therefore, in specific embodiments, polypeptides of the
invention comprise, or alternatively consists of, the following amino acid
sequence:
NERTHLVILGAILLCLGVALTFIFRLR (SEQ ID NO: 119). Moreover, fragments
and variants of these polypeptides (such as, for example, fragments as
described
herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to these polypeptides and polypeptides encoded by the polynucleotide
which
hybridizes, under stringent conditions, to the polynucleotide encoding these
polypeptides ) are encompassed by the invention. Antibodies that bind
polypeptides
of the invention are also encompassed by the invention. Polynucleotides
encoding
these polypeptides are also encompassed by the invention.
As one skilled in the art would understand, the transmembrane domain was
predicted using computer analysis, and the transmembrane domain may vary by
one,
two, three, four, five, six, seven, eight, nine, and/or ten amino acids from
the N and C-
termini of the predicted transmembrane domain.
The B7-H6 gene shares sequence homology with members of the B7 family of
ligands (i.e., B7-1 (See, e.g., Genbank Accession 507873)). These proteins and
their
corresponding receptors play vital roles in the growth, differentiation and
death of T
cells. For example, some members of this family (i.e., B7-H1) are involved in
costimulation of the T cell response, as well as inducing increased cytokine
production. Therefore, antagonists such as antibodies or small molecules
directed
against the B7-H6 gene are useful for treating T cell mediated immune system
disorders.
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The gene encoding the disclosed cDNA is thought to reside on chromosome 9.
Accordingly, polynucleotides related to this invention have uses, such as, for
example, as a marker in linkage analysis for chromosome 9.
An additional preferred embodiment of the invention comprises, or
alternatively consists of, the following allelic-variant of the B7-H6 protein:
MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQ
LDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAAL
QITDVKLQDAGVYRCMISYGGADYKRITVKVNVPYNKINQRILV VDPVTSEH
ELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLR1NTTTN
EIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFR
LRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 123). Moreover,
fragments and variants of these polypeptides (such as, for example, fragments
as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identical to these polypeptides and polypeptides encoded by the
polynucleotide
which hybridizes, under stringent conditions, to the polynucleotide encoding
these
polypeptides ) are encompassed by the invention. Antibodies that bind
polypeptides
of the invention are also encompassed by the invention. Polynucleotides
encoding
these polypeptides are also encompassed by the invention.
It has been discovered that this gene is expressed in dendritic cells,
placental
tissue, and T cells.
In additional nonexclusive embodiments, polypeptides of the invention
comprise, or alternatively consist of, one or more amino acid sequences
selected from
the group: l.) The extracellular domain of the B7-H6 protein:
MRIFAVFIFMTYWHLLNAFTVTVPKDLYV VEYGSNMTIECKFPVEKQLDLAA
LIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDV
KLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQ
AEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCT
FRRLDPEENHTAELVIPELPLAHPP (SEQ ID NO: 120),
2.)The mature extracellular domain of the B7-H6 protein:
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHG
EEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGA
DYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQ
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9
VLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPE
LPLAHPP (SEQ ID N0:121 ), and
3.) The leader sequence of the B7-H6 protein: MRIFAVFIFMTYWHLLNA (SEQ ID
N0:122). Moreover, fragments and variants of these polypeptides (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
Also preferred are polypeptides comprising, or alternatively consisting of,
fragments of the mature extracellular portion of the B7-H6 protein
demonstrating
functional activity (SEQ >D NO: 121). Polynucleotides encoding these
polypeptides
are also encompassed by the invention. By functional activity is meant, a
polypeptide
fragment capable of displaying one or more known functional activities
associated
with the full-length (complete) B7-H6 protein. Such functional activities
include, but
are not limited to, biological activity (e.g., T cell costimulatory activity,
ability to bind
ICOS, and ability to induce or inhibit cytokine production), antigenicity
[ability to
bind (or compete with a B7-H6 polypeptide for binding) to an anti-B7-H6
antibody],
immunogenicity (ability to generate antibody which binds to a B7-H6
polypeptide),
ability to form multimers with B7-H6 polypeptides of the invention, and
ability to
bind to a receptor or ligand for a B7-H6 polypeptide.
Figures lA-D show the nucleotide (SEQ ID NO: 11) and deduced amino acid
sequence (SEQ ID NO: 65) corresponding to this gene. Figure 2 shows an
analysis of
the amino acid sequence (SEQ >Z7 NO: 65). Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible regions;
antigenic
index and surface probability are shown, and all were generated using the
default
settings of the recited computer algorithyms. In the "Antigenic Index or
Jameson-
Wolf' graph, the positive peaks indicate locations of the highly antigenic
regions of
the protein, i.e., regions from which epitope-bearing peptides of the
invention can be
obtained. Polypeptides comprising, or alternatively consisting of, domains
defined by
these graphs are contemplated by the present invention, as are polynucleotides
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encoding these polypeptides. The data presented in Figure 2 are also
represented in
tabular form in Table 6. The columns are labeled with the headings "Res",
"Position",
and Roman Numerals I-XIV. The column headings refer to the following features
of
the amino acid sequence presented in Figure 2, and Table 6: "Res": amino acid
5 residue of SEQ ID NO: 65 and Figures lA-D; "Position": position of the
corresponding residue within SEQ ID NO: 65 and Figures lA-D; I: Alpha, Regions
-
Garnier-Robson; II: Alpha, Regions - Chou-Fasman; III: Beta, Regions - Garnier-
Robson; IV: Beta, Regions - Chou-Fasman; V: Turn, Regions - Gamier-Robson; VI:
Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-Robson; VIII:
10 Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods;
X:
Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions -
Eisenberg; XII: Flexible Regions - Karplus-Schulz; XIII: Antigenic Index -
Jameson-
Wolf; and XIV: Surface Probability Plot - Emini. Preferred embodiments of the
invention in this regard include fragments that comprise, or alternatively
consisting
of, one or more of the following regions: alpha-helix and alpha-helix forming
regions
("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"),
turn
and turn-forming regions ("turn-regions"), coil and coil-forming regions
("coil-
regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta
amphipathic regions, flexible regions, surface-forming regions and high
antigenic
index regions. The data representing the structural or functional attributes
of the
protein set forth in Figure 2 and/or Table 6, as described above, was
generated using
the various modules and algorithms of the DNA*STAR set on default parameters.
In
a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV
of
Table 6 can be used to determine regions of the protein which exhibit a high
degree of
potential for antigenicity. Regions of high antigenicity are determined from
the data
presented in columns VIII, IX, XIII, and/or XIV by choosing values which
represent
regions of the polypeptide which are likely to be exposed on the surface of
the
polypeptide in an environment in which antigen recognition may occur in the
process
of initiation of an immune response. Certain preferred regions in these
regards are set
out in Figure 2, but may, as shown in Table 6, be represented or identified by
using
tabular representations of the data presented in Figure 2. The DNA*STAR
computer
algorithm used to generate Figure 2 (set on the original default parameters)
was used
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11
to present the data in Figure 2 in a tabular format (See Table 6). The tabular
format of
the data in Figure 2 is used to easily determine specific boundaries of a
preferred
region.
The present invention is further directed to fragments of the polynucleotide
sequences described herein. By a fragment of, for example, the polynucleotide
sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:
11,
is intended polynucleotide fragments at least about l5nt, and more preferably
at least
about 20 nt, at least about 25nt, still more preferably at least about 30 nt,
at least about
35nt, and even more preferably, at least about 40 nt in length, at least about
45nt in
length, at least about 50nt in length, at least about 60nt in length, at least
about 70nt in
length, at least about 80nt in length, at least about 90nt in length, at least
about 100nt
in length, at least about 125nt in length, at least about 150nt in length, at
least about
175nt in length, which are useful as diagnostic probes and primers as
discussed
herein. Of course, larger fragments 200-1500 nt in length are also useful
according to
the present invention, as are fragments corresponding to most, if not all, of
the
nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO: 11. By a
fragment at least 20 nt in length, for example, is intended fragments which
include 20
or more contiguous bases from the nucleotide sequence of a deposited cDNA or
the
nucleotide sequence as shown in SEQ ID NO: 11. In this context "about"
includes the
particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2,
or 1)
nucleotides, at either terminus or at both termini. Representative examples of
polynucleotide fragments of the invention include, for example, fragments that
comprise, or alternatively, consist of, a sequence from about nucleotide 1 to
about 50,
from about 51 to about 100, from about 101 to about 150, from about 151 to
about
200, from about 201 to about 250, from about 251 to about 300, from about 301
to
about 350, from about 351 to about 400, from about 401 to about 450, from
about 451
to about 500, and from about 501 to about 550, and from about 551 to about
600,
from about 601 to about 650, from about 651 to about 700, from about 701 to
about
750, from about 751 to about 800, and from about 801 to about 860, of SEQ ID
NO:
11, or the complementary strand thereto, or the cDNA contained in a deposited
clone.
In this context "about" includes the particularly recited ranges, and ranges
larger or
smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both termini.
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12
In additional embodiments, the polynucleotides of the invention encode
functional
attributes of the corresponding protein. Preferred polypeptide fragments of
the
invention comprise, or alternatively consist of, the secreted protein having a
continuous series of deleted residues from the amino or the carboxy terminus,
or both.
Particularly, N-terminal deletions of the polypeptide can be described by the
general formula m-290 where m is an integer from 2 to 284, where m corresponds
to
the position of the amino acid residue identified in SEQ ID NO: 65. More in
particular, the invention provides polynucleotides encoding polypeptides
comprising,
or alternatively consisting of, an amino acid sequence selected from the
group: R-2 to
T-290; I-3 to T-290; F-4 to T-290; A-5 to T-290; V-6 to T-290; F-7 to T-290; I-
8 to
T-290; F-9 to T-290; M-10 to T-290; T-11 to T-290; Y-12 to T-290; W-13 to T-
290;
H-14 to T-290; L-15 to T-290; L-16 to T-290; N-17 to T-290; A-18 to T-290; F-
19 to
T-290; T-20 to T-290; V-21 to T-290; T-22 to T-290; V-23 to T-290; P-24 to T-
290;
K-25 to T-290; D-26 to T-290; L-27 to T-290; Y-28 to T-290; V-29 to T-290; V-
30 to
T-290; E-31 to T-290; Y-32 to T-290; G-33 to T-290; S-34 to T-290; N-35 to T-
290;
M-36 to T-290; T-37 to T-290; I-38 to T-290; E-39 to T-290; C-40 to T-290; K-
41 to
T-290; F-42 to T-290; P-43 to T-290; V-44 to T-290; E-45 to T-290; K-46 to T-
290;
Q-47 to T-290; L-48 to T-290; D-49 to T-290; L-50 to T-290; A-51 to T-290; A-
52 to
T-290; L-53 to T-290; I-54 to T-290; V-55 to T-290; Y-56 to T-290; W-57 to T-
290;
E-58 to T-290; M-59 to T-290; E-60 to T-290; D-61 to T-290; K-62 to T-290; N-
63 to
T-290; I-64 to T-290; I-65 to T-290; Q-66 to T-290; F-67 to T-290; V-68 to T-
290;
H-69 to T-290; G-70 to T-290; E-71 to T-290; E-72 to T-290; D-73 to T-290; L-
74 to
T-290; K-75 to T-290; V-76 to T-290; Q-77 to T-290; H-78 to T-290; S-79 to T-
290;
S-80 to T-290; Y-81 to T-290; R-82 to T-290; Q-83 to T-290; R-84 to T-290; A-
85 to
T-290; R-86 to T-290; L-87 to T-290; L-88 to T-290; K-89 to T-290; D-90 to T-
290;
Q-91 to T-290; L-92 to T-290; S-93 to T-290; L-94 to T-290; G-95 to T-290; N-
96 to
T-290; A-97 to T-290; A-98 to T-290; L-99 to T-290; Q-100 to T-290; I-101 to T-
290; T-102 to T-290; D-103 to T-290; V-104 to T-290; K-105 to T-290; L-106 to
T-
290; Q-107 to T-290; D-108 to T-290; A-109 to T-290; G-110 to T-290; V-111 to
T-
290; Y-112 to T-290; R-113 to T-290; C-114 to T-290; M-115 to T-290; I-116 to
T-
290; S-117 to T-290; Y-118 to T-290; G-119 to T-290; G-120 to T-290; A-121 to
T-
290; D-122 to T-290; Y-123 to T-290; K-124 to T-290; R-125 to T-290; I-126 to
T-
CA 02389722 2002-04-30
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13
290; T-127 to T-290; V-128 to T-290; K-129 to T-290; V-130 to T-290; N-131 to
T-
290; A-132 to T-290; P-133 to T-290; Y-134 to T-290; N-135 to T-290; K-136 to
T-
290; I-137 to T-290; N-138 to T-290; Q-139 to T-290; R-140 to T-290; I-141 to
T-
290; L-142 to T-290; V-143 to T-290; V-144 to T-290; D-145 to T-290; P-146 to
T-
290; V-147 to T-290; T-148 to T-290; S-149 to T-290; E-150 to T-290; H-151 to
T-
290; E-152 to T-290; L-153 to T-290; T-154 to T-290; C-155 to T-290; Q-156 to
T-
290; A-157 to T-290; E-158 to T-290; G-159 to T-290; Y-160 to T-290; P-161 to
T-
290; K-162 to T-290; A-163 to T-290; E-164 to T-290; V-165 to T-290; I-166 to
T-
290; W-167 to T-290; T-168 to T-290; S-169 to T-290; S-170 to T-290; D-171 to
T-
290; H-172 to T-290; Q-173 to T-290; V-174 to T-290; L-175 to T-290; S-176 to
T-
290; G-177 to T-290; K-178 to T-290; T-179 to T-290; T-180 to T-290; T-181 to
T-
290; T-182 to T-290; N-183 to T-290; S-184 to T-290; K-185 to T-290; R-186 to
T-
290; E-187 to T-290; E-188 to T-290; K-189 to T-290; L-190 to T-290; F-191 to
T-
290; N-192 to T-290; V-193 to T-290; T-194 to T-290; S-195 to T-290; T-196 to
T-
290; L-197 to T-290; R-198 to T-290; I-199 to T-290; N-200 to T-290; T-201 to
T-
290; T-202 to T-290; T-203 to T-290; N-204 to T-290; E-205 to T-290; I-206 to
T-
290; F-207 to T-290; Y-208 to T-290; C-209 to T-290; T-210 to T-290; F-211 to
T-
290; R-212 to T-290; R-213 to T-290; L-214 to T-290; D-215 to T-290; P-216 to
T-
290; E-217 to T-290; E-218 to T-290; N-219 to T-290; H-220 to T-290; T-221 to
T-
290; A-222 to T-290; E-223 to T-290; L-224 to T-290; V-225 to T-290; I-226 to
T-
290; P-227 to T-290; E-228 to T-290; L-229 to T-290; P-230 to T-290; L-231 to
T-
290; A-232 to T-290; H-233 to T-290; P-234 to T-290; P-235 to T-290; N-236 to
T-
290; E-237 to T-290; R-238 to T-290; T-239 to T-290; H-240 to T-290; L-241 to
T-
290; V-242 to T-290; I-243 to T-290; L-244 to T-290; G-245 to T-290; A-246 to
T-
290; I-247 to T-290; L-248 to T-290; L-249 to T-290; C-250 to T-290; L-251 to
T-
290; G-252 to T-290; V-253 to T-290; A-254 to T-290; L-255 to T-290; T-256 to
T-
290; F-257 to T-290; I-258 to T-290; F-259 to T-290; R-260 to T-290; L-261 to
T-
290; R-262 to T-290; K-263 to T-290; G-264 to T-290; R-265 to T-290; M-266 to
T-
290; M-267 to T-290; D-268 to T-290; V-269 to T-290; K-270 to T-290; K-271 to
T-
290; C-272 to T-290; G-273 to T-290; I-274 to T-290; Q-275 to T-290; D-276 to
T-
290; T-277 to T-290; N-278 to T-290; S-279 to T-290; K-280 to T-290; K-281 to
T-
290; Q-282 to T-290; S-283 to T-290; D-284 to T-290; and/or T-285 to T-290; of
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
14
SEQ B7 NO: 65. Moreover, fragments and variants of these polypeptides (such
as,
for example, fragments as described herein, polypeptides at least 80%, 85%,
90%,
95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides
encoded by the polynucleotide which hybridizes, under stringent conditions, to
the
polynucleotide encoding these polypeptides ) are encompassed by the invention.
Antibodies that bind polypeptides of the invention are also encompassed by the
invention. Polynucleotides encoding these polypeptides are also encompassed by
the
invention.
Additionally, the invention provides polynucleotides encoding polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the
following group of C-terminal deletions: M-1 to E-289; M-1 to E-288; M-1 to L-
287;
M-1 to H-286; M-1 to T-285; M-1 to D-284; M-1 to S-283; M-1 to Q-282; M-1 to K-
281; M-1 to K-280; M-1 to S-279; M-1 to N-278; M-1 to T-277; M-1 to D-276; M-1
to Q-275; M-1 to I-274; M-1 to G-273; M-1 to C-272; M-1 to K-271; M-1 to K-
270;
M-1 to V-269; M-1 to D-268; M-1 to M-267; M-1 to M-266; M-1 to R-265; M-1 to
G-264; M-1 to K-263; M-1 to R-262; M-1 to L-261; M-1 to R-260; M-1 to F-259; M-
1 to I-258; M-1 to F-257; M-1 to T-256; M-1 to L-255; M-1 to A-254; M-1 to V-
253;
M-1 to G-252; M-1 to L-251; M-1 to C-250; M-1 to L-249; M-1 to L-248; M-1 to I-
247; M-1 to A-246; M-1 to G-245; M-1 to L-244; M-1 to I-243; M-1 to V-242; M-1
to L-241; M-1 to H-240; M-1 to T-239; M-1 to R-238; M-1 to E-237; M-1 to N-
236;
M-1 to P-235; M-1 to P-234; M-1 to H-233; M-1 to A-232; M-1 to L-231; M-1 to P-
230; M-1 to L-229; M-1 to E-228; M-1 to P-227; M-1 to I-226; M-1 to V-225; M-1
to
L-224; M-1 to E-223; M-1 to A-222; M-1 to T-221; M-1 to H-220; M-1 to N-219; M-
1 to E-218; M-1 to E-217; M-1 to P-216; M-1 to D-215; M-1 to L-214; M-1 to R-
213;
M-1 to R-212; M-1 to F-211; M-1 to T-210; M-1 to C-209; M-1 to Y-208; M-1 to F-
207; M-1 to I-206; M-1 to E-205; M-1 to N-204; M-1 to T-203; M-1 to T-202; M-1
to
T-201; M-1 to N-200; M-1 to I-199; M-1 to R-198; M-1 to L-197; M-1 to T-196; M-
1
to S-195; M-1 to T-194; M-1 to V-193; M-1 to N-192; M-1 to F-191; M-1 to L-
190;
M-1 to K-189; M-1 to E-188; M-1 to E-187; M-1 to R-186; M-1 to K-185; M-1 to S-
184; M-1 to N-183; M-1 to T-182; M-1 to T-181; M-1 to T-180; M-1 to T-179; M-1
to K-178; M-1 to G-177; M-1 to S-176; M-1 to L-175; M-1 to V-174; M-1 to Q-
173;
M-1 to H-172; M-1 to D-171; M-1 to S-170; M-1 to S-169; M-1 to T-168; M-1 to W-
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
167; M-1 to I-166; M-1 to V-165; M-1 to E-164; M-1 to A-163; M-1 to K-162; M-1
to P-161; M-1 to Y-160; M-1 to G-159; M-1 to E-158; M-1 to A-157; M-1 to Q-
156;
M-1 to C-155; M-1 to T-154; M-1 to L-153; M-1 to E-152; M-1 to H-151; M-1 to E-
150; M-1 to S-149; M-1 to T-148; M-1 to V-147; M-1 to P-146; M-1 to D-145; M-1
5 to V-144; M-1 to V-143; M-1 to L-142; M-1 to I-141; M-1 to R-140; M-1 to Q-
139;
M-1 to N-138; M-1 to I-137; M-1 to K-136; M-1 to N-135; M-1 to Y-134; M-1 to P-
133; M-1 to A-132; M-1 to N-131; M-1 to V-130; M-1 to K-129; M-1 to V-128; M-1
to T-127; M-1 to I-126; M-1 to R-125; M-1 to K-124; M-1 to Y-123; M-1 to D-
122;
M-1 to A-121; M-1 to G-120; M-1 to G-119; M-1 to Y-118; M-1 to S-117; M-1 to I-
10 116; M-1 to M-115; M-1 to C-114; M-1 to R-113; M-1 to Y-112; M-1 to V-111;
M-1
to G-110; M-1 to A-109; M-1 to D-108; M-1 to Q-107; M-1 to L-106; M-1 to K-
105;
M-1 to V-104; M-1 to D-103; M-1 to T-102; M-1 to I-101; M-1 to Q-100; M-1 to L-
99; M-1 to A-98; M-1 to A-97; M-1 to N-96; M-1 to G-95; M-1 to L-94; M-1 to S-
93;
M-1 to L-92; M-1 to Q-91; M-1 to D-90; M-1 to K-89; M-1 to L-88; M-1 to L-87;
M-
15 1 to R-86; M-1 to A-85; M-1 to R-84; M-1 to Q-83; M-1 to R-82; M-1 to Y-81;
M-1
to S-80; M-1 to S-79; M-1 to H-78; M-1 to Q-77; M-1 to V-76; M-1 to K-75; M-1
to
L-74; M-1 to D-73; M-1 to E-72; M-1 to E-71; M-1 to G-70; M-1 to H-69; M-1 to
V-
68; M-1 to F-67; M-1 to Q-66; M-1 to I-65; M-1 to I-64; M-1 to N-63; M-1 to K-
62;
M-1 to D-61; M-1 to E-60; M-1 to M-59; M-1 to E-58; M-1 to W-57; M-1 to Y-56;
M-1 to V-55; M-1 to I-54; M-1 to L-53; M-1 to A-52; M-1 to A-51; M-1 to L-50;
M-
1 to D-49; M-1 to L-48; M-1 to Q-47; M-1 to K-46; M-1 to E-45; M-1 to V-44; M-
1
to P-43; M-1 to F-42; M-1 to K-41; M-1 to C-40; M-1 to E-39; M-1 to I-38; M-1
to
T-37; M-1 to M-36; M-1 to N-35; M-1 to S-34; M-1 to G-33; M-1 to Y-32; M-1 to
E-
31; M-1 to V-30; M-1 to V-29; M-1 to Y-28; M-1 to L-27; M-1 to D-26; M-1 to K-
25; M-1 to P-24; M-1 to V-23; M-1 to T-22; M-1 to V-21; M-1 to T-20; M-1 to F-
19;
M-1 to A-18; M-1 to N-17; M-1 to L-16; M-1 to L-15; M-1 to H-14; M-1 to W-13;
M-1 to Y-12; M-1 to T-11; M-1 to M-10; M-1 to F-9; M-1 to I-8; and/or M-1 to F-
7;
of SEQ ID NO: 65. Moreover, fragments and variants of these polypeptides (such
as,
for example, fragments as described herein, polypeptides at least 80%, 85%,
90%,
95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides
encoded by the polynucleotide which hybridizes, under stringent conditions, to
the
polynucleotide encoding these polypeptides ) are encompassed by the invention.
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
16
Antibodies that bind polypeptides of the invention are also encompassed by the
invention. Polynucleotides encoding these polypeptides are also encompassed by
the
invention.
Also as mentioned above, even if deletion of one or more amino acids from
the C-terminus of a protein results in modification of loss of one or more
biological
functions of the protein (e.g., ability to inhibit the Mixed Lymphocyte
Reaction),
other functional activities (e.g., biological activities, ability to
multimerize, ability to
bind ligand, ability to generate antibodies, ability to bind antibodies) may
still be
retained. For example, the ability of the shortened polypeptide to induce
and/or bind
to antibodies which recognize the complete or mature forms of the polypeptide
generally will be retained when less than the majority of the residues of the
complete
or mature polypeptide are removed from the C-terminus. Whether a particular
polypeptide lacking C-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a polypeptide
with a
large number of deleted C-terminal amino acid residues may retain some
biological or
immunogenic activities. In fact, peptides composed of as few as six amino acid
residues may often evoke an immune response. Accordingly, the present
invention
further provides polypeptides having one or more residues deleted from the
carboxy
terminus of the amino acid sequence of the polypeptide shown in Figures lA-B
(SEQ
ID N0:65), as described by the general formula 1-n, where n is an integer from
6 to
284, where n corresponds to the position of the amino acid residue identified
in SEQ
ID NO: 65.
More in particular, the invention provides polynucleotides encoding
polypeptides comprising, or alternatively consisting of, an amino acid
sequence
selected from the group of N-terminal deletions of the mature extracellular
portion of
the B7-H6 protein (SEQ ID NO: 121): T-20 to P-235; V-21 to P-235; T-22 to P-
235;
V-23 to P-235; P-24 to P-235; K-25 to P-235; D-26 to P-235; L-27 to P-235; Y-
28 to
P-235; V-29 to P-235; V-30 to P-235; E-31 to P-235; Y-32 to P-235; G-33 to P-
235;
S-34 to P-235; N-35 to P-235; M-36 to P-235; T-37 to P-235; I-38 to P-235; E-
39 to
P-235; C-40 to P-235; K-41 to P-235; F-42 to P-235; P-43 to P-235; V-44 to P-
235;
E-45 to P-235; K-46 to P-235; Q-47 to P-235; L-48 to P-235; D-49 to P-235; L-
50 to
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
17
P-235; A-51 to P-235; A-52 to P-235; L-53 to P-235; I-54 to P-235; V-55 to P-
235;
Y-56 to P-235; W-57 to P-235; E-58 to P-235; M-59 to P-235; E-60 to P-235; D-
61 to
P-235; K-62 to P-235; N-63 to P-235; I-64 to P-235; I-65 to P-235; Q-66 to P-
235; F-
67 to P-235; V-68 to P-235; H-69 to P-235; G-70 to P-235; E-71 to P-235; E-72
to P-
235; D-73 to P-235; L-74 to P-235; K-75 to P-235; V-76 to P-235; Q-77 to P-
235; H-
78 to P-235; S-79 to P-235; S-80 to P-235; Y-81 to P-235; R-82 to P-235; Q-83
to P-
235; R-84 to P-235; A-85 to P-235; R-86 to P-235; L-87 to P-235; L-88 to P-
235; K-
89 to P-235; D-90 to P-235; Q-91 to P-235; L-92 to P-235; S-93 to P-235; L-94
to P-
235; G-95 to P-235; N-96 to P-235; A-97 to P-235; A-98 to P-235; L-99 to P-
235; Q-
100 to P-235; I-101 to P-235; T-102 to P-235; D-103 to P-235; V-104 to P-235;
K-
105 to P-235; L-106 to P-235; Q-107 to P-235; D-108 to P-235; A-109 to P-235;
6-
110 to P-235; V-111 to P-235; Y-112 to P-235; R-113 to P-235; C-114 to P-235;
M-
115 to P-235; I-116 to P-235; S-117 to P-235; Y-118 to P-235; G-119 to P-235;
6-
120 to P-235; A-121 to P-235; D-122 to P-235; Y-123 to P-235; K-124 to P-235;
R-
125 to P-235; I-126 to P-235; T-127 to P-235; V-128 to P-235; K-129 to P-235;
V-
130 to P-235; N-131 to P-235; A-132 to P-235; P-133 to P-235; Y-134 to P-235;
N-
135 to P-235; K-136 to P-235; I-137 to P-235; N-138 to P-235; Q-139 to P-235;
8-
140 to P-235; I-141 to P-235; L-142 to P-235; V-143 to P-235; V-144 to P-235;
D-
145 to P-235; P-146 to P-235; V-147 to P-235; T-148 to P-235; S-149 to P-235;
E-
150 to P-235; H-151 to P-235; E-152 to P-235; L-153 to P-235; T-154 to P-235;
C-
155 to P-235; Q-156 to P-235; A-157 to P-235; E-158 to P-235; G-159 to P-235;
Y-
160 to P-235; P-161 to P-235; K-162 to P-235; A-163 to P-235; E-164 to P-235;
V-
165 to P-235; I-166 to P-235; W-167 to P-235; T-168 to P-235; S-169 to P-235;
S-
170 to P-235; D-171 to P-235; H-172 to P-235; Q-173 to P-235; V-174 to P-235;
L-
175 to P-235; S-176 to P-235; G-177 to P-235; K-178 to P-235; T-179 to P-235;
T-
180 to P-235; T-181 to P-235; T-182 to P-235; N-183 to P-235; S-184 to P-235;
K-
185 to P-235; R-186 to P-235; E-187 to P-235; E-188 to P-235; K-189 to P-235;
L-
190 to P-235; F-191 to P-235; N-192 to P-235; V-193 to P-235; T-194 to P-235;
5-
195 to P-235; T-196 to P-235; L-197 to P-235; R-198 to P-235; I-199 to P-235;
N-
200 to P-235; T-201 to P-235; T-202 to P-235; T-203 to P-235; N-204 to P-235;
E-
205 to P-235; I-206 to P-235; F-207 to P-235; Y-208 to P-235; C-209 to P-235;
T-210
to P-235; F-211 to P-235; R-212 to P-235; R-213 to P-235; L-214 to P-235; D-
215 to
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WO 01/34768 PCT/US00/30039
18
P-235; P-216 to P-235; E-217 to P-235; E-218 to P-235; N-219 to P-235; H-220
to P-
235; T-221 to P-235; A-222 to P-235; E-223 to P-235; L-224 to P-235; V-225 to
P-
235; I-226 to P-235; P-227 to P-235; E-228 to P-235; L-229 to P-235; and/or P-
230 to
P-235; of SEQ ID NO: 65. Moreover, fragments and variants of these
polypeptides
(such as, for example, fragments as described herein, polypeptides at least
80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and
polypeptides
encoded by the polynucleotide which hybridizes, under stringent conditions, to
the
polynucleotide encoding these polypeptides ) are encompassed by the invention.
Antibodies that bind polypeptides of the invention are also encompassed by the
invention. Polynucleotides encoding these polypeptides are also encompassed by
the
invention.
Additionally, the invention provides polynucleotides encoding polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the
group of C-terminal deletions of the mature extracellular portion of the B7-H6
protein
(SEQ ID NO: 121): F-19 to P-234; F-19 to H-233; F-19 to A-232; F-19 to L-231;
F-
19 to P-230; F-19 to L-229; F-19 to E-228; F-19 to P-227; F-19 to I-226; F-19
to V-
225; F-19 to L-224; F-19 to E-223; F-19 to A-222; F-19 to T-221; F-19 to H-
220; F-
19 to N-219; F-19 to E-218; F-19 to E-217; F-19 to P-216; F-19 to D-215; F-19
to L-
214; F-19 to R-213; F-19 to R-212; F-19 to F-211; F-19 to T-210; F-19 to C-
209; F-
19 to Y-208; F-19 to F-207; F-19 to I-206; F-19 to E-205; F-19 to N-204; F-19
to T-
203; F-19 to T-202; F-19 to T-201; F-19 to N-200; F-19 to I-199; F-19 to R-
198; F-19
to L-197; F-19 to T-196; F-19 to S-195; F-19 to T-194; F-19 to V-193; F-19 to
N-
192; F-19 to F-191; F-19 to L-190; F-19 to K-189; F-19 to E-188; F-19 to E-
187; F-
19 to R-186; F-19 to K-185; F-19 to S-184; F-19 to N-183; F-19 to T-182; F-19
to T-
181; F-19 to T-180; F-19 to T-179; F-19 to K-178; F-19 to G-177; F-19 to S-
176; F-
19 to L-175; F-19 to V-174; F-19 to Q-173; F-19 to H-172; F-19 to D-171; F-19
to 5-
170; F-19 to S-169; F-19 to T-168; F-19 to W-167; F-19 to I-166; F-19 to V-
165; F-
19 to E-164; F-19 to A-163; F-19 to K-162; F-19 to P-161; F-19 to Y-160; F-19
to 6-
159; F-19 to E-158; F-19 to A-157; F-19 to Q-156; F-19 to C-155; F-19 to T-
154; F-
19 to L-153; F-19 to E-152; F-19 to H-151; F-19 to E-150; F-19 to S-149; F-19
to T-
148; F-19 to V-147; F-19 to P-146; F-19 to D-145; F-19 to V-144; F-19 to V-
143; F-
19 to L-142; F-19 to I-141; F-19 to R-140; F-19 to Q-139; F-19 to N-138; F-19
to I-
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19
137; F-19 to K-136; F-19 to N-135; F-19 to Y-134; F-19 to P-133; F-19 to A-
132; F-
19 to N-131; F-19 to V-130; F-19 to K-129; F-19 to V-128; F-19 to T-127; F-19
to I-
126; F-19 to R-125; F-19 to K-124; F-19 to Y-123; F-19 to D-122; F-19 to A-
121; F-
19 to G-120; F-19 to G-119; F-19 to Y-118; F-19 to S-117; F-19 to I-116; F-19
to M-
115; F-19 to C-114; F-19 to R-113; F-19 to Y-112; F-19 to V-111; F-19 to G-
110; F-
19 to A-109; F-19 to D-108; F-19 to Q-107; F-19 to L-106; F-19 to K-105; F-19
to V-
104; F-19 to D-103; F-19 to T-102; F-19 to I-101; F-19 to Q-100; F-19 to L-99;
F-19
to A-98; F-19 to A-97; F-19 to N-96; F-19 to G-95; F-19 to L-94; F-19 to S-93;
F-19
to L-92; F-19 to Q-91; F-19 to D-90; F-19 to K-89; F-19 to L-88; F-19 to L-87;
F-19
to R-86; F-19 to A-85; F-19 to R-84; F-19 to Q-83; F-19 to R-82; F-19 to Y-81;
F-19
to S-80; F-19 to S-79; F-19 to H-78; F-19 to Q-77; F-19 to V-76; F-19 to K-75;
F-19
to L-74; F-19 to D-73; F-19 to E-72; F-19 to E-71; F-19 to G-70; F-19 to H-69;
F-19
to V-68; F-19 to F-67; F-19 to Q-66; F-19 to I-65; F-19 to I-64; F-19 to N-63;
F-19 to
K-62; F-19 to D-61; F-19 to E-60; F-19 to M-59; F-19 to E-58; F-19 to W-57; F-
19 to
Y-56; F-19 to V-55; F-19 to I-54; F-19 to L-53; F-19 to A-52; F-19 to A-51; F-
19 to
L-50; F-19 to D-49; F-19 to L-48; F-19 to Q-47; F-19 to K-46; F-19 to E-45; F-
19 to
V-44; F-19 to P-43; F-19 to F-42; F-19 to K-41; F-19 to C-40; F-19 to E-39; F-
19 to
I-38; F-19 to T-37; F-19 to M-36; F-19 to N-35; F-19 to S-34; F-19 to G-33; F-
19 to
Y-32; F-19 to E-31; F-19 to V-30; F-19 to V-29; F-19 to Y-28; F-19 to L-27; F-
19 to
D-26; and/or F-19 to K-25; of SEQ ID NO: 65. Moreover, fragments and variants
of
these polypeptides (such as, for example, fragments as described herein,
polypeptides
at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these
polypeptides and polypeptides encoded by the polynucleotide which hybridizes,
under
stringent conditions, to the polynucleotide encoding these polypeptides ) are
encompassed by the invention. Antibodies that bind polypeptides of the
invention are
also encompassed by the invention. Polynucleotides encoding these polypeptides
are
also encompassed by the invention.
In addition, any of the above listed N- or C-terminal deletions can be
combined to produce a N- and C-terminal deleted polypeptide. The invention
also
provides polypeptides comprising, or alternatively consisting of, one or more
amino
acids deleted from both the amino and the carboxyl termini, which may be
described
generally as having residues m-n of SEQ ID NO: 65, where n and m are integers
as
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described above. Polynucleotides encoding these polypeptides are also
encompassed
by the invention.
The present invention is also directed to proteins containing polypeptides at
least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a
5 polypeptide sequence set forth herein as m-n. In preferred embodiments, the
application is directed to proteins containing polypeptides at least 80%, 85%,
90%,
95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid
sequence of the specific N- and C-terminal deletions recited herein.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
10 Also included are polynucleotide sequences encoding a polypeptide
consisting
of a portion of the complete amino acid sequence encoded by a cDNA clone
contained in ATCC Deposit No. PTA-849, where this portion excludes any integer
of
amino acid residues from 1 to about 284 amino acids from the amino terminus of
the
complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit
1 S No. PTA-849, or any integer of amino acid residues from 1 to about 284
amino acids
from the carboxy terminus, or any combination of the above amino terminal and
carboxy terminal deletions, of the complete amino acid sequence encoded by the
cDNA clone contained in ATCC Deposit No. PTA-849. Polypeptides encoded by
these polynucleotides also are encompassed by the invention.
20 As described herein or otherwise known in the art, the polynucleotides of
the
invention have uses that include, but are not limited to, serving as probes or
primers
in chromosome identification, chromosome mapping, and linkage analysis.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of immune system tissues) or cell types) present
in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, diseases and/or disorders involving immune system activation,
stimulation and/or surveillance, particularly involving T cells and/or
neutrophils, as
well as diseases and/or disorders of the placenta. Similarly, polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s).
Particularly
contemplated are the use of antibodies directed against the extracellular
portion of this
protein which act as antagonists for the activity of the B7-H6 protein. Such
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21
antagonistic antibodies would be useful for the prevention and/or inhibition
of such
biological activities as are disclosed herein (e.g. T cell modulated
activities).
For a number of disorders of the above tissues or cells, particularly of the
immune system, expression of this gene at significantly higher or lower levels
may be
routinely detected in certain tissues or cell types (e.g., immune, placental,
cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression
level in healthy tissue or bodily fluid from an individual not having the
disorder.
The tissue distribution in immune cells (e.g., T-cells, dendritic cells), and
the
homology to members of the B7 family of ligands, indicates that the
polynucleotides
and polypeptides corresponding to this gene are useful for the diagnosis,
detection
and/or treatment of diseases and/or disorders involving immune system
activation,
stimulation and/or surveillance, particularly as relating to T cells and/or
neutrophils.
In particular, the translation product of the B7-H6 gene may be involved in
the
costimulation of T cells, binding to ICOS, and/or may play a role in
modulation of the
expression of particular cytokines, for example.
More generally, the tissue distribution in immune system cells indicates that
this gene product may be involved in the regulation of cytokine production,
antigen
presentation, or other processes that may also suggest a usefulness in the
treatment of
cancer (e.g. by boosting immune responses). Since the gene is expressed in
cells of
lymphoid origin, the gene or protein, as well as, antibodies directed against
the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues. Therefore it may be also used as an agent for
immunological
disorders including arthritis, asthma, immune deficiency diseases such as
AIDS,
leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and
psoriasis. In addition, this gene product may have commercial utility in the
expansion
of stem cells and committed progenitors of various blood lineages, and in the
differentiation and/or proliferation of various cell types. Protein, as well
as, antibodies
directed against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Furthermore, the protein
may also
be used to determine biological activity, to raise antibodies, as tissue
markers, to
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22
isolate cognate ligands or receptors, to identify agents that modulate their
interactions,
in addition to its use as a nutritional supplement.
Specific expression within the placenta suggests that this gene product may
play a role in the proper establishment and maintenance of placental function.
Alternately, this gene product may be produced by the placenta and then
transported
to the embryo, where it may play a crucial role in the development and/or
survival of
the developing embryo or fetus. Expression of this gene product in a vascular-
rich
tissue such as the placenta also suggests that this gene product may be
produced more
generally in endothelial cells or within the circulation. In such instances,
it may play
more generalized roles in vascular function, such as in angiogenesis. It may
also be
produced in the vasculature and have effects on other cells within the
circulation, such
as hematopoietic cells. It may serve to promote the proliferation, survival,
activation,
and/or differentiation of hematopoietic cells, as well as other cells
throughout the
body.
Representative uses are described in the "Immune Activity" and "Infectious
Disease" sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and
elsewhere
herein.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares infant brain 1NIB and to a lesser extent in normalized infant brain
cDNA;
Soares placenta Nb2HP; Soares fetal heart NbHHI9W; Soares NhHMPu_S1;
Human Pancreas Tumor, Reexcision; NCI CGAP_Kidl l; NCI CGAP-Brn23;
Soares melanocyte 2NbHM; Soares-placenta 8to9weeks 2NbHP8to9W;
Soares_fetal liver spleen 1NFLS_S1; Hodgkin's Lymphoma I; NCI CGAP_Lul;
Soares breast 2NbHBst; Normal colon; 12 Week Early Stage Human II, Reexcision;
Human 8 Week Whole Embryo; Soares-parathyroid tumor NbHPA; Human
Cerebellum; Soares total fetus Nb2HF8 9w; Human Fetal Brain, normalized c5-11
26; Kidney Cortex; Rectum tumour; NCI CGAP_KidB; Lung, Cancer (4005313 A3):
Invasive Poorly Differentiated Lung Adenocarcinoma,; Human heart cDNA
(YNakamura); HEL cell line; Human Colon Cancer,re-excision; Human adult
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23
(K.Okubo); Human pancreatic islet; Human Amygdala,re-excision; LNCAP prostate
cell line; NCI CGAP_Lyml2; NCI CLAP-Alvl; Human Neutrophil; KMH2;
Ovary, Cancer(4004650 A3): Well-Differentiated Micropapillary Serous
Carcinoma;
12 Week Old Early Stage Human, II; Human Osteoblasts II; Human Umbilical Vein
Endothelial Cells, uninduced; Human Activated T-Cells; T-Cell PHA 24 hrs;
Macrophage-oxLDL; Human Adipose; Soares NSF F8 9W_OT PA P S 1; Human
Thymus Stromal Cells; Ovarian Tumor 10-3-95; Human Liver, normal;
Hepatocellular Tumor, re-excision; Colon Tumor; Human Testes, Reexcision;
Pancreas normal PCA4 No; Human Amygdala; NCI CGAP_Brn25; Smooth
muscle,control; NCI CGAP_Kid3; NCI CGAP-Kids;
Soares multiple sclerosis 2NbHMSP; Human fetal heart, Lambda ZAP Express;
Soares ovary tumor NbHOT; H. Frontal cortex,epileptic,re-excision; Human
Endometrial Tumor; Activated T-cell(12h)/Thiouridine-re-excision; Osteoblasts;
Keratinocyte; Colon Tumor II; Soares-pregnant uterus NbHPU; Soares testis NHT
and NCI CGAP GCB1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: neurological diseases and/or disorders. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the neural system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily
fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
Predominant expression in infant brain tissue suggests utility for the study
and/or treatment of neurological, developmental and behavioral disorders. The
tissue
distribution suggests that polynucleotides, translation products and
antibodies
corresponding to this clone are useful for the detection and/or treatment of
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24
neurodegenerative disease states and behavioral disorders such as Alzheimers
Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception. In
addition, the
gene or gene product may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, or sexually-
linked
disorders. Additionally, translation products corresponding to this gene, as
well as
antibodies directed against these translation products, may show utility as a
tumor
marker and/or immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
Preferred polypeptides of the invention comprise, or alternatively consist of,
a
polypeptide having the amino acid sequence set out in the sequence listing as
SEQ >17
NO: 124. Moreover, fragments and variants of these polypeptides (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
The gene encoding the disclosed cDNA is thought to reside on chromosome 6.
Accordingly, polynucleotides related to this invention have uses, such as, for
example, as a marker in linkage analysis for chromosome 6.
This gene is expressed primarily in the following tissues/cDNA libraries:
NCI CGAP-GCB 1 and to a lesser extent in Soares~regnant uterus NbHPU;
Weizmann Olfactory Epithelium; NCI CGAP_Kidl l;
Soares multiple sclerosis 2NbHMSP; Soares-parathyroid tumor NbHPA; Human
Fetal Brain, normalized C500HE; Brain pons; Lingual Gyrus; CD34+cells, II,
FRACTION 2; H. Striatum Depression, subt; NCI CGAP-Lul; human colon cancer;
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Salivary Gland; NTERA2 teratocarcinoma cell line+retinoic acid (14 days);
Hepatocellular Tumor,re-excision; Alzheimers, spongy change; NCI CGAP-ColO;
Breast, Cancer: (4004943 AS); Human Chronic Synovitis; H. Kidney Medulla, re-
excision; Human Bone Marrow, re-excision; Clontech human aorta polyA+ mRNA
5 (#6572); Stromal cell TF274; Human Whole Six Week Old Embryo;
NCI CGAP Co3; Human Gall Bladder; Fetal Liver, subtraction II; Colon Normal
II;
Primary Dendritic cells,frac 2; Human Adult Pulmonary,re-excision;
NCI CGAP-Kids; Spleen, Chronic lymphocytic leukemia; H. Frontal
cortex,epileptic,re-excision; NCI CGAP_LuS; Colon Tumor II and
10 Soares total fetus Nb2HF8 9w.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: cancer and/or disorders of the neural system. Similarly,
polypeptides
15 and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the nervous system,
expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily
fluids
20 (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution of this gene suggests that polynucleotides,
translation
25 products and antibodies corresponding to this gene may be useful for the
treatment of
cancer and/or disorders of the neural system.
The tissue distribution suggests that polynucleotides, translation products
and
antibodies corresponding to this clone are useful for the detection and/or
treatment of
neurodegenerative disease states and behavioural disorders such as Alzheimers
Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
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26
including disorders in feeding, sleep patterns, balance, and perception. In
addition, the
gene or gene product may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, or sexually-
linked
disorders. Additionally, translation products corresponding to this gene, as
well as
antibodies directed against these translation products, may show utility as a
tumor
marker and/or immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4
This sequence encodes an open reading frame that is homologous to the
human Delta 1 protein (See Genbank Accession AAB61286). Delta-1 is thought to
have a role in vascular cell growth.
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares senescent fibroblasts NbHSF; Soares total fetus Nb2HF8 9w and to a
lesser extent in Human Fetal Brain, random primed; Glioblastoma;
NCI CGAP-Lym 12; NCI CGAP-Pr2; NCI CGAP-Pr22; NCI CGAP_Brn25;
NCI CGAP-Kid3; NCI CGAP LuS; Keratinocyte and
Soares-pregnant uterus NbHPU.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: diseases and/or disorders of epithelial and vascular cells,
and cancers.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
epithelial and vascular systems, expression of this gene at significantly
higher or
lower levels may be routinely detected in certain tissues or cell types (e.g.,
epithelial,
vascular, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
an individual having such a disorder, relative to the standard gene expression
level,
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27
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution of this gene suggest that this clone or the protein
product of this gene may be useful for the treatment and diagnosis of
disorders of the
epidermal system, the fetal system and cancer. Polynucleotides, translation
products
and antibodies corresponding to this gene may play a role in the growth of
epithelial
cells or vascular cells. Accordingly, antibodies directed against translation
products
corresponding to this gene may be useful in the prevention and/or inhibition
of
cellular growth mediated by translation products corresponding to this gene.
An
application of antagonists of this nature are particularly useful in diseases
and/or
disorders involving epithelial and/or vascular cell growth, such as would be
found in
melanomas or other cancers, for example. Additionally, translation products
corresponding to this gene, as well as antibodies directed against these
translation
products, may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
The gene encoding the disclosed cDNA is thought to reside on chromosome 9.
Accordingly, polynucleotides related to this invention have uses, such as, for
example, as a marker in linkage analysis for chromosome 9.
This gene is expressed primarily in the following tissues/cDNA libraries:
Stratagene lung (#937210); Osteoblasts and to a lesser extent in Colon
Carcinoma;
human tonsils; NCI CGAP_LuS; Soares fetal liver spleen-1NFLS S1;
Soares NFL T GBC_S1; Soares placenta Nb2HP; Soares fetal heart NbHHI9W;
Primary Dendritic Cells, lib 1; Soares fetal liver spleen 1NFLS; Liver HepG2
cell
line.; NCI CGAP_Col2; Human Normal Breast; B Cell lymphoma; Human T-cell
lymphoma,re-excision; Breast, Normal: (4005522:y B2); Liver, Hepatoma; Human
Hippocampus; Human Adrenal Gland Tumor; Human Placenta; Human Testes,
Reexcision; Human Adult Pulmonary,re-excision;
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28
Soares senescent fibroblasts NbIISF; NCI CGAP_Kid3; NCI CGAP Kids;
Human fetal heart, Lambda ZAP Express and Soares infant brain 1NIB.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: pulmonary diseases and/or disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the pulmonary system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., pulmonary, cancerous and
wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy
tissue or bodily fluid from an individual not having the disorder.
The tissue distribution of this sequence predicts that it is useful in the
diagnosis and treatment of lung cancer, emphysema, etc. The tissue
distribution
suggests that polynucleotides, translation products and antibodies
corresponding to
this gene are useful for the detection and treatment of disorders associated
with
developing lungs, particularly in premature infants where the lungs are the
last tissues
to develop. The tissue distribution suggests that the protein product of this
clone is
useful for the diagnosis and intervention of lung tumors, since the gene may
be
involved in the regulation of cell division, particularly since it is
expressed in fetal
tissue. Additionally, translation products corresponding to this gene, as well
as
antibodies directed against these translation products, may show utility as a
tumor
marker and/or immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
This gene is expressed primarily in the following tissues/cDNA libraries:
Keratinocyte and to a lesser extent in NCI CGAP_CLLI; Soares breast 3NbHBst;
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Soares fetal liver spleen-1NFLS S 1; Soares NhHMPu S 1; NCI CGAP Co2;
Lung, Cancer (4005163 B7): Invasive, Poorly Diff. Adenocarcinoma, Metastatic;
Human Lung Cancer,re-excision; NCI CGAP-Co9; Stratagene NT2 neuronal
precursor 937230; NCI CGAP_Utl; Colon Tumor; Pancreas Tumor PCA4 Tu and
Soares fetal liver spleen 1NFLS.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: diseases and/or disorders of epithelial tissues. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of epithelial tissues,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., epithelial, cancerous and wounded tissues) or
bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution of this sequence predicts that it is useful in the
diagnosis and treatment of psoriasis, epidermolysis bullosa, wound repair,
lung
cancer, emphysema. The tissue distribution suggests that the protein product
of this
clone is useful for the treatment, diagnosis, and/or prevention of various
skin
disorders including congenital disorders (i.e. nevi, moles, freckles,
Mongolian spots,
hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses,
Bowen's
disease, basal cell carcinoma, squamous cell carcinoma, malignant melanoma,
Paget's
disease, mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation
of the
skin (i.e.wounds, rashes, prickly heat disorder, psoriasis, dermatitis),
atherosclerosis,
uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupus
erythematosus,
vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus),
keloids, striae, erythema, petechiae, purpura, and xanthelasma. Moreover, such
disorders may predispose increased susceptibility to viral and bacterial
infections of
the skin (i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpes
zoster,
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boils, cellulitis, erysipelas, impetigo, tinea, althletes foot, and ringworm).
Additionally, translation products corresponding to this gene, as well as
antibodies
directed against these translation products, may show utility as a tumor
marker and/or
immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
Preferred polypeptides of the invention comprise, or alternatively consist of,
a
10 polypeptide having the amino acid sequence set out in the sequence listing
as SEQ ID
NO: 125. Moreover, fragments and variants of these polypeptides (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
15 encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
The gene encoding the disclosed cDNA is thought to reside on chromosome 7.
Accordingly, polynucleotides related to this invention have uses, such as, for
20 example, as a marker in linkage analysis for chromosome 7.
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Endometrial Tumor and to a lesser extent in
Soares~regnant uterus NbHPU; Primary Dendritic Cells, lib 1; Chromosome 7
Fetal Brain cDNA Library; Colon Carcinoma; Soares-parathyroid tumor NbHPA;
25 Soares total fetus Nb2HF8 9w; Soares fetal liver spleen-1NFLS_S1; Soares
infant brain 1NIB; Chromosome 7 HeLa cDNA Library; STRATAGENE Human
skeletal muscle cDNA library, cat. #93621 S.; Hemangiopericytoma; Soares
breast
3NbHBst; B-cells (stimulated); Human Fetal Heart; Hodgkin's Lymphoma II;
Soares testis NHT; Subtracted human retinal pigment epithelium (RPE); H.
30 Leukocytes, normalized cot > SOOA; Chromosome 7 Placental cDNA Library;
NCI CGAP-Eso2; Healing Abdomen wound,70&90 min post incision;
NCI CGAP-Lul; H. cerebellum, Enzyme subtracted; Early Stage Human Lung,
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subtracted; Human Soleus; Hepatocellular Tumor; Human Osteosarcoma; Human
endometrial stromal cells; Synovial hypoxia; Healing groin wound, 6.5 hours
post
incision; H. Lymph node breast Cancer; Human Adult Small Intestine;
NCI CGAP_Pr28; Olfactory epithelium,nasalcavity; Human Placenta (re-excision);
Human Thymus; Human Fetal Brain; Soares breast 2NbHBst; NCI CGAP_CoB;
Human Fetal Kidney, Reexcision; Human Synovial Sarcoma; human tonsils;
NCI CGAP_GC6; NCI CGAP Brn25; Spleen, Chronic lymphocytic leukemia; H.
Frontal cortex,epileptic,re-excision; Soares fetal lung NbHLI9W; T cell helper
II;
Soares NhHMPu S 1 and NCI CGAP GCB 1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: endometrial diseases and/or disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
1 S probes for differential identification of the tissues) or cell type(s).
For a number of
disorders of the above tissues or cells, particularly of the endometrium and
female
reproductive system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues or cell types (e.g., endometrial,
female
reproductive, cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
an individual having such a disorder, relative to the standard gene expression
level,
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution of this sequence predicts that it is useful in the
maintenance of a healthy endometrium and pregnant uterus. The tissue
distribution
suggests that the protein product of this clone is useful for treating female
infertility.
The protein product is likely involved in preparation of the endometrium of
implantation and could be administered either topically or orally.
Alternatively, this
gene could be transfected in gene-replacement treatments into the cells of the
endometrium and the protein products could be produced. Similarly, these
treatments
could be performed during artificial insemination for the purpose of
increasing the
likelihood of implantation and development of a healthy embryo. In both cases
this
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32
gene or its gene product could be administered at later stages of pregnancy to
promote
healthy development of the endometrium. Additionally, translation products
corresponding to this gene, as well as antibodies directed against these
translation
products, may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
This gene is expressed primarily in the following tissues/cDNA libraries:
Smooth Muscle- HASTE normalized and to a lesser extent in
Soares fetal heart NbHHI9W; Soares testis NHT; Primary Dendritic Cells, lib 1;
Soares adult brain N2b5HB55Y; Human Fetal Kidney, Reexcision; Soares fetal
liver
spleen 1NFLS; Larynx carcinoma III; Stratagene HeLa cell s3 937216;
NCI CGAP_GCB1; H. Lymph node breast Cancer; Human Fetal Kidney; T-Cell
PHA 24 hrs; Stratagene endothelial cell 937223; NCI CGAP-CoB; Human
Microvascular Endothelial Cells, fract. A; NCI CGAP Brn23;
Soares_multiple sclerosis 2NbHMSP; Soares~lacenta 8to9weeks_2NbHP8to9W;
Activated T-cell(12h)/Thiouridine-re-excision; Osteoblasts;
Soares-parathyroid tumor NbHPA; Soares melanocyte 2NbHM;
Soares fetal lung NbHLI9W; Soares_pregnant uterus NbHPU; Soares placenta
Nb2HP; Soares infant brain 1NIB; Thyroid Normal (SDCA2 No); Lung, Cancer
(4005163 B7): Invasive, Poorly Diff. Adenocarcinoma, Metastatic; Human heart
cDNA (YNakamura); CD40 activated monocyte dendridic cells; NCI CGAP AA1;
Smooth muscle, ILIb induced; Human endometrial stromal cells-treated with
progesterone; Synovial hypoxia; NCI CGAP-Pr22; Macrophage-oxLDL; Human
Placenta (re-excision); NCI CGAP Br2; Human Activated T-Cells, re-excision;
Human Thymus Stromal Cells; Smooth muscle, serum induced,re-exc; Fetal Heart;
Human Testes Tumor; Primary Dendritic cells,frac 2; Human Placenta; Activated
T-
Cell (l2hs)/Thiouridine labelledEco; Human Testes; T cell helper II; Human
Thymus
Tumor; Human Leukocyte, control #2; Human Fetal Brain, normalized C500HE;
NCI CGAP_Mel3; Liver Normal MetSNo; Activated T-Cells, 8 hrs., ligation 2;
Human Prostate, subtracted; KG1-a Lambda Zap Express cDNA library; H Umbilical
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33
Vein Endothelial Cells, frac A, re-excision; NCI CGAP-Br3; NCI CGAP_LymS; H.
Striatum Depression, subt; Human Primary Breast Cancer,re-excision;
NCI CGAP-Pr25; Smooth Muscle Serum Treated, Norm; human colon cancer;
Amniotic Cells - TNF induced; Early Stage Human Lung, subtracted; Human
Quadriceps; NCI CGAP-GC3; Apoptotic T-cell, re-excision; NCI CGAP-Co9;
Human Umbilical Vein, Endo. remake; Human Amygdala,re-excision;
NCI CGAP-ColO; Human Osteosarcoma; LNCAP prostate cell line; Human Colon,
re-excision; Jurkat T-Cell, S phase; Prostate BPH; H. Kidney Medulla, re-
excision;
TF-1 Cell Line GM-CSF Treated; KMH2; B-Cells; Human Brain, Striatum; L428;
Apoptotic T-cell; HUMAN JURKAT MEMBRANE BOUND POLYSOMES; Human
Fetal Dura Mater; Human Activated T-Cells; Human Pancreas Tumor; Stromal cell
TF274; NCI CGAP-Gas4; Human Hypothalmus,Schizophrenia; Human
Hippocampus; Human Chondrosarcoma; Hemangiopericytoma; Bone Marrow
Stromal Cell, untreated; CHME Cell Line,treated S hrs; NTERA2, control;
Hepatocellular Tumor, re-excision; Macrophage-oxLDL, re-excision;
NCI CGAP_Panl; NCI CGAP-Co3; Fetal Liver, subtraction II; Human Substantia
Nigra; Colon Carcinoma; breast lymph node CDNA library; Human Placenta;
NCI CGAP_Kidl l; H Macrophage (GM-CSF treated), re-excision;
NCI CGAP GC4; Human Fetal Lung III; NCI CGAP_GC6; Human Adult
Pulmonary,re-excision; Endothelial cells-control; CD34 depleted Buffy Coat
(Cord
Blood), re-excision; Human Osteoclastoma; Anergic T-cell; NCI CGAP Brn25;
NCI CGAP-Kids; Human Bone Marrow, treated; H. Frontal cortex,epileptic,re-
excision; Human Endometrial Tumor; NCI CGAP-LuS; Keratinocyte and
Soares fetal liver spleen-1NFLS S1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: cardiovascular, gastrointestinal and reproductive system
diseases
and/or disorders. Similarly, polypeptides and antibodies directed to these
polypeptides
are useful in providing immunological probes for differential identification
of the
tissues) or cell type(s). For a number of disorders of the above tissues or
cells,
particularly of the cardiovascular, gastrointestinal and reproductive systems,
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34
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., cardiovascular,
gastrointestinal,
reproductive, cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
an individual having such a disorder, relative to the standard gene expression
level,
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
Expression in smooth muscle, fetal heart and testes suggests utility in the
study and treatment of cardiovascular, gastrointestinal and reproductive
disorders.
The tissue distribution in testes tissue indicates that polynucleotides,
translation
products and antibodies corresponding to this gene are useful for the
treatment and/or
diagnosis of conditions concerning proper testicular function (e.g. endocrine
function,
sperm maturation), as well as cancer. Therefore, polynucleotides, translation
products
and antibodies corresponding to this gene are useful in the treatment of male
infertility and/or impotence. Polynucleotides, translation products and
antibodies
corresponding to this gene are also useful in assays designed to identify
binding
agents, as such agents (antagonists) are useful as male contraceptive agents.
Similarly,
polynucleotides, translation products and antibodies corresponding to this
gene are
useful in the treatment and/or diagnosis of testicular cancer. The testes are
also a site
of active gene expression of transcripts that may be expressed, particularly
at low
levels, in other tissues of the body. Therefore, translation products
corresponding to
this gene may be expressed in other specific tissues or organs where it may
play
related functional roles in other processes, such as hematopoiesis,
inflammation, bone
formation, and kidney function, to name a few possible target indications.
Alternatively, the tissue distribution in smooth muscle and fetal heart
tissues indicates
that the protein product of this gene is useful for the diagnosis and
treatment of
conditions and pathologies of the cardiovascular system, such as heart
disease,
restenosis, atherosclerosis, stroke, angina, thrombosis, and wound healing.
Additionally, translation products corresponding to this gene, as well as
antibodies
directed against these translation products, may show utility as a tumor
marker and/or
immunotherapy targets for the above listed tissues.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 9
This gene is expressed primarily in the following tissues/cDNA libraries: H.
5 Ovarian Tumor, II, OV5232; Stratagene HeLa cell s3 937216; Resting T-Cell
Library,II; Colon Normal II; Primary Dendritic Cells, lib 1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
10 not limited to: diseases and/or disorders of the immune system. Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
immune system,
expression of this gene at significantly higher or lower levels may be
routinely
15 detected in certain tissues or cell types (e.g., immune, cancerous and
wounded tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or sample taken from an individual having such a disorder,
relative to
the standard gene expression level, i.e., the expression level in healthy
tissue or bodily
fluid from an individual not having the disorder.
20 The tissue distribution of this gene suggest that this clone or the protein
product from this gene may be useful for treatment of disorders of the immune
and
haemopoietic system, it may also be useful for treatment and diagnosis of
cancer.
This gene product may be involved in the regulation of cytokine production,
antigen
presentation, or other processes that may also suggest a usefulness in the
treatment of
25 cancer (e.g. by boosting immune responses). Since the gene is expressed in
cells of
lymphoid origin, the gene or protein, as well as, antibodies directed against
the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues. Therefore it may be also used as an agent for
immunological
disorders including arthritis, asthma, immune deficiency diseases such as
AIDS,
30 leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne,
and
psoriasis. In addition, this gene product may have commercial utility in the
expansion
of stem cells and committed progenitors of various blood lineages, and in the
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36
differentiation and/or proliferation of various cell types. Additionally,
translation
products corresponding to this gene, as well as antibodies directed against
these
translation products, may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
Preferred polypeptides of the invention comprise, or alternatively consist of,
a
polypeptide having the amino acid sequence set out in the sequence listing as
SEQ ID
NO: 126. Moreover, fragments and variants of these polypeptides (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
When tested against fibroblast cell lines, supernatants removed from cells
containing this gene activated the EGR1 assay. Thus, it is likely that this
gene
activates fibroblast cells through a signal transduction pathway. Early growth
response 1 (EGR1) is a promoter associated with certain genes that induces
various
tissues and cell types upon activation, leading the cells to undergo
differentiation and
proliferation.
This gene is expressed primarily in the following tissues/cDNA libraries:
Mo7e Cell Line GM-CSF treated (lng/ml); Keratinocyte; T cell helper II;
Soares total fetus Nb2HF8 9w.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: epithelial diseases andlor disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
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37
disorders of the above tissues or cells, particularly of epithelial tissues,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., epithelial, cancerous and wounded tissues) or
bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution of this sequence indicates that polynucleotides and
polypeptides corresponding to this gene are useful in the diagnosis and
treatment of
skin wounds, psoriasis, and other infected skin diseases. The tissue
distribution
suggests that the protein product of this clone is useful for the treatment,
diagnosis,
and/or prevention of various skin disorders including congenital disorders
(i.e. nevi,
moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome),
integumentary
tumors (i.e. keratoses, Bowen's disease, basal cell carcinoma, squamous cell
carcinoma, malignant melanoma, Paget's disease, mycosis fungoides, and
Kaposi's
sarcoma), injuries and inflammation of the skin (i.e.wounds, rashes, prickly
heat
disorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema,
photosensitivity,
autoimmune disorders (i.e. lupus erythematosus, vitiligo, dermatomyositis,
morphea,
scleroderma, pemphigoid, and pemphigus), keloids, striae, erythema, petechiae,
purpura, and xanthelasma. Moreover, such disorders may predispose increased
susceptibility to viral and bacterial infections of the skin (i.e. cold sores,
warts,
chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,
erysipelas,
impetigo, tinea, althletes foot, and ringworm). Additionally, translation
products
corresponding to this gene, as well as antibodies directed against these
translation
products, may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
This gene is expressed primarily in the following tissues/cDNA libraries:
NCI CGAP_GCB1; Soares infant brain 1NIB and to a lesser extent in
Soares NhHMPu Sl; H Female Bladder, Adult; Stratagene lung (#937210);
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38
Soares-placenta 8to9weeks 2NbHP8to9W; Human Bone Marrow, treated;
normalized infant brain cDNA; Soares_pregnant uterus NbHPU; Human Adult
Small Intestine; NCI CGAP_PNS 1; Human Uterus, normal; Healing Abdomen
wound,70&90 min post incision; Frontal lobe,dementia,re-excision; Human Lung;
S Human Quadriceps; Human Colon Cancer,re-excision; Human Prostate Cancer,
Stage
C fraction; Human T-cell lymphoma,re-excision; NCI CGAP_Ewl; human ovarian
cancer; 12 Week Old Early Stage Human, II; Stromal cell TF274; Human Thymus
Stromal Cells; Ovarian Tumor 10-3-95; Human Eosinophils; Brain frontal cortex;
Human Placenta; Normal colon; Human Adult Pulmonary,re-excision; Human
Microvascular Endothelial Cells, fract. A; H. Frontal cortex,epileptic,re-
excision;
Human 8 Week Whole Embryo; Soares melanocyte 2NbHM; Colon Normal III and
Primary Dendritic Cells, lib 1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: neural system diseases and/or disorders. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the neural system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily
fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
Expression in infant brain and other developing or endocrine organs suggests
utility in the study and treatment of reproductive, hormonal and
neurodegenerative
disorders. The tissue distribution suggests that polynucleotides, translation
products
and antibodies corresponding to this clone are useful for the detection and/or
treatment of neurodegenerative disease states and behavioral disorders such as
Alzheimers Disease, Parkinson's Disease, Huntington's Disease, Tourette
Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
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39
disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception. In
addition, the
gene or gene product may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, or sexually-
linked
disorders. Additionally, translation products corresponding to this gene, as
well as
antibodies directed against these translation products, may show utility as a
tumor
marker and/or immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares infant brain 1NIB and to a lesser extent in Soares fetal liver spleen
1NFLS;
Primary Dendritic Cells, lib 1; NCI CGAP-GCB 1; Human Placenta; Human
Cerebellum; Human Neutrophil; Soares retina N2b4HR; Soares melanocyte 2NbHM;
Soares placenta Nb2HP; Soares testis NHT; Smooth muscle-ILb induced; Liver,
Hepatoma; Stratagene hNT neuron (#937233); Soares breast 3NbHBst; Primary
Dendritic cells,frac 2; Human Neutrophil, Activated; NCI CGAP-LuS; normalized
infant brain cDNA; T cell helper II; Soares fetal liver spleen_1NFLS S1; 7
Week
Old Early Stage Human, subtracted; Human Infant Adrenal Gland;
NCI CGAP Kidl2; NCI CGAP-Brn35; HL-60, PMA 4H; Human (HCC) cell line
liver (mouse) metastasis, remake; eosinophil-ILS induced; Human Adult Retina;
Human Neutrophils, Activated, re-excision; Lung, Cancer (4005163 B7):
Invasive,
Poorly Diff. Adenocarcinoma, Metastatic; Early Stage Human Lung, subtracted;
H.
Epididiymus, cauda; Human Whole Brain, re-excision; Human Synovium; Stratagene
placenta (#937225); Human Adipose Tissue, re-excision; LNCAP prostate cell
line;
NCI CGAP_Prl2; Jurkat T-cell G1 phase; Stratagene lung carcinoma 937218;
Human Brain, Striatum; L428; NCI CGAP Pr28; Human
Hypothalmus,Schizophrenia; Human Activated Monocytes; Human Adipose;
Synovial Fibroblasts (control); NCI CGAP_CLL1; CHME Cell Line,treated 5 hrs;
Soares adult brain N2b5HB55Y; Human Ovary; NCI CGAP Panl; Pancreas Islet
Cell Tumor; Human Eosinophils; NCI CGAP-Kidl l; Early Stage Human Brain;
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NCI CGAP_CoB; Human Synovial Sarcoma; Human Ovarian Cancer Reexcision;
Human Adult Pulmonary,re-excision; Endothelial-induced; Endothelial cells-
control;
Smooth muscle,control; Monocyte activated; HUMAN B CELL LYMPHOMA;
Soares-placenta 8to9weeks 2NbHP8to9W; Spleen, Chronic lymphocytic leukemia;
5 Keratinocyte; Soares NFL T GBC_S 1 and Soares NhHMPu S 1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: neural and immune system diseases and/or disorders. Similarly,
10 polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
immune and
neural systems, expression of this gene at significantly higher or lower
levels may be
routinely detected in certain tissues or cell types (e.g., neural, immune,
cancerous and
15 wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial fluid
and spinal fluid) or another tissue or sample taken from an individual having
such a
disorder, relative to the standard gene expression level, i.e., the expression
level in
healthy tissue or bodily fluid from an individual not having the disorder.
Expression in infant brain, fetal liver and spleen tissues suggests utility
for the
20 study and treatment of neurodevelopmental, neurodegenerative, hemapoietic
and
immune disorders and neoplasms. The tissue distribution suggests that
polynucleotides, translation products and antibodies corresponding to this
clone are
useful for the detection and/or treatment of neurodegenerative disease states
and
behavioral disorders such as Alzheimers Disease, Parkinson's Disease,
Huntington's
25 Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia,
obsessive
compulsive disorder, panic disorder, learning disabilities, ALS, psychoses,
autism,
and altered behaviors, including disorders in feeding, sleep patterns,
balance, and
perception. In addition, the gene or gene product may also play a role in the
treatment
and/or detection of developmental disorders associated with the developing
embryo,
30 or sexually-linked disorders. Alternatively, the expression of this gene
product in fetal
liver tissue suggests a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic cell
lineages,
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41
including blood stem cells. This gene product may be involved in the
regulation of
cytokine production, antigen presentation, or other processes that may also
suggest a
usefulness in the treatment of cancer (e.g. by boosting immune responses).
Since the
gene is expressed in cells of lymphoid origin, the gene or protein, as well
as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Therefore it may be also
used as
an agent for immunological disorders including arthritis, asthma, immune
deficiency
diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel
disease,
sepsis, acne, and psoriasis. In addition, this gene product may have
commercial utility
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
Additionally,
translation products corresponding to this gene, as well as antibodies
directed against
these translation products, may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
Preferred polypeptides of the invention comprise, or alternatively consist of,
a
polypeptide having the amino acid sequence set out in the sequence listing as
SEQ ID
NO: 127. Moreover, fragments and variants of these polypeptides (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Rejected Kidney, lib 4; human tonsils; Soares NFL T GBC_S1;
NCI CGAP GCB 1.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
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42
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: diseases and/or disorders of the renal and immune systems.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the renal
and
immune systems, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues or cell types (e.g., renal, immune,
cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or sample taken from an individual
having
such a disorder, relative to the standard gene expression level, i.e., the
expression
level in healthy tissue or bodily fluid from an individual not having the
disorder.
Expression in kidney and tonsils suggests utility for the study and treatment
of
metabolic, excretory, immune and infectious diseases. The tissue distribution
in
kidney suggests that this gene or gene product is useful in the treatment
and/or
detection of kidney diseases including renal failure, nephritus, renal tubular
acidosis,
proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic
syndrome, crush
syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in
addition to
Wilms Tumor Disease, and congenital kidney abnormalities such as horseshoe
kidney, polycystic kidney, and Falconi's syndrome. Alternatively, expression
of this
gene product in tonsils suggests a role in the regulation of the
proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic cell
lineages,
including blood stem cells. This gene product may be involved in the
regulation of
cytokine production, antigen presentation, or other processes that may also
suggest a
usefulness in the treatment of cancer (e.g. by boosting immune responses).
Since the
gene is expressed in cells of lymphoid origin, the gene or protein, as well
as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Therefore it may be also
used as
an agent for immunological disorders including arthritis, asthma, immune
deficiency
diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel
disease,
sepsis, acne, and psoriasis. In addition, this gene product may have
commercial utility
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
Additionally,
CA 02389722 2002-04-30
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43
translation products corresponding to this gene, as well as antibodies
directed against
these translation products, may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
This gene is expressed primarily in Human Placental tissue.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: placental diseases and/or disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the placenta,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., placenta, cancerous and wounded tissues) or
bodily fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
Expression in placenta suggests utility in study and treatment of
reproductive,
metabolic and hormonal disorders. The tissue distribution suggests that
polynucleotides, translation products and antibodies corresponding to this
gene are
useful for the diagnosis and/or treatment of disorders of the placenta.
Specific
expression within the placenta suggests that polynucleotides and translation
products
corresponding to this gene may play a role in the proper establishment and
maintenance of placental function. Alternately, this gene product may be
produced by
the placenta and then transported to the embryo, where it may play a crucial
role in
the development and/or survival of the developing embryo or fetus. Expression
of this
gene product in a vascular-rich tissue such as the placenta also suggests that
this gene
product may be produced more generally in endothelial cells or within the
circulation.
CA 02389722 2002-04-30
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44
In such instances, polynucleotides and translation products corresponding to
this gene
may play more generalized roles in vascular function, such as in angiogenesis.
Polynucleotides and translation products corresponding to this gene may also
be
produced in the vasculature and have effects on other cells within the
circulation, such
S as hematopoietic cells. Polynucleotides and translation products
corresponding to this
gene may serve to promote the proliferation, survival, activation, and/or
differentiation of hematopoietic cells, as well as other cells throughout the
body.
Additionally, translation products corresponding to this gene, as well as
antibodies
directed against these translation products, may show utility as a tumor
marker and/or
immunotherapy targets for the above listed tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15
The gene encoding the disclosed cDNA is thought to reside on chromosome 9.
Accordingly, polynucleotides related to this invention have uses, such as, for
example, as a marker in linkage analysis for chromosome 9.
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares placenta Nb2HP; Soares fetal liver spleen 1NFLS and to a lesser extent
in
Human Placenta; Soares-pregnant uterus NbHPU; Soares adult brain N2b5HB55Y;
Human placenta polyA+ (TFujiwara); Human placenta cDNA (TFujiwara);
Soares fetal liver spleen 1NFLS_S1; Human Placenta; Clontech human aorta
polyA+ mRNA (#6572); Stratagene placenta (#937225); Placenta; Human
endometrial stromal cells-treated with estradiol; Human Placenta (re-
excision);
Human Placenta; Soares~lacenta 8to9weeks 2NbHP8to9W; Human Placenta,
subtracted; pBMC stimulated w/ poly I/C; Soares NSF F8 9W_OT PA P S1;
Human Thymus Stromal Cells; NCI CGAP_Kidl l; Human Osteoclastoma;
Soares-parathyroid tumor NbHPA; Soares NhHMPu S1; NCI CGAP_Schl;
Human Colon; Stratagene neuroepithelium NT2RAMI 937234; Smooth muscle,
control, re-excision; Human adult (K.Okubo); Stratagene ovary (#937217); Human
endometrial stromal cells; Synovial hypoxia; H. Kidney Medulla, re-excision;
Gessler
Wilms tumor; Human Whole Six Week Old Embryo; Adipocytes; Normal colon;
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WO 01/34768 PCT/US00/30039
Smooth muscle,control; Soares senescent fibroblasts NbHSF; NCI CGAP_Kid3;
Soares fetal heart NbHHI9W and Soares testis NHT.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
5 biological sample and for diagnosis of diseases and conditions which include
but are
not limited to: hemapoietic, reproductive, metabolic and hormonal disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
10 immune and reproductive systems, expression of this gene at significantly
higher or
lower levels may be routinely detected in certain tissues or cell types (e.g.,
immune,
reproductive, cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
15 the expression level in healthy tissue or bodily fluid from an individual
not having the
disorder.
Enriched expression in placenta, fetal liver and spleen tissues suggests
utility
for the study and treatment of hemapoietic, reproductive, metabolic and
hormonal
disorders. The tissue distribution suggests that polynucleotides, translation
products
20 and antibodies corresponding to this gene are useful for the diagnosis
and/or treatment
of disorders of the placenta. Specific expression within the placenta suggests
that
polynucleotides and translation products corresponding to this gene may play a
role in
the proper establishment and maintenance of placental function. Alternately,
this gene
product may be produced by the placenta and then transported to the embryo,
where it
25 may play a crucial role in the development and/or survival of the
developing embryo
or fetus. Expression of this gene product in a vascular-rich tissue such as
the placenta
also suggests that this gene product may be produced more generally in
endothelial
cells or within the circulation. In such instances, polynucleotides and
translation
products corresponding to this gene may play more generalized roles in
vascular
30 function, such as in angiogenesis. Polynucleotides and translation products
corresponding to this gene may also be produced in the vasculature and have
effects
on other cells within the circulation, such as hematopoietic cells.
Polynucleotides and
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
46
translation products corresponding to this gene may serve to promote the
proliferation, survival, activation, and/or differentiation of hematopoietic
cells, as
well as other cells throughout the body. Alternatively, this gene product may
be
involved in the regulation of cytokine production, antigen presentation, or
other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the gene or protein, as well as, antibodies directed against the protein may
show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
Therefore it may be also used as an agent for immunological disorders
including
arthritis, asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid
arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this
gene product may have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the differentiation
and/or
proliferation of various cell types. Additionally, translation products
corresponding to
this gene, as well as antibodies directed against these translation products,
may show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
47
0
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CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
48
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CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
49
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CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
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CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
51
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52
Table 1 summarizes the information corresponding to each "Gene No." described
above. The nucleotide sequence identified as "NT SEQ ID NO:X" was assembled
from partially homologous ("overlapping") sequences obtained from the "cDNA
clone ID" identified in Table 1 and, in some cases, from additional related
DNA
clones. The overlapping sequences were assembled into a single contiguous
sequence
of high redundancy (usually three to five overlapping sequences at each
nucleotide
position), resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding
deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits
contain multiple different clones corresponding to the same gene. "Vector"
refers to
the type of vector contained in the cDNA Clone ID.
"Total NT Seq." refers to the total number of nucleotides in the contig
identified by "Gene No." The deposited clone may contain all or most of these
sequences, reflected by the nucleotide position indicated as "5' NT of Clone
Seq."
and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ
ID
NO:X of the putative start codon (methionine) is identified as "5' NT of Start
Codon."
Similarly , the nucleotide position of SEQ ID NO:X of the predicted signal
sequence
is identified as "5' NT of First AA of Signal Pep."
The translated amino acid sequence, beginning with the methionine, is
identified as "AA SEQ ID NO:Y," although other reading frames can also be
easily
translated using known molecular biology techniques. The polypeptides produced
by
these alternative open reading frames are specifically contemplated by the
present
invention.
The first and last amino acid position of SEQ 117 NO:Y of the predicted signal
peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep." The
predicted first amino acid position of SEQ ID NO:Y of the secreted portion is
identified as "Predicted First AA of Secreted Portion." Finally, the amino
acid
position of SEQ ID NO:Y of the last amino acid in the open reading frame is
identified as "Last AA of ORF."
SEQ ID NO:X (where X may be any of the polynucleotide sequences
disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may
be
any of the polypeptide sequences disclosed in the sequence listing) are
sufficiently
CA 02389722 2002-04-30
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53
accurate and otherwise suitable for a variety of uses well known in the art
and
described further below. For instance, SEQ ID NO:X is useful for designing
nucleic
acid hybridization probes that will detect nucleic acid sequences contained in
SEQ ID
NO:X or the cDNA contained in the deposited clone. These probes will also
hybridize to nucleic acid molecules in biological samples, thereby enabling a
variety
of forensic and diagnostic methods of the invention. Similarly, polypeptides
identified from SEQ ID NO:Y may be used, for example, to generate antibodies
which bind specifically to proteins containing the polypeptides and the
secreted
proteins encoded by the cDNA clones identified in Table 1.
Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino
acid sequence. In these cases, the predicted amino acid sequence diverges from
the
actual amino acid sequence, even though the generated DNA sequence may be
greater
than 99.9% identical to the actual DNA sequence (for example, one base
insertion or
deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ ID NO:X and the predicted
translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of
plasmid DNA containing a human cDNA of the invention deposited with the ATCC,
as set forth in Table 1. The nucleotide sequence of each deposited clone can
readily
be determined by sequencing the deposited clone in accordance with known
methods.
The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can
also be directly determined by peptide sequencing or by expressing the protein
in a
suitable host cell containing the deposited human cDNA, collecting the
protein, and
determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be
isolated in accordance with known methods using the sequence information
disclosed
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54
herein. Such methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from appropriate
sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or
species homologs. Procedures known in the art can be used to obtain full-
length
genes, allelic variants, splice variants, full-length coding portions,
orthologs, and/or
species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a
deposited clone, using information from the sequences disclosed herein or the
clones
deposited with the ATCC. For example, allelic variants and/or 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 for allelic
variants and/or
the desired homologue.
Table 2 provides preferred epitopes contained in certain embodiments of the
invention and polynucleotide sequences that may be disclaimed according to
certain
embodiments of the invention. The first column refers to each "Gene No."
described
above in Table 1. The second column provides the sequence identifier, "NT SEQ
>D
NO:X", for polynucleotide sequences disclosed in Table 1. The third column
provides
the sequence identifier, "AA SEQ ID NO:Y", for polypeptide sequences disclosed
in
Table 1. The fourth column provides a unique integer "ntA" where "ntA" is any
integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the
fifth
column provides a unique integer "ntB" where "ntB" is any integer between 15
and
the final nucleotide of SEQ ID NO:X, where both ntA and ntB correspond to the
positions of nucleotide residues shown in SEQ ID NO:X, and where ntB is
greater
than or equal to a + 14. For each of the polynucleotides shown as SEQ ID NO:X,
the
uniquely defined integers can be substituted into the general formula of a-b,
and used
to describe polynucleotides which may be preferably excluded from the
invention.
Column 6 lists residues comprising predicted epitopes contained in the
polypeptides
encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of
potential
immunogenic regions was performed according to the method of Jameson and Wolf
((1988) CABIOS, 4; 181-186); specifically, the Genetics Computer Group (GCG)
implementation of this algorithm, embodied in the program PEPT>DESTRUCTURE
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(Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wisc.). This
method returns a measure of the probability that a given residue is found on
the
surface of the protein. Regions where the antigenic index score is greater
than 0.9
over at least 6 amino acids are indicated in Table 2 as "Preferred Epitopes".
5 Polypeptides of the invention may possess one, two, three, four, five or
more
antigenic epitopes comprising residues described in Table 2. It will be
appreciated
that depending on the analytical criteria used to predict antigenic
determinants, the
exact address of the determinant may vary slightly.
10 Table 3 summarizes the expression profile of polynucleotides corresponding
to the clones disclosed in Table 1. The first column provides a unique clone
identifier, "Clone D7", for a cDNA clone related to each contig sequence
disclosed in
Table 1. Column 2, "Library Codes" shows the expression profile of tissue
and/or cell
line libraries which express the polynucleotides of the invention. Each
Library Code
15 in column 2 represents a tissue/cell source identifier code corresponding
to the
Library Code and Library description provided in Table 4. Expression of these
polynucleotides was not observed in the other tissues and/or cell libraries
tested. One
of skill in the art could routinely use this information to identify tissues
which show a
predominant expression pattern of the corresponding polynucleotide of the
invention
20 or to identify polynucleotides which show predominant and/or specific
tissue
expression.
Table 4 provides a key to the Library Code disclosed in Table 3. Column 1
provides the Library Code disclosed in Table 3, column 2. Column 2 provides a
description of the tissue or cell source from which the corresponding library
was
25 derived. Library codes corresponding to diseased Tissues are indicated in
column 3
with the word "disease".
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Table 2
.. ..
1 11 65 1 - 15 - Gly-70 to Lys-75
3554 3568
Ser-79 to Ala-85
Gly-119 to Ile-126
Pro-133 to Asn-138
Lys-178 to Lys-189
Arg-212 to His-220
Gl -273 to Thr-290.
1 26 80 1 - 15 - Gly-70 to Lys-75
877 891
Ser-79 to Ala-85.
1 27 81 1 - 15 -
2341 2355
1 28 82 1 - 15 -
1062 1076
2 12 66 1 - 15 - Asp-31 to Pro-36
810 824
Ser-88 to Gln-95.
2 29 83 1 - 15 -
3927 3941
2 30 84 1 - 15 - Asp-31 to Pro-36
2520 2534
Ser-88 to Gln-95
Ala-163 to Glu-171.
3 13 67 1 - 15 - Arg-17 to Ser-22
4428 4442
Lys-100 to Asp-109
Gln-124 to Ser-130
Glu-186 to Met-203
Asp-237 to Arg-247
Tyr-281 to Cys-287
Glu-297 to Ile-311
Glu-328 to Ser-341
Ala-355 to Ser-363
Pro-382 to His-391
Gln-444 to Pro-450.
3 31 85 1 - 15 - Arg-17 to Ser-22
552 566
Lys-100 to Asp-109
Gln-124 to As -129.
3 32 86 1 - 15 -
1750 1764
3 33 87 1 - 15 - Ar -83 to Asn-96.
946 960
3 34 88 1 - 15 -
1294 1308
3 35 89 1 - 15 -
695 709
4 14 68 1 - 15 - Val-24 to Ser-31
1646 1660
Gly-44 to Glu-53
Pro-64 to Trp-75
Phe-87 to Cys-95
Ser-99 to Gly-105
His-124 to Ala-132
Pro-134 to Gly-139
Pro-141 to Gly-153
Pro-220 to Asp-233
Pro-254 to Val-260
Ala-331 to Val-336
Pro-351 to Glu-357
Ar -371 to Lys-379.
4 36 90 1 - 15 - Val-24 to C s-29.
466 480
4 37 91 1 - 15 -
1827 1841
15 69 1 - 15 - Met-1 to T -6
3085 3099
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Ala-8 to Arg-13
Gln-49 to Lys-54
Gly-65 to Gln-78
Asn-87 to Gly-96
Gly-105 to Thr-114
Tyr-156 to Ser-173
Trp-227 to His-233
Asn-238 to Glu-244
Lys-284 to Thr-291
Gln-315 to Lys-324
Lys-332 to Pro-338
Glu-340 to Glu-345
Leu-413 to Gly-421
Ser-545 to Glu-551
Arg-607 to Gly-614
Phe-632 to Ala-639
Tyr-676 to Tyr-681
Pro-684 to Asp-690
Glu-700 to Cys-709.
38 92 1 - 15 - Met-1 to Trp-6
409 423
Ala-8 to Arg-13.
5 39 93 1 - 15 -
1652 1666
5 40 94 1 - 15 - Pro-1 to Ala-10.
1124 1138
6 16 70 1 - 15 - Ser-5 to Ttp-10
535 549
Ala-30 to Glu-39
Arg-66 to Trp-72
Glu-84 to Arg-97.
6 41 95 1 - 15 - Ser-5 to Trp-10
1106 1120
Ala-30 to Glu-39
Arg-66 to Trp-72
Glu-84 to Arg-97
Glu-159 to Gly-176
Ile-189 to Glu-197
Glu-206 to Arg-215
Arg-218 to His-226.
6 42 96 1 - 15 - T -2 to Met-16.
1879 1893
6 43 97 1 - 15 - Gln-24 to Gly-31
1173 1187
Pro-33 to Ala-38.
7 17 71 1 - 15 - Ala-32 to L s-55.
2598 2612
7 44 98 1 - 15 - Ala-32 to Lys-55.
1111 1125
7 45 99 1 - 15 - Pro-13 to His-21
2283 2297
Val-25 to Gl -33.
7 46 100 1 - 15 - Ser-17 to Cys-29
468 482
Ar -32 to Arg-38.
8 18 72 1 - 15 -
1083 1097
8 47 101 1 - 15 -
754 768
8 48 102 1 - 15 - Arg-1 to Asn-9
2073 2087
Pro-24 to Ile-32
Val-95 to C s-106.
8 49 103 1 - 15 - Glu-1 to Glu-8
2082. 2096
Pro-38 to Gly-45
Leu-53 to Gly-60
Glu-112 to Arg-117
Lys-153 to Lys-163
Trp-245 to Ala-251
Phe-259 to Gly-273.
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9 19 73 1 - 15 - Ser-31 to L s-37.
1423 1437
9 50 104 1 - 15 - Ser-31 to L s-37.
558 572
9 51 105 1 - 15 - Arg-36 to Arg-43
1714 1728 Ar -78 to L s-83.
9 52 106 1 - 15 - Val-2 to L s-9.
911 925
20 74 I - 15 - Ser-51 to Thr-57.
987 1001
10 53 107 1 - 15 - Ser-51 to Thr-57.
987 1001
10 54 108 1 - 15 - Gln-23 to As -28.
655 669
11 21 75 1 - 15 -
1754 1768
11 55 109 1 - 15 -
1786 1800
11 56 110 1 - 15 -
913 927
11 57 1 I 1 1 - 15 -
650 664
12 22 76 1 - 15 -
2293 2307
12 58 112 1 - 15 -
459 473
12 59 113 I - 15 -
1862 1876
12 60 114 1 - 15 -
430 444
12 61 115 1 - 15 - Phe-16 to Gl -23.
2476 2490
13 23 77 1 - 15 - Ser-33 to Ala-40.
1699 1713
14 24 78 1 - 15 - Arg-18 to Trp-33
799 813 Pro-36 to Ser-47.
14 62 116 1 - 15 - Arg-18 to Trp-33
631 645 Pro-36 to Ser-47.
25 79 1 - 15 -
7630 7644
15 63 117 1 - 15 -
3384 3398
15 64 118 1 - 15 -
1783 1797
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Table 3
r
HDPAP35 H0069 H0265 H0422 H0521 H0555 H0635 H0638
H0641 H0667 L1290
50002 S0132 S0426 T0039
HAMFC93 H0013 H0046 H0052 H0069 H0171 H0179 H0413
H0423 H0427 H0435
H0485 H0494 H0497 H0510 H0551 H0556 H0560
H0562 H0574 H0596
H0616 H0622 H0624 H0641 H0646 H0659 H0670
H0703 L1290 S0010
S0014 50028 50126 S0142 S0150 50222 S0250
50328 S0346 50360
50374 50376 50378 50406
HDPMS H0014 H0263 H0309 H0342 H0393 H0421 H0431
12 H0445 H0455 H0459
H0486 H0522 H0547 H0571 H0575 H0632 H0656
H0661 L1290 50026
S0030 50222 S0328 50354 50360 S0392 50422
S6024
HKAAV61 H0172 H0351 H0494 H0545 H0690 L1290 S0328
HDPCJ43 H0033 H0039 H0040 H0051 H0123 H0135 H0156
H0188 H0250 H0264
H0271 H0295 H0405 H0424 H0457 H0509 H0521
H0522 H0543 H0547
H0549 H0553 H0575 H0581 H0583 H0591 H0616
H0647 H0662 H0684
H0688 H0702 L1290 S0001 50002 S0040 50046
S0126 50152 50280
50340 S0356 50360
HKACM93 H0355 H0494 H0544 H0594 H0647 H0692 L1290
S0376 S0380
HHEMM74 H0009 H0036 H0046 H0050 H0063 H0087 H0135
H0208 H0252 H0261
H0264 H0288 H0331 H0333 H0445 H0455 H0486
H0521 H0542 H0543
H0544 H0551 H0556 H0580 H0586 H0600 H0609
H0618 H0620 H0635
H0637 H0644 H0657 H0660 H0688 H0699 L1290
50002 50194 50222
S0312 50314 S0356 50366
HSYAZ50 H0012 H0013 H0024 H0036 H0040 H0046 H0051
H0056 H0058 H0063
H0081 H0135 H0212 H0251 H0252 H0253 H0265
H0266 H0267 H0294
H0318 H0333 H0341 H0351 H0370 H0402 H0422
H0423 H0424 H0431
H0433 H0435 H0436 H0445 H0457 H0494 H0519
H0520 H0521 H0529
H0538 H0539 H0543 H0545 H0551 H0553 H0556
H0560 H0561 H0563
H0574 H0575 H0581 H0587 H0592 H0597 H0606
H0618 H0619 H0620
H0623 H0628 H0629 H0638 H0645 H0650 H0651
H0656 H0658 H0662
H0663 H0687 L1290 S0002 50003 50026 50036
S0037 50044 S0045
S0049 S0051 50114 S0116 50132 50142 50150
S0152 S0194 50222
50250 S0276 S0278 50280 S0342 50344 S0346
S0364 S0372 50420
S0424 S0438 S3014 T0002 T0023 T0042 T0082
HDPAQ55 H0415 H0436 H0521 50354
HKAFT66 H0494 H0543 H0580 50422 S0474
HLWAZ70 H0037 H0266 H0328 H0553 L1290 T0069
HLWBG83 H0052 H0071 H0135 H0149 H0163 H0179 H0208
H0250 H0257 H0271
H0318 H0416 H0422 H0427 H0428 H0445 H0457
H0494 H0509 H0521
H0522 H0539 H0543 H0550 H0551 H0553 H0561
H0575 H0586 H0615
H0638 H0641 H0647 H0658 H0696 L1290 50002
50007 S0028 50032
S0045 S0046 50049 50051 S0140 S0150 S0330
50364 S0366 T0041
T0082
HRABS65 H0014 H0024 H0030 H0031 H0046 H0051 H0052
H0056 H0059 H0063
H0083 H0087 HO100 H0124 H0135 H0156 H0172
H0179 H0194 H0201
H0251 H0264 H0265 H0266 H0268 H0286 H0290
H0309 H0318 H0327
H0328 H0329 H0333 H0341 H0351 H0393 H0410
H0423 H0428 H0435
H0457 H0494 H0509 H0519 H0520 H0521 H0522
H0529 H0539 H0540
H0542 H0543 H0544 H0545 H0547 H0551 H0555
H0556 H0574 H0575
H0580 H0581 H0583 H0592 H0615 H0619 H0620
H0623 H0628 H0632
H0634 H0635 H0637 H0638 H0641 H0645 H0646
H0650 H0656 H0657
H0658 H0659 H0660 H0662 H0672 H0675 H0687
H0689 H0698 H0702
L1290 S0001 50002 50011 50022 S0027 S0028
50031 S0037 S0038
S0040 50045 50046 S0048 50050 S0052 50053
50114 50134 S0144
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SO150 50152 50208 S0212 S0216 50242 S0278 50282
50328 S0344
50356 S0358 S0364 50374 50376 50380 S0418 S0420
S0426 53012
T0010 T0039 T0041 T0042 T0048 T0068
HLWBH18 H0553
HLWAX42 H0553
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Table 4
.. ~ . . ~ . .
H0009 Human Fetal Brain
H0012 Human Fetal Kidney
H0013 Human 8 Week Whole Embryo
H0014 Human Gall Bladder
H0024 Human Fetal Lun III
H0030 Human Placenta
H0031 Human Placenta
H0033 Human Pituitar
H0036 Human Adult Small Intestine
H0037 Human Adult Small Intestine
H0039 Human Pancreas Tumor disease
H0040 Human Testes Tumor disease
H0046 Human Endometrial Tumor disease
H0050 Human Fetal Heart
H0051 Human Hippocam us
H0052 Human Cerebellum
H0056 Human Umbilical Vein, Endo. remake
H0058 Human Th us Tumor disease
H0059 Human Uterine Cancer disease
H0063 Human Th us
H0069 Human Activated T-Cells
H0071 Human Infant Adrenal Gland
H0081 Human Fetal E ithelium (Skin)
H0083 HUMAN JURKAT MEMBRANE BOUND POLYSOMES
H0087 Human Th us
HO100 Human Whole Six Week Old Emb o
H0123 Human Fetal Dura Mater
H0124 Human Rhabdomyosarcoma disease
H0135 Human Synovial Sarcoma
H0149 7 Week Old Earl Sta a Human, subtracted
H0156 Human Adrenal Gland Tumor disease
H0163 Human Synovium
H0171 12 Week Old Early Stage Human, II
H0172 Human Fetal Brain, random rimed
H0179 Human Neutro hil
H0188 Human Normal Breast
H0194 Human Cerebellum, subtracted
H0201 Human Hi ocam us, subtracted
H0208 Earl Sta a Human Lun , subtracted
H0212 Human Prostate, subtracted
H0250 Human Activated Monoc tes
H0251 Human Chondrosarcoma disease
H0252 Human Osteosarcoma disease
H0253 Human adult testis, lar a inserts
H0257 HL-60, PMA 4H
H0261 H. cerebellum, Enz me subtracted
I H0263 human colon cancer disease
H0264 human tonsils
I H0265 Activated T-Cell (l2hs)/Thiouridine labelledEco
H0266 Human Microvascular Endothelial Cells,
fract. A
H0267 Human Microvascular Endothelial Cells,
fract. B
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H0268 Human Umbilical Vein Endothelial Cells,
fract. A
H0271 Human Neutro hil, Activated
H0286 Human OB MG63 treated 10 nM E2) fraction
I
H0288 Human OB HOS control fraction I
H0290 Human OB HOS treated ( 1 nM E2 fraction
I
H0294 Amniotic Cells - TNF induced
H0295 Amniotic Cells - Primary Culture
H0309 Human Chronic Synovitis disease
H0318 HUMAN B CELL LYMPHOMA disease
H0327 human co us colosum
H0328 human ovarian cancer disease
H0329 Dermatofibrosarcoma Protuberance disease
H0331 He atocellular Tumor disease
H0333 Hemangio eric toms disease
H0341 Bone Marrow Cell Line RS4,11)
H0342 Lingual G s
H0351 Glioblastoma disease
H0355 Human Liver
H0370 H. L m h node breast Cancer disease
H0393 Fetal Liver, subtraction II
H0402 CD34 de leted Buffy Coat (Cord Blood),
re-excision
H0405 Human Pituitary, subtracted VI
H0410 H. Male bladder, adult
H0413 Human Umbilical Vein Endothelial Cells,
uninduced
H0415 H. Ovarian Tumor, II, OV5232 disease
H0416 Human Neutro hils, Activated, re-excision
H0421 Human Bone Marrow, re-excision
H0422 T-Cell PHA 16 hrs
H0423 T-Cell PHA 24 hrs
H0424 Human Pituitar , subt IX
H0427 Human Adi ose
H0428 Human Ova
H0431 H. Kidne Medulla, re-excision
H0433 Human Umbilical Vein Endothelial cells,
frac B, re-excision
H0435 Ovarian Tumor 10-3-95
H0436 Resting T-Cell Librar ,II
H0445 S leen, Chronic 1 m hoc is leukemia disease
H0455 H. Striatum De ression, subt
H0457 Human Eosino hits
H0459 CD34+cells, II, FRACTION 2
H0485 Hodgkin's L homy I disease
H0486 Hod kin's L homa II disease
H0494 Keratinoc a
H0497 HEL cell line
H0509 Liver, He atoma disease
H0510 Human Liver, normal
H0519 NTERA2, control
H0520 NTERA2 + retinoic acid, 14 days
H0521 Primary Dendritic Cells, lib 1
H0522 Primar Dendritic cells,frac 2
H0529 M oloid Pro enitor Cell Line
H0538 Merkel Cells
H0539 rPancreas Islet Cell Tumor disease
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H0540 Skin, burned
H0542 T Cell hel er I
H0543 T cell hel er II
H0544 Human endometrial stromal cells
H0545 Human endometrial stromal cells-treated
with ro esterone
H0547 NTERA2 teratocarcinoma cell line+retinoic
acid 14 da s)
H0549 H. Epididiymus, caput & co us
H0550 H. Epididiymus, cauda ~'"
H0551 Human Thymus Stromal Cells
H0553 Human Placenta
H0555 Re'ected Kidne , lib 4 disease
H0556 Activated T-cell(12h)/Thiouridine-re-excision
H0560 KMH2
H0561 L428
H0562 Human Fetal Brain, normalized c5-11-26
H0563 Human Fetal Brain, normalized 50021F
H0571 Human Fetal Brain, normalized C500HE
H0574 He atocellular Tumor, re-excision disease
H0575 Human Adult Pulmonar ,re-excision
H0580 Dendritic cells, pooled
H0581 Human Bone Marrow, treated
H0583 B Cell 1 phoma disease
H0586 Healing groin wound, 6.5 hours ost incisiondisease
H0587 Healin roin wound, 7.5 hours ost incisiondisease
H0591 Human T-cell lym homa,re-excision disease
H0592 Healin roin wound - zero hr ost-incisiondisease
control)
H0594 Human Lung Cancer,re-excision disease
H0596 Human Colon Cancer,re-excision
H0597 Human Colon, re-excision
H0600 Healing Abdomen wound,70&90 min ost incisiondisease
H0606 Human Prima Breast Cancer,re-excision disease
H0609 H. Leukocytes, normalized cot > 500A
H0615 Human Ovarian Cancer Reexcision disease
H0616 Human Testes, Reexcision
H0618 Human Adult Testes, Large Inserts, Reexcision
H0619 Fetal Heart
H0620 Human Fetal Kidne , Reexcision
H0622 Human Pancreas Tumor, Reexcision disease
H0623 Human Umbilical Vein, Reexcision
H0624 12 Week Earl Stage Human II, Reexcision
H0628 Human Pre-Differentiated Adi ocytes
H0629 Human Leukoc e, control #2
H0632 He atocellular Tumor,re-excision
H0634 Human Testes Tumor, re-excision disease
H0635 Human Activated T-Cells, re-excision
H0637 Dendritic Cells From CD34 Cells
H0638 CD40 activated monocyte dendridic cells
H0641 LPS activated derived dendritic cells
H0644 Human Placenta (re-excision)
H0645 Fetal Heart, re-excision
H0646 Lung, Cancer (4005313 A3): Invasive Poorly
Differentiated Lung
Adenocarcinoma,
H0647 Lung, Cancer (4005163 B7): Invasive, disease
Poorly Dif
Adenocarcinoma, Metastatic
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H0650 B-Cells
H0651 Ovar , Normal: 9805C040R)
H0656 B-cells unstimulated
H0657 B-cells (stimulated)
H0658 Ovar , Cancer 9809C332 : Poorl differentiateddisease
adenocarcinoma
H0659 Ovar , Cancer 15395A1F : Grade II Pa disease
illa Carcinoma
H0660 Ovary, Cancer: (15799A1F) Poorl differentiateddisease
carcinoma
H0661 Breast, Cancer: (4004943 A5) disease
H0662 Breast, Normal: (400552282)
H0663 Breast, Cancer: 4005522 A2) disease
H0667 Stromal cells(HBM3.18)
H0670 Ovary, Cancer(4004650 A3): Well-Differentiated
Micropapillary
Serous Carcinoma
H0672 Ovar , Cancer: (4004576 A8)
H0675 Colon, Cancer: (9808C064R)
H0684 Ovarian cancer, Serous Pa illary Adenocarcinoma
H0687 Human normal ovar #96106215)
H0688 Human Ovarian Cancer(#98076017)
H0689 Ovarian Cancer
H0690 Ovarian Cancer, # 97026001
H0692 BL S Rece for from Ex ression Cloning
H0696 Prostate Adenocarcinoma
H0698 NK CellsYao20 IL2 treated for 48 hrs
H0699 NKyaol9(Control)
H0702 NK15(IL2 treated for 48 hours)
H0703 NKYA019 IL2 TREATED FOR 72 HOURS)
L1290 Stratagene lung (#937210)
S0001 Brain frontal cortex
50002 Monoc a activated
S0003 Human Osteoclastoma disease
S0007 Earl Stage Human Brain
S0010 Human Am dala
S0011 STROMAL -OSTEOCLASTOMA disease
S0014 Kidne Cortex
50022 Human Osteoclastoma Stromal Cells - unam
lifted
S0026 Stromal cell TF274
50027 Smooth muscle, serum treated
S0028 Smooth muscle,control
S0030 Brain pons
S0031 S final cord
S0032 Smooth muscle-ILb induced
S0036 Human Substantia Nigra
S0037 Smooth muscle, ILlb induced
S0038 Human Whole Brain #2 - Oli o dT > l.SKb
S0040 Adi oc es
S0044 Prostate BPH disease
S0045 Endothelial cells-control
S0046 Endothelial-induced
S0048 Human H othalamus, Alzheimer's disease
S0049 Human Brain, Striatum
50050 Human Frontal Cortex, Schizophrenia disease
50051 Human Hypothalmus,Schizophrenia disease
S0052 neutrophils control
S0053 ~ Neutrophils IL-1 and LPS induced
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50114 Aner is T-cell
50116 Bone marrow
50126 Osteoblasts
S0132 E ithelial-TNFa and INF induced
50134 A o totic T-cell
S0140 eosino hil-IL5 induced
S0142 Macro hage-oxLDL
S0144 Macro hage (GM-CSF treated)
S0150 LNCAP rostate cell line
50152 PC3 Prostate cell line
S0194 S novial h oxia
S0208 Messangial cell, fray 1
S0212 Bone Marrow Stromal Cell, untreated
50216 Neutro hils IL-1 and LPS induced
S0222 H. Frontal cortex,e ile tic,re-excision disease
S0242 S novial Fibroblasts (Ill/TNF , subt
50250 Human Osteoblasts II disease
S0276 S novial h oxia-RSF subtracted
S0278 H Macro hage (GM-CSF treated , re-excision
S0280 Human Adipose Tissue, re-excision
S0282 Brain Frontal Cortex, re-excision
S0312 Human osteoarthritic,fraction II disease
S0314 Human osteoarthritis,fraction I disease
50328 Palate carcinoma disease
50330 Palate normal
S0340 Human Osteoarthritic Cartila a Fraction disease
IV
S0342 Adi oc tes,re-excision
S0344 Macro ha e-oxLDL, re-excision
S0346 Human Am gdala,re-excision
S0354 Colon Normal II
S0356 Colon Carcinoma disease
S0358 Colon Normal III
S0360 Colon Tumor II disease
50364 Human Quadriceps
50366 Human Soleus
50372 Lar x carcinoma III disease
S0374 Normal colon
50376 Colon Tumor disease
50378 Pancreas normal PCA4 No
S0380 Pancreas Tumor PCA4 Tu disease
S0392 Salivar Gland
S0406 Rectum tumour
S0418 CHME Cell Line,treated 5 hrs
S0420 CHME Cell Line,untreated
S0422 Mo7e Cell Line GM-CSF treated lng/ml)
S0424 TF-1 Cell Line GM-CSF Treated
S0426 Monocyte activated, re-excision
S0438 Liver Normal MetSNo
S0474 Human blood latelets
S3012 Smooth Muscle Serum Treated, Norm
53014 Smooth muscle, serum induced,re-exc
S6024 Alzheimers, s on change disease
T0002 Activated T-cells
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T0010 Human Infant Brain
T0023 Human Pancreatic Carcinoma disease
T0039 HSA 172 Cells '
T0041 Jurkat T-cell G 1 hase
T0042 Jurkat T-Cell, S hase
T0048 Human Aortic Endothelium
T0068 Normal Ovar , Premeno ausal
T0069 Human Uterus, normal
T0082 Human Adult Retina
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The polypeptides of the invention can be prepared in any suitable manner.
Such polypeptides include isolated naturally occurring polypeptides,
recombinantly
produced polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means for preparing such
polypeptides
are well understood in the art.
The polypeptides may be in the form of the secreted protein, including the
mature form, or may be a part of a larger protein, such as a fusion protein
(see below).
It is often advantageous to include an additional amino acid sequence which
contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification ,
such as multiple histidine residues, or an additional sequence for stability
during
recombinant production.
The polypeptides of the present invention are preferably provided in an
isolated form, and preferably are substantially purified. A recombinantly
produced
version of a polypeptide, including the secreted polypeptide, can be
substantially
purified using techniques described herein or otherwise known in the art, such
as, for
example, by the one-step method described in Smith and Johnson, Gene 67:31-40
(1988). Polypeptides of the invention also can be purified from natural,
synthetic or
recombinant sources using techniques described herein or otherwise known in
the art,
such as, for example, antibodies of the invention raised against the secreted
protein.
The present invention provides a polynucleotide comprising, or alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA
contained
in ATCC deposit Z. The present invention also provides a polypeptide
comprising, or
alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides
encoding a polypeptide comprising, or alternatively consisting of the
polypeptide
sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA
contained in ATCC deposit Z are also encompassed by the invention.
Signal Seguences
The present invention also encompasses mature forms of the polypeptide
having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence
encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature
forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or
the
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68
polynucleotide sequence contained in the cDNA of a deposited clone) are also
encompassed by the invention. According to the signal hypothesis, proteins
secreted
by mammalian cells have a signal or secretary leader sequence which is cleaved
from
the mature protein once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and even insect
cells cleave secreted proteins with the same specificity. However, in some
cases,
cleavage of a secreted protein is not entirely uniform, which results in two
or more
mature species of the protein. Further, it has long been known that cleavage
specificity of a secreted protein is ultimately determined by the primary
structure of
the complete protein, that is, it is inherent in the amino acid sequence of
the
polypeptide.
Methods for predicting whether a protein has a signal sequence, as well as the
cleavage point for that sequence, are available. For instance, the method of
McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-
terminal
charged region and a subsequent uncharged region of the complete (uncleaved)
protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses
the
information from the residues surrounding the cleavage site, typically
residues -13 to
+2, where +1 indicates the amino terminus of the secreted protein. The
accuracy of
predicting the cleavage points of known mammalian secretory proteins for each
of
these methods is in the range of 75-80%. (von Heinje, supra.) However, the two
methods do not always produce the same predicted cleavage points) for a given
protein.
In the present case, the deduced amino acid sequence of the secreted
polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen
et
al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location
of a
protein based on the amino acid sequence. As part of this computational
prediction of
localization, the methods of McGeoch and von Heinje are incorporated. The
analysis
of the amino acid sequences of the secreted proteins described herein by this
program
provided the results shown in Table 1.
As one of ordinary skill would appreciate, however, cleavage sites sometimes
vary from organism to organism and cannot be predicted with absolute
certainty.
Accordingly, the present invention provides secreted polypeptides having a
sequence
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shown in SEQ ID NO:Y which have an N-terminus beginning within S residues
(i.e.,
+ or - 5 residues) of the predicted cleavage point. Similarly, it is also
recognized that
in some cases, cleavage of the signal sequence from a secreted protein is not
entirely
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polypeptides, are contemplated by the present
invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally occurring signal sequence may be further upstream from the predicted
signal
sequence. However, it is likely that the predicted signal sequence will be
capable of
directing the secreted protein to the ER. Nonetheless, the present invention
provides
the mature protein produced by expression of the polynucleotide sequence of
SEQ >D
NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited
clone, in a mammalian cell (e.g., COS cells, as desribed below). These
polypeptides,
and the polynucleotides encoding such polypeptides, are contemplated by the
present
invention.
Polynucleotide and Polypeptide Variants
The present invention is directed to variants of the polynucleotide sequence
disclosed in SEQ )D NO:X, the complementary strand thereto, and/or the cDNA
sequence contained in a deposited clone.
The present invention also encompasses variants of the polypeptide sequence
disclosed in SEQ >D NO:Y and/or encoded by a deposited clone.
"Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential
properties thereof. Generally, variants are overall closely similar, and, in
many
regions, identical to the polynucleotide or polypeptide of the present
invention.
The present invention is also directed to nucleic acid molecules which
comprise, or alternatively consist of, a nucleotide sequence which is at least
80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide
coding sequence in SEQ ID NO:X or the complementary strand thereto, the
nucleotide coding sequence contained in a deposited cDNA clone or the
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complementary strand thereto, a nucleotide sequence encoding the polypeptide
of
SEQ ~ NO:Y, a nucleotide sequence encoding the polypeptide encoded by the
cDNA contained in a deposited clone, and/or polynucleotide fragments of any of
these nucleic acid molecules (e.g., those fragments described herein).
5 Polynucleotides which hybridize to these nucleic acid molecules under
stringent
hybridization conditions or lower stringency conditions are also encompassed
by the
invention, as are polypeptides encoded by these polynucleotides.
The present invention is also directed to polypeptides which comprise, or
alternatively consist of, an amino acid sequence which is at least 80%, 85%,
90%,
10 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence
shown in SEQ ~ NO:Y, the polypeptide sequence encoded by the cDNA contained
in a deposited clone, and/or polypeptide fragments of any of these
polypeptides (e.g.,
those fragments described herein).
By a nucleic acid having a nucleotide sequence at least, for example, 95%
1 S "identical" to a reference nucleotide sequence of the present invention,
it is intended
that the nucleotide sequence of the nucleic acid is identical to the reference
sequence
except that the nucleotide sequence may include up to five point mutations per
each
100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In
other words, to obtain a nucleic acid having a nucleotide sequence at least
95%
20 identical to a reference nucleotide sequence, up to 5% of the nucleotides
in the
reference sequence may be deleted or substituted with another nucleotide, or a
number of nucleotides up to 5% of the total nucleotides in the reference
sequence may
be inserted into the reference sequence. The query sequence may be an entire
sequence shown inTable 1, the ORF (open reading frame), or any fragment
specified
25 as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
a
nucleotide sequence of the presence invention can be determined conventionally
using known computer programs. A preferred method for determining the best
30 overall match between a query sequence (a sequence of the present
invention) and a
subject sequence, also referred to as a global sequence alignment, can be
determined
using the FASTDB computer program based on the algorithm of Brutlag et al.
(Comp.
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App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject
sequences are both DNA sequences. An RNA sequence can.be compared by
converting U's to T's. The result of said global sequence alignment is in
percent
identity. Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject
nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the FASTDB program does nvt account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
sequences truncated at the S' or 3' ends, relative to the query sequence, the
percent
identity is corrected by calculating the number of bases of the query sequence
that are
5' and 3' of the subject sequence, which are not matched/aligned, as a percent
of the
total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent identity
score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
subject sequence is compared with a 100 base query sequence. This time the
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72
deletions are internal deletions so that there are no bases on the 5' or 3' of
the subject
sequence which are not matched/aligned with the query. In this case the
percent
identity calculated by FASTDB is not manually corrected. Once again, only
bases 5'
and 3' of the subject sequence which are not matched/aligned with the query
sequence
are manually corrected for. No other manual corrections are to made for the
purposes
of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other
words, to obtain a polypeptide having an amino acid sequence at least 95%
identical
to a query amino acid sequence, up to 5% of the amino acid residues in the
subject
sequence may be inserted, deleted, (indels) or substituted with another amino
acid.
These alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those
terminal positions, interspersed either individually among residues in the
reference
sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%,
85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid
sequences shown in Table 1 (SEQ ID NO:Y) or to the amino acid sequence encoded
by cDNA contained in a deposited clone can be determined conventionally using
known computer programs. A preferred method for determing the best overall
match
between a query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be determined
using
the FASTDB computer program based on the algorithm of Brutlag et al. (Comp.
App.
Biosci. 6:237-245(1990)). In a sequence alignment the query and subject
sequences
are either both nucleotide sequences or both amino acid sequences. The result
of said
global sequence alignment is in percent identity. Preferred parameters used in
a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
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Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence due to N- or C-
terminal deletions, not because of internal deletions, a manual correction
must be
made to the results. This is because the FASTDB program does not account for N-
and C-terminal truncations of the subject sequence when calculating global
percent
identity. For subject sequences truncated at the N- and C-termini, relative to
the
query sequence, the percent identity is corrected by calculating the number of
residues
of the query sequence that are N- and C-terminal of the subject sequence,
which are
not matched/aligned with a corresponding subject residue, as a percent of the
total
bases of the query sequence. Whether a residue is matched/aligned is
determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from
the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score
is what is used for the purposes of the present invention. Only residues to
the N- and
C-termini of the subject sequence, which are not matched/aligned with the
query
sequence, are considered for the purposes of manually adjusting the percent
identity
score. That is, only query residue positions outside the farthest N- and C-
terminal
residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues at the N-
and C-
termini not matched/total number of residues in the query sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the
remaining 90 residues were perfectly matched the final percent identity would
be
90%. In another example, a 90 residue subject sequence is compared with a 100
residue query sequence. This time the deletions are internal deletions so
there are no
residues at the N- or C-termini of the subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually corrected. Once again, only residue positions outside the N- and C-
terminal
ends of the subject sequence, as displayed in the FASTDB alignment, which are
not
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matched/aligned with the query sequnce are manually corrected for. No other
manual
corrections are to made for the purposes of the present invention.
The variants may contain alterations in the coding regions, non-coding
regions, or both. Especially preferred are polynucleotide variants containing
alterations which produce silent substitutions, additions, or deletions, but
do not alter
the properties or activities of the encoded polypeptide. Nucleotide variants
produced
by silent substitutions due to the degeneracy of the genetic code are
preferred.
Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted,
deleted, or
added in any combination are also preferred. Polynucleotide variants can be
produced
for a variety of reasons, e.g., to optimize codon expression for a particular
host
(change codons in the human mRNA to those preferred by a bacterial host such
as E.
coli).
Naturally occurring variants are called "allelic variants," and refer to one
of
several alternate forms of a gene occupying a given locus on a chromosome of
an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).)
These
allelic variants can vary at either the polynucleotide and/or polypeptide
level and are
included in the present invention. Alternatively, non-naturally occurring
variants may
be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics
of the
polypeptides of the present invention. For instance, one or more amino acids
can be
deleted from the N-terminus or C-terminus of the secreted protein without
substantial
loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988
(1993), reported variant KGF proteins having heparin binding activity even
after
deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma
exhibited up to ten times higher activity after deleting 8-10 amino acid
residues from
the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-
216
(1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity similar to that of the naturally occurring protein. For example,
Gayle and
coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive
mutational
analysis of human cytokine IL-la. They used random mutagenesis to generate
over
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3,500 individual IL-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible amino acid position. The investigators found that "[m]ost of the
molecule
could be altered with little effect on either [binding or biological
activity]." (See,
5 Abstract.) In fact, only 23 unique amino acid sequences, out of more than
3,500
nucleotide sequences examined, produced a protein that significantly differed
in
activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or
C-terminus of a polypeptide results in modification or loss of one or more
biological
10 functions, other biological activities may still be retained. For example,
the ability of
a deletion variant to induce and/or to bind antibodies which recognize the
secreted
form will likely be retained when less than the majority of the residues of
the secreted
form are removed from the N-terminus or C-terminus. Whether a particular
polypeptide lacking N- or C-terminal residues of a protein retains such
immunogenic
15 activities can readily be determined by routine methods described herein
and
otherwise known in the art.
Thus, the invention further includes polypeptide variants which show
substantial biological activity. Such variants include deletions, insertions,
inversions, repeats, and substitutions selected according to general rules
known in the
20 art so as have little effect on activity. For example, guidance concerning
how to make
phenotypically silent amino acid substitutions is provided in Bowie et al.,
Science
247:1306-1310 (1990), wherein the authors indicate that there are two main
strategies
for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
25 selection during the process of evolution. By comparing amino acid
sequences in
different species, conserved amino acids can be identified. These conserved
amino
acids are likely important for protein function. In contrast, the amino acid
positions
where substitutions have been tolerated by natural selection indicates that
these
positions are not critical for protein function. Thus, positions tolerating
amino acid
30 substitution could be modified while still maintaining biological activity
of the
protein.
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The second strategy uses genetic engineering to introduce amino acid changes
at specific positions of a cloned gene to identify regions critical for
protein function.
For example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction
of single alanine mutations at every residue in the molecule) can be used.
(Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant
molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and
Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the
acidic
residues Asp and Glu; replacement of the amide residues Asn and Gln,
replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic residues
Phe, Tyr,
and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,
and Gly.
Besides conservative amino acid substitution, variants of the present
invention
include (i) substitutions with one or more of the non-conserved amino acid
residues,
where the substituted amino acid residues may or may not be one encoded by the
genetic code, or (ii) substitution with one or more of amino acid residues
having a
substituent group, or (iii) fusion of the mature polypeptide with another
compound,
such as a compound to increase the stability and/or solubility of the
polypeptide (for
example, polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or leader or
secretory
sequence, or a sequence facilitating purification or (v) fusion of the
polypeptide with
another compound, such as albumin (including, but not limited to, recombinant
albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent
0 413
622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated
by
reference in their entirety)). Such variant polypeptides are deemed to be
within the
scope of those skilled in the art from the teachings herein.
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For example, polypeptide variants containing amino acid substitutions of
charged amino acids with other charged or neutral amino acids may produce
proteins
with improved characteristics, such as less aggregation. Aggregation of
pharmaceutical formulations both reduces activity and increases clearance due
to the
aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-
340
(1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit.
Rev.
Therapeutic Drug Carner Systems 10:307-377 (1993).)
A further embodiment of the invention relates to a polypeptide which
comprises the amino acid sequence of the present invention having an amino
acid
sequence which contains at least one amino acid substitution, but not more
than 50
amino acid substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and
still even more preferably, not more than 20 amino acid substitutions. Of
course, in
order of ever-increasing preference, it is highly preferable for a peptide or
polypeptide
to have an amino acid sequence which comprises the amino acid sequence of the
present invention, which contains at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3,
2 or 1 amino acid substitutions. In specific embodiments, the number of
additions,
substitutions, and/or deletions in the amino acid sequence of the present
invention or
fragments thereof (e.g., the mature form and/or other fragments described
herein), is
1-5, 5-10, S-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions
are
preferable.
Polynucleotide and Polypentide Fragments
The present invention is also directed to polynucleotide fragments of the
polynucleotides of the invention.
In the present invention, a "polynucleotide fragment" refers to a short
polynucleotide having a nucleic acid sequence which: is a portion of that
contained in
a deposited clone, or encoding the polypeptide encoded by the cDNA in a
deposited
clone; is a portion of that shown in SEQ ID NO:X or the complementary strand
thereto, or is a portion of a polynucleotide sequence encoding the polypeptide
of SEQ
ID NO:Y. The nucleotide fragments of the invention are preferably at least
about 1 S
nt, and more preferably at least about 20 nt, still more preferably at least
about 30 nt,
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and even more preferably, at least about 40 nt, at least about 50 nt, at least
about 75
nt, or at least about 150 nt in length. A fragment "at least 20 nt in length,"
for
example, is intended to include 20 or more contiguous bases from the cDNA
sequence contained in a deposited clone or the nucleotide sequence shown in
SEQ ID
NO:X. In this context "about" includes the particularly recited value, a value
larger
or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both
termini. These nucleotide fragments have uses that include, but are not
limited to, as
diagnostic probes and primers as discussed herein. Of course, larger fragments
(e.g.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the
invention, include, for example, fragments comprising, or alternatively
consisting of,
a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-
250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-
750,
751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,
1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,
1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,
1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the
complementary strand thereto, or the cDNA contained in a deposited clone. In
this
context "about" includes the particularly recited ranges, and ranges larger or
smaller
by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both
termini.
Preferably, these fragments encode a polypeptide which has biological
activity. More
preferably, these polynucleotides can be used as probes or primers as
discussed
herein. Polynucleotides which hybridize to these nucleic acid molecules under
stringent hybridization conditions or lower stringency conditions are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
In the present invention, a "polypeptide fragment" refers to an amino acid
sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the
cDNA contained in a deposited clone. Protein (polypeptide) fragments may be
"free-
standing," or comprised within a larger polypeptide of which the fragment
forms a
part or region, most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, include, for example,
fragments
comprising, or alternatively consisting of, from about amino acid number 1-20,
21-40,
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41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding
region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70,
80, 90,
100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about"
includes the particularly recited ranges or values, and ranges or values
larger or
smaller by several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at
both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Preferred polypeptide fragments include the secreted protein as well as the
mature form. Further preferred polypeptide fragments include the secreted
protein or
the mature form having a continuous series of deleted residues from the amino
or the
carboxy terminus, or both. For example, any number of amino acids, ranging
from 1
60, can be deleted from the amino terminus of either the secreted polypeptide
or the
mature form. Similarly, any number of amino acids, ranging from 1-30, can be
deleted from the carboxy terminus of the secreted protein or mature form.
Furthermore, any combination of the above amino and carboxy terminus deletions
are
preferred. Similarly, polynucleotides encoding these polypeptide fragments are
also
preferred.
Also preferred are polypeptide and polynucleotide fragments characterized by
structural or functional domains, such as fragments that comprise alpha-helix
and
alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn
and turn-
forming regions, coil and coil-forming regions, hydrophilic regions,
hydrophobic
regions, alpha amphipathic regions, beta amphipathic regions, flexible
regions,
surface-forming regions, substrate binding region, and high antigenic index
regions.
Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are
specifically contemplated by the present invention. Moreover, polynucleotides
encoding these domains are also contemplated.
Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
identical, to an activity of the polypeptide of the present invention. The
biological
activity of the fragments may include an improved desired activity, or a
decreased
undesirable activity. Polynucleotides encoding these polypeptide fragments are
also
encompassed by the invention.
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Preferably, the polynucleotide fragments of the invention encode a
polypeptide which demonstrates a functional activity. By a polypeptide
demonstrating a "functional activity" is meant, a polypeptide capable of
displaying
one or more known functional activities associated with a full-length
(complete)
5 polypeptide of invention protein. Such functional activities include, but
are not
limited to, biological activity, antigenicity [ability to bind (or compete
with a
polypeptide of the invention for binding) to an antibody to the polypeptide of
the
invention], immunogenicity (ability to generate antibody which binds to a
polypeptide
of the invention), ability to form multimers with polypeptides of the
invention, and
10 ability to bind to a receptor or ligand for a polypeptide of the invention.
The functional activity of polypeptides of the invention, and fragments,
variants derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or compete with full-length polypeptide of the invention for binding to an
antibody of
15 the polypeptide of the invention, various immunoassays known in the art can
be used,
including but not limited to, competitive and non-competitive assay systems
using
techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ immunoassays (using
20 colloidal gold, enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays, immunofluorescence
assays,
protein A assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary antibody. In
another
25 embodiment, the primary antibody is detected by detecting binding of a
secondary
antibody or reagent to the primary antibody. In a further embodiment, the
secondary
antibody is labeled. Many means are known in the art for detecting binding in
an
immunoassay and are within the scope of the present invention.
In another embodiment, where a ligand for a polypeptide of the invention
30 identified, or the ability of a polypeptide fragment, variant or derivative
of the
invention to multimerize is being evaluated, binding can be assayed, e.g., by
means
well-known in the art, such as, for example, reducing and non-reducing gel
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chromatography, protein affinity chromatography, and affinity blotting. See
generally, Phizicky, E., et al., 1995, lVlicrobiol. Rev. 59:94-123. In another
embodiment, physiological correlates of binding of a polypeptide of the
invention to
its substrates (signal transduction) can be assayed.
In addition, assays described herein (see Examples) and otherwise known in
the art may routinely be applied to measure the ability of polypeptides of the
invention and fragments, variants derivatives and analogs thereof to elicit
related
biological activity related to that of the polypeptide of the invention
(either in vitro or
in vivo). Other methods will be known to the skilled artisan and are within
the scope
of the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ ID
1 S NO:Y, or an epitope of the polypeptide sequence encoded by a
polynucleotide
sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:X or contained in
ATCC deposit No. Z under stringent hybridization conditions or lower
stringency
hybridization conditions as defined supra. The present invention further
encompasses
polynucleotide sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID NO:X),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence
encoding an epitope of the invention, and polynucleotide sequences which
hybridize
to the complementary strand under stringent hybridization conditions or lower
stringency hybridization conditions defined supra.
The term "epitopes," as used herein, refers to portions of a polypeptide
having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably in a human. In a preferred embodiment, the present invention
encompasses a polypeptide comprising an epitope, as well as the polynucleotide
encoding this polypeptide. An "immunogenic epitope," as used herein, is
defined as
a portion of a protein that elicits an antibody response in an animal, as
determined by
any method known in the art, for example, by the methods for generating
antibodies
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described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is
defined as a
portion of a protein to which an antibody can immunospecifically bind its
antigen as
determined by any method well known in the art, for example, by the
immunoassays
described herein. Immunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985),
further described in U.S. Patent No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 20,
at least 25, at
least 30, at least 40, at least 50, and, most preferably, between about 15 to
about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Additional non-exclusive preferred antigenic
epitopes
include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. Preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as any combination of two, three, four,
five or more
of these antigenic epitopes. Antigenic epitopes can be used as the target
molecules in
immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984);
Sutcliffe et
al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe
et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-
914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred
immunogenic
epitopes include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic epitopes.
The
polypeptides comprising one or more immunogenic epitopes may be presented for
eliciting an antibody. response together with a carrier protein, such as an
albumin, to
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an animal system (such as rabbit or mouse), or, if the polypeptide is of
sufficient
length (at least about 25 amino acids), the polypeptide may be presented
without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino
acids
have been shown to be sufficient to raise antibodies capable of binding to, at
the very
S least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to,
in vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-
2354 (1985). If in vivo immunization is used, animals may be immunized with
free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide
to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus
toxoid. For instance, peptides containing cysteine residues may be coupled to
a
carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester
(MBS),
while other peptides may be coupled to carriers using a more general linking
agent
such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized
with
either free or carrier- coupled peptides, for instance, by intraperitoneal
and/or
intradermal injection of emulsions containing about 100 pg of peptide or
Garner
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune response. Several booster injections may be needed, for instance, at
intervals of about two weeks, to provide a useful titer of anti-peptide
antibody which
can be detected, for example, by ELISA assay using free peptide adsorbed to a
solid
surface. The titer of anti-peptide antibodies in serum from an immunized
animal may
be increased by selection of anti-peptide antibodies, for instance, by
adsorption to the
peptide on a solid support and elution of the selected antibodies according to
methods
well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention comprising an immunogenic or antigenic
epitope can be fused to other polypeptide sequences. For example, the
polypeptides
of the present invention may be fused with the constant domain of
immunoglobulins
(IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof), or albumin (including but not limited to
recombinant
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albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent
0 413
622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated
by
reference in their entirety)), resulting in chimeric polypeptides. Such fusion
proteins
may facilitate purification and may increase half life in vivo. This has been
shown
for chimeric proteins consisting of the first two domains of the human CD4-
polypeptide and various domains of the constant regions of the heavy or light
chains
of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al.,
Nature,
331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barner
to the
immune system has been demonstrated for antigens (e.g., insulin) conjugated to
an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications
WO
96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG portion desulfide bonds have also been found
to be
more efficient in binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J.
Biochem.,
270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be
recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin
("HA")
tag or flag tag) to aid in detection and purification of the expressed
polypeptide. For
example, a system described by Janknecht et al. allows for the ready
purification of
non-denatured fusion proteins expressed in human cell lines (Janknecht et al.,
1991,
Proc. Natl. Acad. Sci. USA 88:8972- 897). In this system, the gene of interest
is
subcloned into a vaccinia recombination plasmid such that the open reading
frame of
the gene is translationally fused to an amino-terminal tag consisting of six
histidine
residues. The tag serves as a matrix binding domain for the fusion protein.
Extracts
from cells infected with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can be
selectively
eluted with imidazole-containing buffers.
Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to
modulate the activities of polypeptides of the invention, such methods can be
used to
generate polypeptides with altered activity, as well as agonists and
antagonists of the
polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721;
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5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-
33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
(1998) (each of these patents and publications are hereby incorporated by
reference in
5 its entirety). In one embodiment, alteration of polynucleotides
corresponding to SEQ
ID NO:X and the polypeptides encoded by these polynucleotides may be achieved
by
DNA shuffling. DNA shuffling involves the assembly of two or more DNA
segments by homologous or site-specific recombination to generate variation in
the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention,
10 or the encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior
to recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of
the
invention may be recombined with one or more components, motifs, sections,
parts,
15 domains, fragments, etc. of one or more heterologous molecules.
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment,
20 or variant of SEQ 1D NO:Y, and/or an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying specific
antibody-
antigen binding). Antibodies of the invention include, but are not limited to,
polyclonal, monoclonal, multispecific, human, humanized or chimeric
antibodies,
single chain antibodies, Fab fragments, F(ab') fragments, fragments produced
by a
25 Fab expression library, anti-idiotypic (anti-Id) antibodies (including,
e.g., anti-Id
antibodies to antibodies of the invention), and epitope-binding fragments of
any of
the above. The term "antibody," as used herein, refers to immunoglobulin
molecules
and immunologically active portions of immuiloglobulin molecules, i.e.,
molecules
that contain an antigen binding site that immunospecifically binds an antigen.
The
30 immunoglobulin molecules of the invention can be of any type (e.g., IgG,
IgE, IgM,
IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and IgA2) or
subclass
of immunoglobulin molecule. In preferred embodiments, the immunoglobulin
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86
molecules of the invention are IgGI. In other preferred embodiments, the
immunoglobulin molecules of the invention are IgG4.
Most preferably the antibodies are human antigen-binding antibody fragments
of the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd,
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv)
and
fragments comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the variable
regions)
alone or in combination with the entirety or a portion of the following: hinge
region,
CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CH1, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human immunoglobulin and include antibodies isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of
the epitope(s) or portions) of a polypeptide of the present invention which
they
recognize or specifically bind. The epitope(s) or polypeptide portions) may be
specified as described herein, e.g., by N-terminal and C-terminal positions,
by size in
contiguous amino acid residues, or listed in the Tables and Figures.
Antibodies which
specifically bind any epitope or polypeptide of the present invention may also
be
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excluded. Therefore, the present invention includes antibodies that
specifically bind
polypeptides of the present invention, and allows for the exclusion of the
same.
Antibodies of the present invention may also be described or specified in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least
SS%, and at
least 50% identity (as calculated using methods known in the art and described
herein) to a polypeptide of the present invention are also included in the
present
invention. In specific embodiments, antibodies of the present invention cross-
react
with murine, rat and/or rabbit homologs of human proteins and the
corresponding
epitopes thereof. Antibodies that do not bind polypeptides with less than 95%,
less
than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less
than 65%,
less than 60%, less than 55%, and less than SO% identity (as calculated using
1 S methods known in the art and described herein) to a polypeptide of the
present
invention are also included in the present invention. In a specific
embodiment, the
above-described cross-reactivity is with respect to any single specific
antigenic or
immunogenic polypeptide, or combinations) of 2, 3, 4, 5, or more of the
specific
antigenic and/or immunogenic polypeptides disclosed herein. Further included
in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides
which hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the present
invention
may also be described or specified in terms of their binding affinity to a
polypeptide
of the invention. Preferred binding affinities include those with a
dissociation
constant or Kd less than 5 X 10-2 M, 10-Z M, S X 10-3 M, 10-3 M, 5 X 10-4 M,
10-4 M,
5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-~ M, 107 M, 5 X 10-8 M, 10-g M,
5 X
10-~ M, 109 M, 5 X 10-' ° M, 10-' ° M, 5 X 10-" M, 10-" M, 5 X
10-' 2 M, ' °-' Z M, 5 X
10-' 3 M, 10-' 3 M, 5 X 10-' 4 M, 10-' 4 M, 5 X 10-' S M, or 10-' S M.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art
for determining competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively inhibits binding
to the
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epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least
75%, at least
70%, at least 60%, or at least SO%.
Antibodies of the present invention may act as agonists or antagonists of the
polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. Preferrably, antibodies of the present
invention
bind an antigenic epitope disclosed herein, or a portion thereof. The
invention
features both receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor-specific antibodies which do not prevent
ligand
binding but prevent receptor activation. Receptor activation (i.e., signaling)
may be
determined by techniques described herein or otherwise known in the art. For
example, receptor activation can be determined by detecting the
phosphorylation
(e.g., tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example, as
described
supra). In specific embodiments, antibodies are provided that inhibit ligand
activity
or receptor activity by at least 95%, at least 90%, at least 85%, at least
80%, at least
75%, at least 70%, at least 60%, or at least 50% of the activity in absence of
the
antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
well as antibodies which bind the ligand, thereby preventing receptor
activation, but
do not prevent the ligand from binding the receptor. Further included in the
invention
are antibodies which activate the receptor. These antibodies may act as
receptor
agonists, i.e., potentiate or activate either all or a subset of the
biological activities of
the ligand-mediated receptor activation, for example, by inducing dimerization
of the
receptor. The antibodies may be specified as agonists, antagonists or inverse
agonists
for biological activities comprising the specific biological activities of the
peptides of
the invention disclosed herein. The above antibody agonists can be made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
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5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu
et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-
3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et
al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et
al.,
Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998);
Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference
herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited
to, to purify, detect, and target the polypeptides of the present invention,
including
both in vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and quantitatively
measuring
levels of the polypeptides of the present invention in biological samples.
See, e.g.,
1 S Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may
be used either alone or in combination with other compositions. The antibodies
may
further be recombinantly fused to a heterologous polypeptide at the N- or C-
terminus
or chemically conjugated (including covalently and non-covalently
conjugations) to
polypeptides or other compositions. For example, antibodies of the present
invention
may be recombinantly fused or conjugated to molecules useful as labels in
detection
assays and effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO
91/14438;
WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e, by
the covalent attachment of any type of molecule to the antibody such that
covalent
attachment does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation, acetylation,
pegylation,
phosphylation, amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any
of
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numerous chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage, acetylation,
formylation,
metabolic synthesis of tunicamycin, etc. Additionally, the derivative may
contain
one or more non-classical amino acids.
5 The antibodies of the present invention may be generated by any suitable
method known in the art. Polyclonal antibodies to an antigen-of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of
the invention can be administered to various host animals including, but not
limited
to, rabbits, mice, rats, etc. to induce the production of sera containing
polyclonal
10 antibodies specific for the antigen. Various adjuvants may be used to
increase the
immunological response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
15 potentially useful human adjuvants such as BCG (bacille Calmette-Guerin)
and
corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be
20 produced using hybridoma techniques including those known in the art and
taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and
T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated
by
reference in their entireties). The term "monoclonal antibody" as used herein
is not
25 limited to antibodies produced through hybridoma technology. The term
"monoclonal antibody" refers to an antibody that is derived from a single
clone,
including any eukaryotic, prokaryotic, or phage clone, and not the method by
which it
is produced.
Methods for producing and screening for specific antibodies using hybridoma
30 technology are routine and well known in the art and are discussed in
detail in the
Examples (e.g., Example 16). In a non-limiting example, mice can be immunized
with a polypeptide of the invention or a cell expressing such peptide. Once an
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immune response is detected, e.g., antibodies specific for the antigen are
detected in
the mouse serum, the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by well known techniques to any suitable myeloma
cells,
for example cells from cell line SP20 available from the ATCC. Hybridomas are
S selected and cloned by limited dilution. The hybridoma clones are then
assayed by
methods known in the art for cells that secrete antibodies capable of binding
a
polypeptide of the invention. Ascites fluid, which generally contains high
levels of
antibodies, can be generated by immunizing mice with positive hybridoma
clones.
Accordingly, the present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method comprising
culturing a hybridoma cell secreting an antibody of the invention wherein,
preferably,
the hybridoma is generated by fusing splenocytes isolated from a mouse
immunized
with an antigen of the invention with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that secrete an
antibody
able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such
as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region
and the
CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
using various phage display methods known in the art. In phage display
methods,
functional antibody domains are displayed on the surface of phage particles
which
carry the polynucleotide sequences encoding them. In a particular embodiment,
such
phage can be utilized to display antigen binding domains expressed from a
repertoire
or combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or
identified
with antigen, e.g., using labeled antigen or antigen bound or captured to a
solid
surface or bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab, Fv or
disulfide stabilized Fv antibody domains recombinantly fused to either the
phage
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gene III or gene VIII protein. Examples of phage display methods that can be
used to
make the antibodies of the present invention include those disclosed in
Brinkman et
al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic
et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;
5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI
34:26-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston
et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including
in vivo use of antibodies in humans and in vitro detection assays, it may be
preferable
to use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule
in which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
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Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entirety. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known in the art, e.g., by modeling of the interactions of the CDR and
framework
residues to identify framework residues important for antigen binding and
sequence
comparison to identify unusual framework residues at particular positions.
(See, e.g.,
Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323
(1988),
which are incorporated herein by reference in their entireties.) Antibodies
can be
humanized using a variety of techniques known in the art including, for
example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al.,
Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973
(1994)),
and chain shuffling (U.5. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes. For example, the human heavy and light
chain immunoglobulin gene complexes may be introduced randomly or by
homologous recombination into mouse embryonic stem cells. Alternatively, the
human variable region, constant region, and diversity region may be introduced
into
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mouse embryonic stem cells in addition to the human heavy and light chain
genes.
The mouse heavy and light chain immunoglobulin genes may be rendered non-
functional separately or simultaneously with the introduction of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of the JH region prevents endogenous antibody production. The
modified
embryonic stem cells are expanded and microinjected into blastocysts to
produce
chimeric mice. The chimeric mice are then bred to produce homozygous offspring
which express human antibodies. The transgenic mice are immunized in the
normal
fashion with a selected antigen, e.g., all or a portion of a polypeptide of
the invention.
Monoclonal antibodies directed against the antigen can be obtained from the
immunized, transgenic mice using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange during B
cell
differentiation, and subsequently undergo class switching and somatic
mutation.
Thus, using such a technique, it is possible to produce therapeutically useful
IgG, IgA,
IgM and IgE antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a
detailed discussion of this technology for producing human antibodies and
human '
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which
are
incorporated by reference herein in their entirety. In addition, companies
such as
Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to
provide human antibodies directed against a selected antigen using technology
similar
to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Biotechnology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized
to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using
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techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies which bind to and competitively inhibit
polypeptide
multimerization and/or binding of a polypeptide of the invention to a ligand
can be
5 used to generate anti-idiotypes that "mimic" the polypeptide multimerization
and/or
binding domain and, as a consequence, bind to and neutralize polypeptide
and/or its
ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-
idiotypes can
be used in therapeutic regimens to neutralize polypeptide ligand. For example,
such
anti-idiotypic antibodies can be used to bind a polypeptide of the invention
and/or to
10 bind its ligands/receptors, and thereby block its biological activity.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
1 S invention also encompasses polynucleotides that hybridize under stringent
or lower
stringency hybridization conditions, e.g., as defined supra, to
polynucleotides that
encode an antibody, preferably, that specifically binds to a polypeptide of
the
invention, preferably, an antibody that binds to a polypeptide having the
amino acid
sequence of SEQ ID NO:Y.
20 The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described
in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the
25 synthesis of overlapping oligonucleotides containing portions of the
sequence
encoding the antibody, annealing and ligating of those oligonucleotides, and
then
amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from
nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a
30 particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoglobulin may be chemically
synthesized
or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA
library
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generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any
tissue
or cells expressing the antibody, such as hybridoma cells selected to express
an
antibody of the invention) by PCR amplification using synthetic primers
hybridizable
to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide
probe
specific for the particular gene sequence to identify, e.g., a cDNA clone from
a
cDNA library that encodes the antibody. Amplified nucleic acids generated by
PCR
may then be cloned into replicable cloning vectors using any method well known
in
the art.
Once the nucleotide sequence and corresponding amino acid sequence of the
antibody is determined, the nucleotide sequence of the antibody may be
manipulated
using methods well known in the art for the manipulation of nucleotide
sequences,
e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see,
for
example, the techniques described in Sambrook et al., 1990, Molecular Cloning,
A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John
Wiley & Sons, NY, which are both incorporated by reference herein in their
entireties ), to generate antibodies having a different amino acid sequence,
for
example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light
chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the
art, e.g., by comparison to known amino acid sequences of other heavy and
light
chain variable regions to determine the regions of sequence hypervariability.
Using
routine recombinant DNA techniques, one or more of the CDRs may be inserted
within framework regions, e.g., into human framework regions to humanize a non-
human antibody, as described supra. The framework regions may be naturally
occurring or consensus framework regions, and preferably human framework
regions
(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of
human
framework regions). Preferably, the polynucleotide generated by the
combination of
the framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the. framework regions, and, preferably, the
amino
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acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
alterations to the polynucleotide are encompassed by the present invention and
within
the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing
genes
from a mouse antibody molecule of appropriate antigen specificity together
with
genes from a human antibody molecule of appropriate biological activity can be
used.
As described supra, a chimeric antibody is a molecule in which different
portions are
derived from different animal species, such as those having a variable region
derived
from a murine mAb and a human immunoglobulin constant region, e.g., humanized
antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et
al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature
334:544-54 (1989)) can be adapted to produce single chain antibodies. Single
chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al.,
Science 242:1038- 1041 (1988)).
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the
art for the synthesis of antibodies, in particular, by chemical synthesis or
preferably,
by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, (e.g., a heavy or light chain of an antibody of
the
invention or a single chain antibody of the invention), requires construction
of an
expression vector containing a polynucleotide that encodes the antibody. Once
a
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polynucleotide encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or light chain
variable
domain), of the invention has been obtained, the vector for the production of
the
antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding nucleotide
sequence are
described herein. Methods which are well known to those skilled in the art can
be
used to construct expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals. These methods
include,
for example, in vitro recombinant DNA techniques, synthetic techniques, and in
vivo
genetic recombination. The invention, thus, provides replicable vectors
comprising a
nucleotide sequence encoding an antibody molecule of the invention, or a heavy
or
light chain thereof, or a heavy or light chain variable domain, operably
linked to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant
region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of
the antibody may be cloned into such a vector for expression of the entire
heavy or
light chain.
The expression vector is transferred to a host cell by conventional techniques
and the transfected cells are then cultured by conventional techniques to
produce an
antibody of the invention. Thus, the invention includes host cells containing
a
polynucleotide encoding an antibody of the invention, or a heavy or light
chain
thereof, or a single chain antibody of the invention, operably linked to a
heterologous
promoter. In preferred embodiments for the expression of double-chained
antibodies,
vectors encoding both the heavy and light chains may be co-expressed in the
host cell
for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced and
subsequently
purified, but also represent cells which may, when transformed or transfected
with
the appropriate nucleotide coding sequences, express an antibody molecule of
the
invention in situ. These include but are not limited to microorganisms such as
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bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant
yeast
expression vectors containing antibody coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g., baculovirus)
containing
antibody coding sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus,
TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid)
containing antibody coding sequences; or mammalian cell systems (e.g., COS,
CHO,
BHK, 293, 3T3 cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia
coli,
and more preferably, eukaryotic cells, especially for the expression of whole
recombinant antibody molecule, are used for the expression of a recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells (CHO), in conjunction with a vector such as the major intermediate early
gene
promoter element from human cytomegalovirus is an effective expression system
for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2
(1990)).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule being
expressed.
For example, when a large quantity of such a protein is to be produced, for
the
generation of pharmaceutical compositions of an antibody molecule, vectors
which
direct the expression of high levels of fusion protein products that are
readily purified
may be desirable. Such vectors include, but are not limited, to the E. coli
expression
vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding
sequence may be ligated individually into the vector in frame with the lac Z
coding
region so that a fusion protein is produced; pIN vectors (Inouye & Inouye,
Nucleic
Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-
5509 (1989)); and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such
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fusion proteins are soluble and can easily be purified from lysed cells by
adsorption
and binding to matrix glutathione-agarose beads followed by elution in the
presence
of free glutathione. The pGEX vectors are designed to include thrombin or
factor Xa
protease cleavage sites so that the cloned target gene product can be released
from the
GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin gene) of
the virus
and placed under control of an AcNPV promoter (for example the polyhedrin
promoter).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region
E1 or E3) will result in a recombinant virus that is viable and capable of
expressing
the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad.
Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required
for
efficient translation of inserted antibody coding sequences. These signals
include the
ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding sequence to
ensure
translation of the entire insert. These exogenous translational control
signals and
initiation codons can be of a variety of origins, both natural and synthetic.
The
efficiency of expression may be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc. (see Bittner
et al.,
Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression
of the inserted sequences, or modifies and processes the gene product in the
specific
fashion desired. Such modifications (e.g., glycosylation) and processing
(e.g.,
cleavage) of protein products may be important for the function of the
protein.
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Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins and gene products.
Appropriate
cell lines or host systems can be chosen to ensure the correct modification
and
processing ofthe foreign protein expressed. To this end, eukaryotic host cells
which
possess the cellular machinery for proper processing of the primary
transcript,
glycosylation, and phosphorylation of the gene product may be used. Such
mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with DNA
controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to
grow for 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the
herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc.
Natl.
Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et
al.,
Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells,
respectively.
Also, antimetabolite resistance can be used as the basis of selection for the
following
genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl.
Acad. Sci.
USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981));
gpt,
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which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad.
Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside
6-
418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers
resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in
the art of recombinant DNA technology may be routinely applied to select the
desired
recombinant clone, and such methods are described, for example, in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.
Biol. 1 S0:1 ( 1981 ), which are incorporated by reference herein in their
entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second
vector encoding a light chain derived polypeptide. The two vectors may contain
identical selectable markers which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes, and is
capable of expressing, both heavy and light chain polypeptides. In such
situations,
the light chain should be placed before the heavy chain to avoid an excess of
toxic
free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad.
Sci.
USA 77:2197 (1980)). The coding sequences for the heavy and light chains may
comprise cDNA or genomic DNA.
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Once an antibody molecule of the invention has been produced by an animal,
chemically synthesized, or recombinantly expressed, it may be purified by any
method known in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity, particularly by
affinity for
the specific antigen after Protein A, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard technique
for the
purification of proteins. In addition, the antibodies of the present invention
or
fragments thereof can be fused to heterologous polypeptide sequences described
herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations)
to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50,
60, 70, 80,
90 or 100 amino acids of the polypeptide) of the present invention to generate
fusion
proteins. The fusion does not necessarily need to be direct, but may occur
through
linker sequences. The antibodies may be specific for antigens other than
polypeptides
(or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or
100 amino
acids of the polypeptide) of the present invention. For example, antibodies
may be
used to target the polypeptides of the present invention to particular cell
types, either
in vitro or in vivo, by fusing or conjugating the polypeptides of the present
invention
to antibodies specific for particular cell surface receptors. Antibodies fused
or
conjugated to the polypeptides of the present invention may also be used in in
vitro
immunoassays and purification methods using methods known in the art. See
e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et
al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al.,
PNAS
89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are
incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any
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combination of whole domains or portions thereof. The polypeptides may also be
fused or conjugated to the above antibody portions to form multimers. For
example,
Fc portions fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher multimeric forms can
be
made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing
or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad. Sci.
USA 89:11337- 11341(1992) (said references incorporated by reference in their
entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated
to
the above antibody portions to increase the in vivo half life of the
polypeptides or for
use in immunoassays using methods known in the art. Further, the polypeptides
corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody
portions to facilitate purification. One reported example describes chimeric
proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The
polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964
(1995)). In
many cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and
thus can result in, for example, improved pharmacokinetic properties. (EP A
232,262). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
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identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition
8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can be
fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311), among others, many of which are commercially available. As described
in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for convenient purification of the fusion protein. Other
peptide tags
useful for purification include, but are not limited to, the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson
et al., Cell 37:767 (1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given
treatment regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include various
enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, radioactive materials, positron emitting metals using various
positron
emission tomographies, and nonradioactive paramagnetic metal ions. The
detectable
substance may be coupled or conjugated either directly to the antibody (or
fragment
thereof) or indirectly, through an intermediate (such as, for example, a
linker known
in the art) using techniques known in the art. See, for example, U.S. Patent
No.
4,741,900 for metal ions which can be conjugated to antibodies for use as
diagnostics
according to the present invention. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin
and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material
includes luminol; examples of bioluminescent materials include luciferase,
luciferin,
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and aequorin; and examples of suitable radioactive material include 125I,
131I, 111In
or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or
a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells. Examples
include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to
classical chemical therapeutic agents. For example, the drug moiety may be a
protein
or polypeptide possessing a desired biological activity. Such proteins may
include,
for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria
toxin; a protein such as tumor necrosis factor, a-interferon,13-interferon,
nerve growth
factor, platelet derived growth factor, tissue plasminogen activator, an
apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand
(Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International
Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent,
e.g.,
angiostatin or endostatin; or, biological response modifiers such as, for
example,
lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6
("IL-6"),
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granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte
colony
stimulating factor ("G-CSF"), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly
useful for immunoassays or purification of the target antigen. Such solid
supports
include, but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene,
polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld
et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),
pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Garners Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis,
Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980,
which
is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of
cell lines and biological samples. The translation product of the gene of the
present
invention may be useful as a cell specific marker, or more specifically as a
cellular
marker that is differentially expressed at various stages of differentiation
and/or
maturation of particular cell types. Monoclonal antibodies directed against a
specific
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epitope, or combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be utilized using
monoclonal antibodies to screen for cellular populations expressing the
marker(s), and
include magnetic separation using antibody-coated magnetic beads, "panning"
with
antibody attached to a solid matrix (i.e., plate), and flow cytometry (See,
e.g., U.S.
Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells,
such as might be found with hematological malignancies (i.e. minimal residual
disease (MRD) in acute leukemic patients) and "non-self' cells in
transplantations to
prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for
the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood.
Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding
by any method known in the art. The immunoassays which can be used include but
are not limited to competitive and non-competitive assay systems using
techniques
such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions,
gel diffusion precipitin reactions, immunodiffusion assays, agglutination
assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
protein A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular
Biology, Vol. l, John Wiley & Sons, Inc., New York, which is incorporated by
reference herein in its entirety). Exemplary immunoassays are described
briefly
below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells
in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium
deoxycholate, 0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1
Trasylol) supplemented with protein phosphatase and/or protease inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C,
adding protein A
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and/or protein G sepharose beads to the cell lysate, incubating for about an
hour or
more at 4° C, washing the beads in lysis buffer and resuspending the
beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a
particular antigen can be assessed by, e.g., western blot analysis. One of
skill in the
art would be knowledgeable as to the parameters that can be modified to
increase the
binding of the antibody to an antigen and decrease the background (e.g., pre-
clearing
the cell lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transfernng the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-
fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking
the membrane with primary antibody (the antibody of interest) diluted in
blocking
buffer, washing the membrane in washing buffer, blocking the membrane with a
secondary antibody (which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase
or
alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in
blocking
buffer, washing the membrane in wash buffer, and detecting the presence of the
antigen. One of skill in the art would be knowledgeable as to the parameters
that can
be modified to increase the signal detected and to reduce the background
noise. For
further discussion regarding western blot protocols see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New
York
at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate with the antigen, adding the antibody of interest conjugated to a
detectable
compound such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline
phosphatase) to the well and incubating for a period of time, and detecting
the
presence of the antigen. In ELISAs the antibody of interest does not have to
be
conjugated to a detectable compound; instead, a second antibody (which
recognizes
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the antibody of interest) conjugated to a detectable compound may be added to
the
well. Further, instead of coating the well with the antigen, the antibody may
be
coated to the well. In this case, a second antibody conjugated to a detectable
compound may be added following the addition of the antigen of interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that
can be modified to increase the signal detected as well as other variations of
ELISAs
known in the art. For further discussion regarding ELISAs see, e.g., Ausubel
et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc.,
New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody bound to the labeled antigen. The affinity of the antibody of
interest for a
particular antigen and the binding off rates can be determined from the data
by
scatchard plot analysis. Competition with a second antibody can also be
determined
using radioimmunoassays. In this case, the antigen is incubated with antibody
of
interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence
of
increasing amounts of an unlabeled second antibody.
Therapeutic Uses
The present invention is further directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, preferably a
mammal,
and most preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or conditions. Therapeutic compounds of the invention
include,
but are not limited to, antibodies of the invention (including fragments,
analogs and
derivatives thereof as described herein) and nucleic acids encoding antibodies
of the
invention (including fragments, analogs and derivatives thereof and anti-
idiotypic
antibodies as described herein). The antibodies of the invention can be used
to treat,
inhibit or prevent diseases, disorders or conditions associated with aberrant
expression
and/or activity of a polypeptide of the invention, including, but not limited
to, any
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one or more of the diseases, disorders, or conditions described herein. The
treatment
and/or prevention of diseases, disorders, or conditions associated with
aberrant
expression and/or activity of a polypeptide of the invention includes, but is
not
limited to, alleviating symptoms associated with those diseases, disorders or
conditions. Antibodies of the invention may be provided in pharmaceutically
acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may
be used therapeutically includes binding polynucleotides or polypeptides of
the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed with the
teachings provided herein, one of ordinary skill in the art will know how to
use the
antibodies of the present invention for diagnostic, monitoring or therapeutic
purposes
without undue experimentation.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and II,-7), for
example, which
serve to increase the number or activity of effector cells which interact with
the
antibodies.
The antibodies of the invention may be administered alone or in combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal
therapy, immunotherapy and anti-tumor agents). Generally, administration of
products of a species origin or species reactivity (in the case of antibodies)
that is the
same species as that of the patient is preferred. Thus, in a preferred
embodiment,
human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered
to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of disorders related to polynucleotides or polypeptides, including
fragments
thereof, of the present invention. Such antibodies, fragments, or regions,
will
preferably have an affinity for polynucleotides or polypeptides of the
invention,
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including fragments thereof. Preferred binding affinities include those with a
dissociation constant or Kd less than 5 X 10-2 M, 10-Z M, 5 X 10-3 M, 10-3 M,
5 X 10-
4 M, 10-4 M, 5 X 10-5 M, 105 M, 5 X 10-G M, 10-6 M, 5 X 10-~ M, 10-~ M, 5 X 10-
$ M,
10-g M, 5 X 10-~ M, 10-9 M, 5 X 10-' ° M, 10-' ° M, 5 X 10-" M,
10-'' M, 5 X 10-' Z M,
10-' 2 M, 5 X 10-' 3 M, 10-' 3 M, S X 10-' 4 M, 10-' 4 M, 5 X 10-' S M, and 10-
' S M.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
1 S Any of the methods for gene therapy available in the art can be used
according
to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art
of recombinant DNA technology which can be used are described in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
and
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990).
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors
that express the antibody or fragments or chimeric proteins or heavy or light
chains
thereof in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or
constitutive, and, optionally, tissue- specific. In another particular
embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other
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desired sequences are .flanked by regions that promote homologous
recombination at a
desired site in the genome, thus providing for intrachromosomal expression of
the
antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci.
USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific
embodiments, the expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy
and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case
the patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or
indirect, in which case, cells are first transformed with the nucleic acids in
vitro, then
transplanted into the patient. These two approaches are known, respectively,
as in
vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered
in vivo, where it is expressed to produce the encoded product. This can be
accomplished by any of numerous methods known in the art, e.g., by
constructing
them as part of an appropriate nucleic acid expression vector and
administering it so
that they become intracellular, e.g., by infection using defective or
attenuated
retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by
direct
injection of naked DNA, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
agents, encapsulation in liposomes, microparticles, or microcapsules, or by
administering them in linkage to a peptide which is known to enter the
nucleus, by
administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic
acid-ligand complexes can be formed in which the ligand comprises a fusogenic
viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO
93/20221 ). Alternatively, the nucleic acid can be introduced intracellularly
and
incorporated within host cell DNA for expression, by homologous recombination
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(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al.,
Nature 342:435-438 (1989)).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can
be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These
retroviral
vectors contain the components necessary for the correct packaging of the
viral
genome and integration into the host cell DNA. The nucleic acid sequences
encoding
the antibody to be used in gene therapy are cloned into one or more vectors,
which
facilitates delivery of the gene into a patient. More detail about retroviral
vectors can
be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem cells in
order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-
651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,
Human
Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in
Genetics
and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of
being capable of infecting non-dividing cells. Kozarsky and Wilson, Current
Opinion in Genetics and Development 3:499-503 (1993) present a review of
adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory
epithelia of rhesus monkeys. Other instances of the use of adenoviruses in
gene
therapy can be found in Rosenfeld et al., Science 252:431-434 (1991);
Rosenfeld et
al., Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234
(1993);
PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995).
In
a preferred embodiment, adenovirus vectors are used.
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Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
Another approach to gene therapy involves transferring a gene to cells in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes the
transfer of a selectable marker to the cells. The cells are then placed under
selection
to isolate those cells that have taken up and are expressing the transferred
gene.
Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method known in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
containing the nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes into
cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,
Meth.
Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be
used in accordance with the present invention, provided that the necessary
developmental and physiological functions of the recipient cells are not
disrupted.
The technique should provide for the stable transfer of the nucleic acid to
the cell, so
that the nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
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neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of
the present invention (see e.g. PCT Publication WO 94/08598; Stemple and
Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980);
and
Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region,
such that expression of the nucleic acid is controllable by controlling the
presence or
absence of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo for the desired therapeutic or
prophylactic
activity, prior to use in humans. For example, in vitro assays to demonstrate
the
therapeutic or prophylactic utility of a compound or pharmaceutical
composition
include, the effect of a compound on a cell line or a patient tissue sample.
The effect
of the compound or composition on the cell line and/or tissue sample can be
determined utilizing techniques known to those of skill in the art including,
but not
limited to, rosette formation assays and cell lysis assays. In accordance with
the
invention, in vitro assays which can be used to determine whether
administration of a
specific compound is indicated, include in vitro cell culture assays in which
a patient
tissue sample is grown in culture, and exposed to or otherwise administered a
compound, and the effect of such compound upon the tissue sample is observed.
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TherapeuticlProphylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention, preferably an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired side-effects). The
subject is
preferably an animal, including but not limited to animals such as cows, pigs,
horses,
chickens, cats, dogs, etc., and is preferably a mammal, and most preferably
human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor-
mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432
(1987)),
construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of
introduction include but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes. The
compounds or
compositions may be administered by any convenient route, for example by
infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered
together
with other biologically active agents. Administration can be systemic or
local. In
addition, it may be desirable to introduce the pharmaceutical compounds or
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such
as an Ommaya reservoir. Pulmonary administration can also be employed, e.g.,
by
use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical
compounds or compositions of the invention locally to the area in need of
treatment;
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this may be achieved by, for example, and not by way of limitation, local
infusion
during surgery, topical application, e.g., in conjunction with a wound
dressing after
surgery, by injection, by means of a catheter, by means of a suppository, or
by means
of an implant, said implant being of a porous, non-porous, or gelatinous
material,
including membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention, care must be
taken to
use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in
a controlled release system. In one embodiment, a pump may be used (see
Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974);
Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen
and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.
Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During
et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105
(1989)). In yet
another embodiment, a controlled release system can be placed in proximity of
the
therapeutic target, i.e., the brain, thus requiring only a fraction of the
systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release, supra,
vol. 2, pp.
115-138 (1984)).
Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic
acid expression vector and administering it so that it becomes intracellular,
e.g., by
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use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct
injection, or by
use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating
with lipids or cell-surface receptors or transfecting agents, or by
administering it in
linkage to a homeobox- like peptide which is known to enter the nucleus (see
e.g.,
S Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc.
Alternatively, a
nucleic acid can be introduced intracellularly and incorporated within host
cell DNA
for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal
or a state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carner" refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic
is administered. Such pharmaceutical carriers can be sterile liquids, such as
water
and oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
agents. These compositions can take the form of solutions, suspensions,
emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The
composition can be formulated as a suppository, with traditional binders and
Garners
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical Garners are described in "Remington's Pharmaceutical Sciences"
by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of
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the compound, preferably in purified form, together with a suitable amount of
carrier
so as to provide the form for proper administration to the patient. The
formulation
should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an
infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition
is administered by injection, an ampoule of sterile water for injection or
saline can be
provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with canons such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by
standard clinical techniques. In addition, in vitro assays may optionally be
employed
to help identify optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and the
seriousness of
the disease or disorder, and should be decided according to the judgment of
the
practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
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For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a
patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
S antibodies have a longer half life within the human body than antibodies
from other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
dosage and frequency of administration of antibodies of the invention may be
reduced by enhancing uptake and tissue penetration (e.g., into the brain) of
the
antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically
bind to a polypeptide of interest can be used for diagnostic purposes to
detect,
diagnose, or monitor diseases, disorders, and/or conditions associated with
the
aberrant expression and/or activity of a polypeptide of the invention. The
invention
provides for the detection of aberrant expression of a polypeptide of
interest,
comprising (a) assaying the expression of the polypeptide of interest in cells
or body
fluid of an individual using one or more antibodies specific to the
polypeptide interest
and (b) comparing the level of gene expression with a standard gene expression
level,
whereby an increase or decrease in the assayed polypeptide gene expression
level
compared to the standard expression level is indicative of aberrant
expression.
The invention provides a diagnostic assay for diagnosing a disorder,
comprising (a) assaying the expression of the polypeptide of interest in cells
or body
fluid of an individual using one or more antibodies specific to the
polypeptide interest
and (b) comparing the level of gene expression with a standard gene expression
level,
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whereby an increase or decrease in the assayed polypeptide gene expression
level
compared to the standard expression level is indicative of a particular
disorder. With
respect to cancer, the presence of a relatively high amount of transcript in
biopsied
tissue from an individual may indicate a predisposition for the development of
the
disease, or may provide a means for detecting the disease prior to the
appearance of
actual clinical symptoms. A more definitive diagnosis of this type may allow
health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et
al., J. Cell .
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting
protein gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S),
tritium (3H),
indium (112In), and technetium (99Tc); luminescent labels, such as luminol;
and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of interest in
an animal,
preferably a mammal and most preferably a human. In one embodiment, diagnosis
comprises: a) administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled molecule
which
specifically binds to the polypeptide of interest; b) waiting for a time
interval
following the administering for permitting the labeled molecule to
preferentially
concentrate at sites in the subject where the polypeptide is expressed (and
for
unbound labeled molecule to be cleared to background level); c) determining
background level; and d) detecting the labeled molecule in the subject, such
that
detection of labeled molecule above the background level indicates that the
subject
has a particular disease or disorder associated with aberrant expression of
the
polypeptide of interest. Background level can be determined by various methods
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including, comparing the amount of labeled molecule detected to a standard
value
previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
S diagnostic images. In the case of a radioisotope moiety, for a human
subject, the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode
of administration, the time interval following the administration for
permitting the
labeled molecule to preferentially concentrate at sites in the subject and for
unbound
labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24
hours or
6 to 12 hours. In another embodiment the time interval following
administration is 5
to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carned out by
repeating the method for diagnosing the disease or disease, for example, one
month
after initial diagnosis, six months after initial diagnosis, one year after
initial
diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods
known in the art for in vivo scanning. These methods depend upon the type of
label
used. Skilled artisans will be able to determine the appropriate method for
detecting a
particular label. Methods and devices that may be used in the diagnostic
methods of
the invention include, but are not limited to, computed tomography (CT), whole
body
scan such as position emission tomography (PET), magnetic resonance imaging
(MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et
al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is
labeled with
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a fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patent using positron emission-
tomography. In
yet another embodiment, the molecule is labeled with a paramagnetic label and
is
S detected in a patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In
one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the first antibody
may
be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic
kit for use in screening serum containing antibodies specific against
proliferative
and/or cancerous polynucleotides and polypeptides. Such a kit may include a
control
antibody that does not react with the polypeptide of interest. Such a kit may
include a
substantially isolated polypeptide antigen comprising an epitope which is
specifically
immunoreactive with at least one anti-polypeptide antigen antibody. Further,
such a
kit includes means for detecting the binding of said antibody to the antigen
(e.g., the
antibody may be conjugated to a fluorescent compound such as fluorescein or
rhodamine which can be detected by flow cytometry). In specific embodiments,
the
kit may include a recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to a solid
support.
In a more specific embodiment the detecting means of the above-described kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may
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also include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can be detected
by
binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The detecting means of the kit may include a second,
labeled
monoclonal antibody. Alternatively, or in addition, the detecting means may
include
a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
bound anti-antigen antibody on the solid support. The reagent is again washed
to
remove unbound labeled antibody, and the amount of reporter associated with
the
reagent is determined. Typically, the reporter is an enzyme which is detected
by
incubating the solid phase in the presence of a suitable fluorometric,
luminescent or
colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques
for attaching protein material to solid support material, such as polymeric
beads, dip
sticks, 96-well plate or filter material. These attachment methods generally
include
non-specific adsorption of the protein to the support or covalent attachment
of the
protein, typically through a free amine group, to a chemically reactive group
on the
solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used iri conjunction with
biotinylated
antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic method. The kit generally includes a support with surface- bound
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recombinant antigens, and a reporter-labeled anti-human antibody for detecting
surface-bound anti-antigen antibody.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion
proteins. For example, the polypeptide of the present invention, when fused to
a
second protein, can be used as an antigenic tag. Antibodies raised against the
polypeptide of the present invention can be used to indirectly detect the
second
protein by binding to the polypeptide. Moreover, because secreted proteins
target
cellular locations based on trafficking signals, the polypeptides of the
present
invention can be used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present
invention include not only heterologous signal sequences, but also other
heterologous
functional regions. The fusion does not necessarily need to be direct, but may
occur
through linker sequences.
Moreover, fusion proteins may also be engineered to improve characteristics
of the polypeptide of the present invention. For instance, a region of
additional amino
acids, particularly charged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and persistence during purification from the
host cell
or subsequent handling and storage. Also, peptide moieties may be added to the
polypeptide to facilitate purification. Such regions may be removed prior to
final
preparation of the polypeptide. The addition of peptide moieties to facilitate
handling
of polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and
specifically epitopes, can be combined with parts of the constant domain of
immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and
any
combination thereof, including both entire domains and portions thereof),
resulting in
chimeric polypeptides. These fusion proteins facilitate purification and show
an
increased half life in vivo. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).)
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Fusion proteins having disulfide-linked dimeric structures (due to the IgG)
can also be
more efficient in binding and neutralizing other molecules, than the monomeric
secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 (1995).) Polynucleotides comprising or alternatively consisting
of
S nucleic acids which encode these fusion proteins are also encompassed by the
invention.
Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins comprising various portions of constant region of immunoglobulin
molecules
together with another human protein or part thereof. In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for
example, human proteins, such as hIL-5, have been fused with Fc portions for
the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
(See,
D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et
al., J. Biol.
Chem. 270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide.
In preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 91311 ), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
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The present invention also relates to vectors containing the polynucleotide of
the present invention, host cells, and the production of polypeptides by
recombinant
techniques. The vector may be, for example, a phage, plasmid, viral, or
retroviral
vector. Retroviral vectors may be replication competent or replication
defective. In
the latter case, viral propagation generally will occur only in complementing
host
cells.
The polynucleotides may be joined to a vector containing a selectable marker
for propagation in a host. Generally, a plasmid vector is introduced in a
precipitate,
such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the
vector is a virus, it may be packaged in vitro using an appropriate packaging
cell line
and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
resistance for eukaryotic cell culture and tetracycline, kanamycin or
ampicillin
resistance genes for culturing in E. coli and other bacteria. Representative
examples
of appropriate hosts include, but are not limited to, bacterial cells, such as
E. coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells
(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
201178));
insect cells such as Drosophila S2 and Spodoptera Sf~ cells; animal cells such
as
CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known in the
art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,
pNHBA,
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pNHl6a, pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. Preferred expression vectors for use in yeast systems include,
but are
not limited to pYES2, pYDI, pTEFI/Zeo, pYES2/GS, pPICZ,pGAPZ, pGAPZaIph,
pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, pPIC9K, and PA081 S (all
available
from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent
to the
skilled artisan.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection, or other methods. Such
methods
are described in many standard laboratory manuals, such as Davis et al., Basic
Methods In Molecular Biology (1986). It is specifically contemplated that the
polypeptides of the present invention may in fact be expressed by a host cell
lacking a
recombinant vector.
A polypeptide of this invention can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or canon exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification.
Polypeptides of the present invention, and preferably the secreted form, can
also be recovered from: products purified from natural sources, including
bodily
fluids, tissues and cells, whether directly isolated or cultured; products of
chemical
synthetic procedures; and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial, yeast,
higher plant,
insect, and mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may be
glycosylated
or may be non-glycosylated. In addition, polypeptides of the invention may
also
include an initial modified methionine residue, in some cases as a result of
host-
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mediated processes. Thus, it is well known in the art that the N-terminal
methionine
encoded by the translation initiation codon generally is removed with high
efficiency
from any protein after translation in all eukaryotic cells. While the N-
terminal
methionine on most proteins also is efficiently removed in most prokaryotes,
for some
proteins, this prokaryotic removal process is inefficient, depending on the
nature of
the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express the
polypeptide of the present invention in a eukaryotic system. Pichia pastoris
is a
methylotrophic yeast which can metabolize methanol as its sole carbon source.
A
main step in the methanol metabolization pathway is the oxidation of methanol
to
formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol
oxidase. In
order to metabolize methanol as its sole carbon source, Pichia pastoris must
generate
high levels of alcohol oxidase due, in part, to the relatively low affinity of
alcohol
oxidase for Oz. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXl ) is
highly active. In the presence of methanol, alcohol oxidase produced from the
AOXI
gene comprises up to approximately 30% of the total soluble protein in Pichia
pastoris. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz,
P.J, et al.,
Yeast 5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76
(1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide
of the
present invention, under the transcriptional regulation of all or part of the
AOXl
regulatory sequence is expressed at exceptionally high levels in Pichia yeast
grown in
the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding
a Polypeptide of the invention, as set forth herein, in a Pichea yeast system
essentially
as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins
and
J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector
allows
expression and secretion of a protein of the invention by virtue of the strong
AOXl
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory
signal
peptide (i.e., leader) located upstream of a multiple cloning site.
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Many other yeast vectors could be used in place of pPIC9K, such as, pYES2,
pYDI, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would
readily
appreciate, as long as the proposed expression construct provides
appropriately
located signals for transcription, translation, secretion (if desired), and
the like,
including an in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding
sequence, such as, for example, a polynucleotide of the present invention, may
be
achieved by cloning the heterologous polynucleotide of the invention into an
expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide
sequences) that is operably associated with the polynucleotides of the
invention, and
which activates, alters, and/or amplifies endogenous polynucleotides. For
example,
techniques known in the art may be used to operably associate heterologous
control
regions (e.g., promoter and/or enhancer) and endogenous polynucleotide
sequences
via homologous recombination, resulting in the formation of a new
transcription unit
(see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; U.S. Patent No.
5,733,761, issued March 31, 1998; International Publication No. WO 96/29411,
published September 26, 1996; International Publication No. WO 94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-
8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of
each of
which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and
Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al.,
Nature,
310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of
a
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polypeptide sequence of the invention can be synthesized by use of a peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
amino acid
analogs can be introduced as a substitution or addition into the polypeptide
sequence.
Non-classical amino acids include, but are not limited to, to the D-isomers of
the
common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-
aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid,
Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-
butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,
fluoro-
amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl
amino
acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore,
the
amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses polypeptides which are differentially modified
during or after translation, e.g., by glycosylation, acetylation,
phosphorylation,
1 S amidation, derivatization by known protecting/blocking groups, proteolytic
cleavage,
linkage to an antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carned out by known techniques, including but
not
limited, to specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin,
papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic
host cell expression. The polypeptides may also be modified with a detectable
label,
such as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
Also provided by the invention are chemically modified derivatives of the
polypeptides of the invention which may provide additional advantages such as
increased solubility, stability and circulating time of the polypeptide, or
decreased
immunogenicity (see U.S. Patent NO: 4,179,337). The chemical moieties for
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derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,
dextran, polyvinyl alcohol and the like. The polypeptides may be modified at
random
positions within the molecule, or at predetermined positions within the
molecule and
may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic protein or analog). For example, the polyethylene glycol may have
an
average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000,
3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500,
15,000,
15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
20,000,
25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,
75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et
al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al.,
Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are incorporated
herein by
reference.
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic
domains of the protein. There are a number of attachment methods available to
those
skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG
to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
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be covalently bound through amino acid residues via a reactive group, such as,
a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene glycol molecule may be bound. The amino acid residues having a
free
amino group may include lysine residues and the N-terminal amino acid
residues;
those having a free carboxyl group may include aspartic acid residues glutamic
acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used
as a reactive group for attaching the polyethylene glycol molecules. Preferred
for
therapeutic purposes is attachment at an amino group, such as attachment at
the
N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via
linkage to any of a number of amino acid residues. For example, polyethylene
glycol
can be linked to a proteins via covalent bonds to lysine, histidine, aspartic
acid,
glutamic acid, or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid residues (e.g.,
lysine,
histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to
more than one
type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic
acid,
cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one
may
select from a variety of polyethylene glycol molecules (by molecular weight,
branching, etc.), the proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation reaction
to be
performed, and the method of obtaining the selected N-terminally pegylated
protein.
The method of obtaining the N-terminally pegylated preparation (i.e.,
separating this
moiety from other monopegylated moieties if necessary) may be by purification
of the
N-terminally pegylated material from a population of pegylated protein
molecules.
Selective proteins chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential reactivity of
different
types of primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate reaction
conditions,
substantially selective derivatization of the protein at the N-terminus with a
carbonyl
group containing polymer is achieved.
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As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the protein either directly or by an intervening linker.
Linkerless systems
for attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of
Hematol.
68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO
95/06058;
and WO 98/32466, the disclosures of each of which are incorporated herein by
reference.
One system for attaching polyethylene glycol directly to amino acid residues
of proteins without an intervening linker employs tresylated MPEG, which is
produced by the modification of morunethoxy polyethylene glycol (MPEG) using
tresylchloride (C1SOZCHZCF3). Upon reaction of protein with tresylated MPEG,
polyethylene glycol is directly attached to amine groups of the protein. Thus,
the
invention includes protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule having a
2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of
different intervening linkers. For example, U.S. Patent No. 5,612,460, the
entire
disclosure of which is incorporated herein by reference, discloses urethane
linkers for
connecting polyethylene glycol to proteins. Protein-polyethylene glycol
conjugates
wherein the polyethylene glycol is attached to the protein by a linker can
also be
produced by reaction of proteins with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-
succinate derivatives. A number additional polyethylene glycol derivatives and
reaction chemistries for attaching polyethylene glycol to proteins are
described in
WO 98/32466, the entire disclosure of which is incorporated herein by
reference.
Pegylated protein products produced using the reaction chemistries set out
herein are
included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12,
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15, 17, 20, or more polyethylene glycol molecules. Similarly, the average
degree of
substitution within ranges such as 1-3, 2-4, 3-S, 4-6, 5-7, 6-8, 7-9, 8-10, 9-
11, 10-12,
11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per protein molecule. Methods for determining the degree of
substitution are
discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.
9:249-
304 (1992).
The polypeptides of the invention may be in monomers or multimers (i.e.,
dimers, trimers, tetramers and higher multimers). Accordingly, the present
invention
relates to monomers and multimers of the polypeptides of the invention, their
preparation, and compositions (preferably, Therapeutics) containing them. In
specific
embodiments, the polypeptides of the invention are monomers, dimers, trimers
or
tetramers. In additional embodiments, the multimers of the invention are at
least
dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer, refers to a multimer containing only
polypeptides
corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA
contained in a deposited clone (including fragments, variants, splice
variants, and
fusion proteins, corresponding to these polypeptides as described herein).
These
homomers may contain polypeptides having identical or different amino acid
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only polypeptides having an identical amino acid sequence. In
another
specific embodiment, a homomer of the invention is a multimer containing
polypeptides having different amino acid sequences. In specific embodiments,
the
multimer of the invention is a homodimer (e.g., containing polypeptides having
identical or different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid sequences). In
additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at
least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or
more heterologous polypeptides (i. e., polypeptides of different proteins) in
addition to
the polypeptides of the invention. In a specific embodiment, the multimer of
the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional
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embodiments, the heteromeric multimer of the invention is at least a
heterodimer, at
least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked, by for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as,
for example, homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of
the invention, such as, for example, heterotrimers or heterotetramers, are
formed
when polypeptides of the invention contact antibodies to the polypeptides of
the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, multimers of the
invention are formed by covalent associations with and/or between the
polypeptides
of the invention. Such covalent associations may involve one or more amino
acid
residues contained in the polypeptide sequence ( e.g., that recited in the
sequence
listing, or contained in the polypeptide encoded by a deposited clone). In one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring) polypeptide. In another instance, the covalent associations are the
consequence of chemical or recombinant manipulation. Alternatively, such
covalent
associations may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence
contained in a fusion protein of the invention (see, e.g., US Patent Number
5,478,925). In a specific example, the covalent associations are between the
heterologous sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent associations of
fusion
proteins of the invention are between heterologous polypeptide sequence from
another protein that is capable of forming covalently associated multimers,
such as for
example, oseteoprotegerin (see, e.g., International Publication NO: WO
98/49305, the
contents of which are herein incorporated by reference in its entirety). In
another
embodiment, two or more polypeptides of the invention are joined through
peptide
linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627
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(hereby incorporated by reference). Proteins comprising multiple polypeptides
of the
invention separated by peptide linkers may be produced using conventional
recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves
use of polypeptides of the invention fused to a leucine zipper or isoleucine
zipper
polypeptide sequence. Leucine zipper and isoleucine zipper domains are
polypeptides
that promote multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins (Landschulz
et al.,
Science 240:1759, (1988)), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurnng peptides and
derivatives thereof that dimerize or trimerize. Examples of leucine zipper
domains
suitable for producing soluble multimeric proteins of the invention are those
described
in PCT application WO 94/10308, hereby incorporated by reference. Recombinant
fusion proteins comprising a polypeptide of the invention fused to a
polypeptide
sequence that dimerizes or trimerizes in solution are expressed in suitable
host cells,
and the resulting soluble multimeric fusion protein is recovered from the
culture
supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are
those that preferentially form trimers. One example is a leucine zipper
derived from
lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters
344:191,
(1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by
reference. Other peptides derived from naturally occurnng trimeric proteins
may be
employed in preparing trimeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions
between Flag~ polypeptide sequence contained in fusion proteins of the
invention
containing Flag~ polypeptide seuqence. In a further embodiment, associations
proteins of the invention are associated by interactions between heterologous
polypeptide sequence contained in Flag~ fusion proteins of the invention and
anti-
Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in the art. For example, polypeptides desired to be contained in the
multimers
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of the invention may be chemically cross-linked using linker molecules and
linker
molecule length optimization techniques known in the art (see, e.g., US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques
known in
the art to form one or more inter-molecule cross-links between the cysteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). Further, polypeptides of the invention may be
routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the
polypeptide components desired to be contained in the multimer of the
invention (see,
1 S e.g., US Patent Number 5,478,925, which is herein incorporated by
reference in its
entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In a
specific embodiment, polynucleotides coding for a homodimer of the invention
are
generated by ligating a polynucleotide sequence encoding a polypeptide of the
invention to a sequence encoding a linker polypeptide and then further to a
synthetic
polynucleotide encoding the translated product of the polypeptide in the
reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). In another embodiment, recombinant techniques
described
herein or otherwise known in the art are applied to generate recombinant
polypeptides
of the invention which contain a transmembrane domain (or hyrophobic or signal
peptide) and which can be incorporated by membrane reconstitution techniques
into
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liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety).
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes
known techniques.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are presently available. Each polynucleotide of the present
invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be
selected using computer analysis so that primers do not span more than one
predicted
exon in the genomic DNA. These primers are then used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only those
hybrids
containing the human gene corresponding to the SEQ ID NO:X will yield an
amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycner. Moreover, sublocalization of the
polynucleotides
can be achieved with panels of specific chromosome fragments. Other gene
mapping
strategies that can be used include in situ hybridization, prescreening with
labeled
flow-sorted chromosomes, preselection by hybridization to construct chromosome
specific-cDNA libraries and computer mapping techniques (See, e.g., Shiner,
Trends
Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its
entirety)..
Precise chromosomal location of the polynucleotides can also be achieved
using fluorescence in situ hybridization (FISH) of a metaphase chromosomal
spread.
This technique uses polynucleotides as short as 500 or 600 bases; however,
polynucleotides 2,000-4,000 by are preferred. For a review of this technique,
see
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Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon
Press, New York (1988).
For chromosome mapping, the polynucleotides can be used individually (to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes).
The polynucleotides of the present invention would likewise be useful for
radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For
a review of these techniques and others known in the art, see, e.g., Dear,
"Genome
Mapping: A Practical Approach," IRL Press at Oxford University Press, London
(1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry
3:483-
492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al.,
Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 ( 1999)
each of
which is hereby incorporated by reference in its entirety.
Once a polynucleotide has been mapped to a precise chromosomal location,
the physical position of the polynucleotide can be used in linkage analysis.
Linkage
analysis establishes coinheritance between a chromosomal location and
presentation
of a particular disease. (Disease mapping data are found, for example, in V.
McKusick, Mendelian Inheritance in Man (available on line through Johns
Hopkins
University Welch Medical Library) .) Assuming 1 megabase mapping resolution
and
one gene per 20 kb, a cDNA precisely localized to a chromosomal region
associated
with the disease could be one of SO-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and
the corresponding gene between affected and unaffected individuals can be
examined.
First, visible structural alterations in the chromosomes, such as deletions or
translocations, are examined in chromosome spreads or by PCR. If no structural
alterations exist, the presence of point mutations are ascertained. Mutations
observed
in some or all affected individuals, but not in normal individuals, indicates
that the
mutation may cause the disease. However, complete sequencing of the
polypeptide
and the corresponding gene from several normal individuals is required to
distinguish
the mutation from a polymorphism. If a new polymorphism is identified, this
polymorphic polypeptide can be used for further linkage analysis.
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Furthermore, increased or decreased expression of the gene in affected
individuals as compared to unaffected individuals can be assessed using
polynucleotides of the present invention. Any of these alterations (altered
expression,
chromosomal rearrangement, or mutation) can be used as a diagnostic or
prognostic
S marker.
Thus, the invention also provides a diagnostic method useful during diagnosis
of a disorder, involving measuring the expression level of polynucleotides of
the
present invention in cells or body fluid from an individual and comparing the
measured gene expression level with a standard level of polynucleotide
expression
level, whereby an increase or decrease in the gene expression level compared
to the
standard is indicative of a disorder.
In still another embodiment, the invention includes a kit for analyzing
samples
for the presence of proliferative and/or cancerous polynucleotides derived
from a test
subject. In a general embodiment, the kit includes at least one polynucleotide
probe
containing a nucleotide sequence that will specifically hybridize with a
polynucleotide of the present invention and a suitable container. In a
specific
embodiment, the kit includes two polynucleotide probes defining an internal
region of
the polynucleotide of the present invention, where each probe has one strand
containing a 31'mer-end internal to the region. In a further embodiment, the
probes
may be useful as primers for polyrnerase chain reaction amplification.
Where a diagnosis of a disorder, has already been made according to
conventional methods, the present invention is useful as a prognostic
indicator,
whereby patients exhibiting enhanced or depressed polynucleotide of the
present
invention expression will experience a worse clinical outcome relative to
patients
expressing the gene at a level nearer the standard level.
By "measuring the expression level of polynucleotide of the present
invention" is intended qualitatively or quantitatively measuring or estimating
the level
of the polypeptide of the present invention or the level of the mRNA encoding
the
polypeptide in a first biological sample either directly (e.g., by determining
or
estimating absolute protein level or mRNA level) or relatively (e.g., by
comparing to
the polypeptide level or m.RNA level in a second biological sample).
Preferably, the
polypeptide level or mRNA level in the first biological sample is measured or
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estimated and compared to a standard polypeptide level or mRNA level, the
standard
being taken from a second biological sample obtained from an individual not
having
the disorder or being determined by averaging levels from a population of
individuals
not having a disorder. As will be appreciated in the art, once a standard
polypeptide
level or mRNA level is known, it can be used repeatedly as a standard for
comparison.
By "biological sample" is intended any biological sample obtained from an
individual, body fluid, cell line, tissue culture, or other source which
contains the
polypeptide of the present invention or mRNA. As indicated, biological samples
include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and
spinal fluid) which contain the polypeptide of the present invention, and
other tissue
sources found to express the polypeptide of the present invention. Methods for
obtaining tissue biopsies and body fluids from mammals are well known in the
art.
Where the biological sample is to include mRNA, a tissue biopsy is the
preferred
source.
The methods) provided above may preferrably be applied in a diagnostic
method and/or kits in which polynucleotides andJor polypeptides are attached
to a
solid support. In one exemplary method, the support may be a "gene chip" or a
"biological chip" as described in US Patents 5,837,832, 5,874,219, and
5,856,174.
Further, such a gene chip with polynucleotides of the present invention
attached may
be used to identify polymorphisms between the polynucleotide sequences, with
polynucleotides isolated from a test subject. The knowledge of such
polymorphisms
(i.e. their location, as well as, their existence) would be beneficial in
identifying
disease loci for many disorders, including cancerous diseases and conditions.
Such a
method is described in US Patents 5,858,659 and 5,856,104. The US Patents
referenced supra are hereby incorporated by reference in their entirety
herein.
The present invention encompasses polynucleotides of the present invention
that are chemically synthesized, or reproduced as peptide nucleic acids (PNA),
or
according to other methods known in the art. The use of PNAs would serve as
the
preferred form if the polynucleotides are incorporated onto a solid support,
or gene
chip. For the purposes of the present invention, a peptide nucleic acid (PNA)
is a
polyamide type of DNA analog and the monomeric units for adenine, guanine,
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thymine and cytosine are available commercially (Perceptive Biosystems).
Certain
components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose
derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.
Egholm, R. H.
Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt,
L.Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim,
B.
Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and
tightly to complementary DNA strands and are not degraded by nucleases. In
fact,
PNA binds more strongly to DNA than DNA itself does. This is probably because
there is no electrostatic repulsion between the two strands, and also the
polyamide
backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider
range of stringency conditions than DNA/DNA duplexes, making it easier to
perform
multiplex hybridization. Smaller probes can be used than with DNA due to the
strong
binding. In addition, it is more likely that single base mismatches can be
determined
with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (Tm) by 8°-20° C, vs. 4°-
16° C for the DNA/DNA 15-
mer duplex. Also, the absence of charge groups in PNA means that hybridization
can
be done at low ionic strengths and reduce possible interference by salt during
the
analysis.
The present invention is useful for detecting cancer in mammals. In particular
the invention is useful during diagnosis of pathological cell proliferative
neoplasias
which include, but are not limited to: acute myelogenous leukemias including
acute
monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,
acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic
leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous
leukemias including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs,
horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative diseases, disorders, and/or conditions are
often
associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P.
et al.,
"The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology," in
Neoplastic Diseases of the Blood, Vol l., Wiernik, P. H. et al. eds., 161-182
(1985)).
Neoplasias are now believed to result from the qualitative alteration of a
normal
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cellular gene product, or from the quantitative modification of gene
expression by
insertion into the chromosome of a viral sequence, by chromosomal
translocation of a
gene to a more actively transcribed region, or by some other mechanism.
(Gelmann
et al., supra) It is likely that mutated or altered expression of specific
genes is
involved in the pathogenesis of some leukemias, among other tissues and cell
types.
(Gelmann et al., supra) Indeed, the human counterparts of the oncogenes
involved in
some animal neoplasias have been amplified or translocated in some cases of
human
leukemia and carcinoma. (Gelmann et al., supra)
For example, c-myc expression is highly amplified in the non-lymphocytic
leukemia
cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation, the
level of c-myc is found to be downregulated. (International Publication Number
WO
91/15580) However, it has been shown that exposure of HL-60 cells to a DNA
construct that is complementary to the 5' end of c-myc or c-myb blocks
translation of
the corresponding mRNAs which downregulates expression of the c-myc or c-myb
proteins and causes arrest of cell proliferation and differentiation of the
treated cells.
(International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl.
Acad.
Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)).
However,
the skilled artisan would appreciate the present invention's usefulness would
not be
limited to treatment of proliferative diseases, disorders, and/or conditions
of
hematopoietic cells and tissues, in light of the numerous cells and cell types
of
varying origins which are known to exhibit proliferative phenotypes.
In addition to the foregoing, a polynucleotide can be used to control gene
expression through triple helix formation or antisense DNA or RNA. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRCPress,
Boca
Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et
al.,
Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and
Dervan et al., Science 251: 1360 ( 1991 ). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques, preferred
polynucleotides are usually oligonucleotides 20 to 40 bases in length and
complementary to either the 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
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(1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself
(antisense
- Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides 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 are effective in model systems, and the
information
disclosed herein can be used to design antisense or triple helix
polynucleotides in an
effort to treat or prevent disease.
Polynucleotides of the present invention are also useful in gene therapy. One
goal of gene therapy is to insert a normal gene into an organism having a
defective
gene, in an effort to correct the genetic defect. The polynucleotides
disclosed in the
present invention offer a means of targeting such genetic defects in a highly
accurate
manner. Another goal is to insert a new gene that was not present in the host
genome,
thereby producing a new trait in the host cell.
The polynucleotides are also useful for identifying individuals from minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel.
In this technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of
"Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
The polynucleotides of the present invention can also be used as an
alternative
to RFLP, by determining the actual base-by-base DNA sequence of selected
portions
of an individual's genome. These sequences can be used to prepare PCR primers
for
amplifying and isolating such selected DNA, which can then be sequenced. Using
this technique, individuals can be identified because each individual will
have a
unique set of DNA sequences. Once an unique ID database is established for an
individual, positive identification of that individual, living or dead, can be
made from
extremely small tissue samples.
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Forensic biology also benefits from using DNA-based identification
techniques as disclosed herein. DNA sequences taken from very small biological
samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, semen,
synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or
surfactant,urine,fecal matter, etc., can be amplified using PCR. In one prior
art
technique, gene sequences amplified from polymorphic loci, such as DQa class
II
HLA gene, are used in forensic biology to identify individuals. (Erlich, H.,
PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are
amplified, they are digested with one or more restriction enzymes, yielding an
identifying set of bands on a Southern blot probed with DNA corresponding to
the
DQa class II HLA gene. Similarly, polynucleotides of the present invention can
be
used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a
particular tissue. Such need arises, for example, in forensics when presented
with
tissue of unknown origin. Appropriate reagents can comprise, for example, DNA
probes or primers specific to particular tissue prepared from the sequences of
the
present invention. Panels of such reagents can identify tissue by species
and/or by
organ type. In a similar fashion, these reagents can be used to screen tissue
cultures
for contamination.
In the very least, the polynucleotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences
in the process of discovering novel polynucleotides, for selecting and making
oligomers for attachment to a "gene chip" or other support, to raise anti-DNA
antibodies using DNA immunization techniques, and as an antigen to elicit an
immune response.
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The
following description should be considered exemplary and utilizes known
techniques.
A polypeptide of the present invention can be used to assay protein levels in
a
biological sample using antibody-based techniques. For example, protein
expression
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in tissues can be studied with classical immunohistological methods.
(Jalkanen, M.,
et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell .
Biol. 105:3087-
3096 (1987).) Other antibody-based methods useful for detecting protein gene
expression include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium
(112In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
In addition to assaying secreted protein levels in a biological sample,
proteins
can also be detected in vivo by imaging. Antibody labels or markers for in
vivo
imaging of protein include those detectable by X-radiography, NMR or ESR. For
X-
radiography, suitable labels include radioisotopes such as barium or cesium,
which
emit detectable radiation but are not overtly harmful to the subject. Suitable
markers
for NMR and ESR include those with a detectable characteristic spin, such as
deuterium, which may be incorporated into the antibody by labeling of
nutrients for
the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
131I,
1 121n, 99mTc), a radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally, subcutaneously,
or
intraperitoneally) into the mammal. It will be understood in the art that the
size of the
subject and the imaging system used will determine the quantity of imaging
moiety
needed to produce diagnostic images. In the case of a radioisotope moiety, for
a
human subject, the quantity of radioactivity injected will normally range from
about 5
to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will
then
preferentially accumulate at the location of cells which contain the specific
protein.
In vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics
of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. (1982).)
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Thus, the invention provides a diagnostic method of a disorder, which
involves (a) assaying the expression of a polypeptide of the present invention
in cells
or body fluid of an individual; (b) comparing the level of gene expression
with a
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard expression level is
indicative of a disorder. With respect to cancer, the presence of a relatively
high
amount of transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical symptoms. A
more
definitive diagnosis of this type may allow health professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
Moreover, polypeptides of the present invention can be used to treat, prevent,
and/or diagnose disease. For example, patients can be administered a
polypeptide of
the present invention in an effort to replace absent or decreased levels of
the
polypeptide (e.g., insulin), to supplement absent or decreased levels of a
different
polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair
proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or
tumor
supressor), to activate the activity of a polypeptide (e.g., by binding to a
receptor), to
reduce the activity of a membrane bound receptor by competing with it for free
ligand
(e.g., soluble TNF receptors used in reducing inflammation), or to bring about
a
desired response (e.g., blood vessel growth inhibition, enhancement of the
immune
response to proliferative cells or tissues).
Similarly, antibodies directed to a polypeptide of the present invention can
also be used to treat, prevent, and/or diagnose disease. For example,
administration of
an antibody directed to a polypeptide of the present invention can bind and
reduce
overproduction of the polypeptide. Similarly, administration of an antibody
can
activate the polypeptide, such as by binding to a polypeptide bound to a
membrane
(receptor).
At the very least, the polypeptides of the present invention can be used as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art. Polypeptides
can also
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be used to raise antibodies, which in turn are used to measure protein
expression from
a recombinant cell, as a way of assessing transformation of the host cell.
Moreover,
the polypeptides of the present invention can be used to test the following
biological
activities.
Gene Therapy Methods
Another aspect of the present invention is to gene therapy methods for
treatingor preventing disorders, diseases and conditions. The gene therapy
methods
relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA)
sequences into an animal to achieve expression of a polypeptide of the present
invention. This method requires a polynucleotide which codes for a polypeptide
of the
invention that operatively linked to a promoter and any other genetic elements
necessary for the expression of the polypeptide by the target tissue. Such
gene therapy
and delivery techniques are known in the art, see, for example, W090/11092,
which
is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) comprising a promoter operably linked to a
polynucleotide of the invention ex vivo, with the engineered cells then being
provided
to a patient to be treated with the polypeptide. Such methods are well-known
in the
art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993);
Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J.
Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229
(1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et
al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-
1255
(1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are
herein
incorporated by reference. In one embodiment, the cells which are engineered
are
arterial cells. The arterial cells may be reintroduced into the patient
through direct
injection to the artery, the tissues surrounding the artery, or through
catheter injection.
As discussed in more detail below, the polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells of an
animal,
such as, injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver,
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and the like). The polynucleotide constructs may be delivered in a
pharmaceutically
acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
that are free from any delivery vehicle that acts to assist, promote or
facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations,
lipofectin or precipitating agents and the like. However, the polynucleotides
of the
invention can also be delivered in liposome formulations and lipofectin
formulations
and the like can be prepared by methods well known to those skilled in the
art. Such
methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466,
and
5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs of the invention used in the gene
therapy method are preferably constructs that will not integrate into the host
genome
nor will they contain sequences that allow for replication. Appropriate
vectors
include pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene;
pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI/V5,
pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will
be
readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving
the expression of polynucleotide sequence of the invention. Suitable promoters
include adenoviral promoters, such as the adenoviral major late promoter; or
heterologous promoters, such as the cytomegalovirus (CMV) promoter; the
respiratory syncytial virus (RSV) promoter; inducible promoters, such as the
MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter;
the ApoAI promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thyrnidine kinase promoter; retroviral LTRs; the b-
actin
promoter; and human growth hormone promoters. The promoter also may be the
native promoter for the polynucleotides of the invention.
Unlike other gene therapy techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have shown that non-replicating
DNA
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sequences can be introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
The polynucleotide construct of the invention can be delivered to the
interstitial
space of tissues within the an animal, including of muscle, skin, brain, lung,
liver,
spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney,
gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye,
gland, and connective tissue. Interstitial space of the tissues comprises the
intercellular,
fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues,
elastic
fibers in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that
same matrix within connective tissue ensheathing muscle cells or in the
lacunae of
bone. It is similarly the space occupied by the plasma of the circulation and
the lymph
fluid of the lymphatic channels. Delivery to the interstitial space of muscle
tissue is
preferred for the reasons discussed below. They may be conveniently delivered
by
injection into the tissues comprising these cells. They are preferably
delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression may be achieved in non-differentiated or less completely
differentiated
cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells
are particularly competent in their ability to take up and express
polynucleotides.
For the nakednucleic acid sequence injection, an effective dosage amount of
DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about
50
mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to
about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mglkg: Of course,
as
the artisan of ordinary skill will appreciate, this dosage will vary according
to the
tissue site of injection. The appropriate and effective dosage of nucleic acid
sequence
can readily be determined by those of ordinary skill in the art and may depend
on the
condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection
into the interstitial space of tissues. However, other parenteral routes may
also be
used, such as, inhalation of an aerosol formulation particularly for delivery
to lungs or
bronchial tissues, throat or mucous membranes of the nose. In addition, naked
DNA
constructs can be delivered to arteries during angioplasty by the catheter
used in the
procedure.
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The naked polynucleotides are delivered by any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, and so-called "gene
guns". These
delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents, etc.
Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs of the invention are
complexed in a liposome preparation. Liposomal preparations for use in the
instant
invention include cationic (positively charged), anionic (negatively charged)
and
neutral preparations. However, cationic liposomes are particularly preferred
because a
tight charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci.
USA ,
84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone
et
al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein
incorporated
by reference); and purified transcription factors (Debs et al., J. Biol.
Chem.,
265:10189-10192 (1990), which is herein incorporated by reference), in
functional
form.
Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly useful and are available under the trademark Lipofectin, from
GIBCO
BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
,
84:7413-7416 (1987), which is herein incorporated by reference). Other
commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
Other cationic liposomes can be prepared from readily available materials
using techniques well known in the art. See, e.g. PCT Publication NO: WO
90/11092
(which is herein incorporated by reference) for a description of the synthesis
of
DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation
of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al.,
Proc.
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Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference.
Similar methods can be used to prepare liposomes from other cationic lipid
materials.
Similarly, anionic and neutral liposomes are readily available, such as from
Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using
readily
available materials. Such materials include phosphatidyl, choline,
cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE),
among others. These materials can also be mixed with the DOTMA and DOTAP
starting materials in appropriate ratios. Methods for making liposomes using
these
materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can be used in various combinations to make conventional liposomes,
with or
without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be
prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas
into a sonication vial. The sample is placed under a vacuum pump overnight and
is
hydrated the following day with deionized water. The sample is then sonicated
for 2
hours in a capped vial, using a Heat Systems model 350 sonicator equipped with
an
inverted cup (bath type) probe at the maximum setting while the bath is
circulated at
15EC. Alternatively, negatively charged vesicles can be prepared without
sonication
to produce multilamellar vesicles or by extrusion through nucleopore membranes
to
produce unilamellar vesicles of discrete size. Other methods are known and
available
to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SLTVs), or large unilamellar vesicles (LIlVs), with SUVs being
preferred.
The various liposome-nucleic acid complexes are prepared using methods well
known
in the art. See, e.g., Straubinger et al., Methods of Immunology , 101:512-527
(1983),
which is herein incorporated by reference. For example, MLVs containing
nucleic
acid can be prepared by depositing a thin film of phospholipid on the walls of
a glass
tube and subsequently hydrating with a solution of the material to be
encapsulated.
SLTVs are prepared by extended sonication of MLVs to produce a homogeneous
population of unilamellar liposomes. The material to be entrapped is added to
a
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suspension of preformed MLVs and then sonicated. When using liposomes
containing
cationic lipids, the dried lipid film is resuspended in an appropriate
solution such as
sterile water or an isotonic buffer solution such as 10 mM Tris/NaCI,
sonicated, and
then the preformed liposomes are mixed directly with the DNA. The liposome and
DNA form a very stable complex due to binding of the positively charged
liposomes
to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are
prepared by a number of methods, well known in the art. Commonly used methods
include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta,
394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer
et al.,
Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.
Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348
(1979));
detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979));
and
reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431
(1980);
Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et
al.,
Science, 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about
1:10. Preferably, the ration will be from about 5:1 to about 1:5. More
preferably, the
ration will be about 3:1 to about 1:3. Still more preferably, the ratio will
be about 1:1.
U.S. Patent NO: 5,676,954 (which is herein incorporated by reference) reports
on the injection of genetic material, complexed with cationic liposomes
Garners, into
mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466,
5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469
(which are herein incorporated by reference) provide cationic lipids for use
in
transfecting DNA into cells and mammals. U.5. Patent Nos. 5,589,466,
5,693,622,
5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are
herein incorporated by reference) provide methods for delivering DNA-cationic
lipid
complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a
retroviral particle containing RNA which comprises a sequence encoding
polypeptides of the invention. Retroviruses from which the retroviral plasmid
vectors
may be derived include, but are not limited to, Moloney Murine Leukemia Virus,
spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian
leukosis
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virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form producer cell lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-
19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described
in Miller, Human Gene Therapy , 1:5-14 ( 1990), which is incorporated herein
by
reference in its entirety. The vector may transduce the packaging cells
through any
means known in the art. Such means include, but are not limited to,
electroporation,
the use of liposomes, and CaP04 precipitation. In one alternative, the
retroviral
plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and
then
administered to a host.
The producer cell line generates infectious retroviral vector particles which
include polynucleotide encoding polypeptides of the invention. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or in
vivo. The transduced eukaryotic cells will express polypeptides of the
invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with
polynucleotides of the invention contained in an adenovirus vector. Adenovirus
can
be manipulated such that it encodes and expresses polypeptides of the
invention, and
at the same time is inactivated in terms of its ability to replicate in a
normal lytic viral
life cycle. Adenovirus expression is achieved without integration of the viral
DNA
into the host cell chromosome, thereby alleviating concerns about insertional
mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines
for
many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir.
Dis.,
109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and
CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991);
Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to
attempt
to establish adenovirus as a causative agent in human cancer were uniformly
negative
(Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
Suitable adenoviral vectors useful in the present invention are described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993);
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Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther.,
4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2 is useful
and can
be grown in human 293 cells. These cells contain the El region of adenovirus
and
constitutively express Ela and Elb, which complement the defective
adenoviruses by
providing the products of the genes deleted from the vector. In addition to
Ad2, other
varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the
present
invention.
Preferably, the adenoviruses used in the present invention are replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus and/or
packaging cell line to form infectious particles. The resulting virus is
capable of
infecting cells and can express a polynucleotide of interest which is operably
linked to
a promoter, but cannot replicate in most cells. Replication deficient
adenoviruses
may be deleted in one or more of all or a portion of the following genes: Ela,
Elb,
E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
Curr.
Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few
viruses that
may integrate its DNA into non-dividing cells. Vectors containing as little as
300 base
pairs of AAV can be packaged and can integrate, but space for exogenous DNA is
limited to about 4.5 kb. Methods for producing and using such AAVs are known
in
the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will
include all the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The polynucleotide construct containing polynucleotides of the
invention
is inserted into the AAV vector using standard cloning methods, such as those
found
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989). The recombinant AAV vector is then transfected into packaging
cells
which are infected with a helper virus, using any standard technique,
including
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lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper
viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses.
Once the packaging cells are transfected and infected, they will produce
infectious
AAV viral particles which contain the polynucleotide construct of the
invention.
These viral particles are then used to transduce eukaryotic cells, either ex
vivo or in
vivo. The transduced cells will contain the polynucleotide construct
integrated into its
genome, and will express the desired gene product.
Another method of gene therapy involves operably associating heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding the
polypeptide sequence of interest) via homologous recombination (see, e.g.,
U.S.
Patent NO: 5,641,670, issued June 24, 1997; International Publication NO: WO
96/2941 l, published September 26, 1996; International Publication NO: WO
94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This
method
involves the activation of a gene which is present in the target cells, but
which is not
normally expressed in the cells, or is expressed at a lower level than
desired.
Polynucleotide constructs are made, using standard techniques known in the
art, which contain the promoter with targeting sequences flanking the
promoter.
Suitable promoters are described herein. The targeting sequence is
sufficiently
complementary to an endogenous sequence to permit homologous recombination of
the promoter-targeting sequence with the endogenous sequence. The targeting
sequence will be sufficiently near the 5' end of the desired endogenous
polynucleotide sequence so the promoter will be operably linked to the
endogenous
sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5'
and 3' ends. Preferably, the 3' end of the first targeting sequence contains
the same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the
second targeting sequence contains the same restriction site as the 3' end of
the
amplified promoter. The amplified promoter and targeting sequences are
digested
and ligated together.
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The promoter-targeting sequence construct is delivered to the cells, either as
naked polynucleotide, or in conjunction with transfection-facilitating agents,
such as
liposomes, viral sequences, viral particles, whole viruses, lipofection,
precipitating
agents, etc., described in more detail above. The P promoter-targeting
sequence can
be delivered by any method, included direct needle injection, intravenous
injection,
topical administration, catheter infusion, particle accelerators, etc. The
methods are
described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous
recombination between the construct and the endogenous sequence takes place,
such
that an endogenous sequence is placed under the control of the promoter. The
promoter then drives the expression of the endogenous sequence.
The polynucleotides encoding polypeptides of the present invention may be
administered along with other polynucleotides encoding other angiongenic
proteins.
Angiogenic proteins include, but are not limited to, acidic and basic
fibroblast growth
factors, VEGF-l, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor
alpha and beta, platelet-derived endothelial cell growth factor, platelet-
derived growth
factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like
growth
factor, colony stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
Preferably, the polynucleotide encoding a polypeptide of the invention
contains a secretory signal sequence that facilitates secretion of the
protein.
Typically, the signal sequence is positioned in the coding region of the
polynucleotide
to be expressed towards or at the 5' end of the coding region. The signal
sequence
may be homologous or heterologous to the polynucleotide of interest and may be
homologous or heterologous to the cells to be transfected. Additionally, the
signal
sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect. This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available
depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial
solid
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(tablet or pill) pharmaceutical formulations, and decanting or topical
applications
during surgery. For example, direct injection of naked calcium
phosphate-precipitated plasmid into rat liver and rat spleen or a protein-
coated
plasmid into the portal vein has resulted in gene expression of the foreign
gene in the
rat livers. (Kaneda et al., Science, 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably,
a
recombinant molecule of the present invention complexed with a delivery
vehicle is
administered by direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to
injecting
the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide
construct of the present invention in or around a surgical wound. For example,
a
patient can undergo surgery and the polynucleotide construct can be coated on
the
surface of tissue inside the wound or the construct can be injected into areas
of tissue
inside the wound.
Therapeutic compositions useful in systemic administration, include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
administration comprise liposomes comprising ligands for targeting the vehicle
to a
particular site.
Preferred methods of systemic administration, include intravenous injection,
aerosol, oral and percutaneous (topical) delivery. Intravenous injections can
be
performed using methods standard in the art. Aerosol delivery can also be
performed
using methods standard in the art (see, for example, Stribling et al., Proc.
Natl. Acad.
Sci. USA , 189:11277-11281 (1992), which is incorporated herein by reference).
Oral
delivery can be performed by complexing a polynucleotide construct of the
present
invention to a carrier capable of withstanding degradation by digestive
enzymes in the
gut of an animal. Examples of such carriers, include plastic capsules or
tablets, such
as those known in the art. Topical delivery can be performed by mixing a
polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
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Determining an effective amount of substance to be delivered can depend
upon a number of factors including, for example, the chemical stricture and
biological activity of the substance, the age and weight of the animal, the
precise
condition requiring treatment and its severity, and the route of
administration. The
frequency of treatments depends upon a number of factors, such as the amount
of
polynucleotide constructs administered per dose, as well as the health and
history of
the subject. The precise amount, number of doses, and timing of doses will be
determined by the attending physician or veterinarian. Therapeutic
compositions of
the present invention can be administered to any animal, preferably to mammals
and
birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle,
horses and pigs, with humans being particularly
Biological Activities
The polynucleotides or polypeptides, or agonists or antagonists of the present
invention can be used in assays to test for one or more biological activities.
If these
polynucleotides and polypeptides do exhibit activity in a particular assay, it
is likely
that these molecules may be involved in the diseases associated with the
biological
activity. Thus, the polynucleotides or polypeptides, or agonists or
antagonists could
be used to treat the associated disease.
Immune Activity
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be useful in treating, preventing, and/or diagnosing
diseases,
disorders, and/or conditions of the immune system, by, for example, activating
or
inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of
immune
cells. Immune cells develop through a process called hematopoiesis, producing
myeloid (platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B
and T lymphocytes) cells from pluripotent stem cells. The etiology of these
immune
diseases, disorders, and/or conditions may be genetic, somatic, such as cancer
and
some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or
infectious.
Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
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the present invention can be used as a marker or detector of a particular
immune
system disease or disorder.
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be useful in treating, preventing, and/or diagnosing
diseases,
disorders, and/or conditions of hematopoietic cells. Polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention could be
used to
increase differentiation and proliferation of hematopoietic cells, including
the
pluripotent stem cells, in an effort to treat or prevent those diseases,
disorders, and/or
conditions associated with a decrease in certain (or many) types hematopoietic
cells.
Examples of immunologic deficiency syndromes include, but are not limited to:
blood protein diseases, disorders, and/or conditions (e.g.,
agammaglobulinemia,
dysgammaglobulinemia), ataxia telangiectasia, common variable
immunodeficiency,
Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe
combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia, or hemoglobinuria.
Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention could also be used to modulate hemostatic
(the
stopping of bleeding) or thrombolytic activity (clot formation). For example,
by
increasing hemostatic or thrombolytic activity, polynucleotides or
polypeptides,
and/or agonists or antagonists of the present invention could be used to treat
or
prevent blood coagulation diseases, disorders, and/or conditions (e.g.,
afibrinogenemia, factor deficiencies), blood platelet diseases, disorders,
and/or
conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery,
or
other causes. Alternatively, polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention that can decrease hemostatic or
thrombolytic
activity could be used to inhibit or dissolve clotting. These molecules could
be
important in the treatment or prevention of heart attacks (infarction),
strokes, or
scarring.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be useful in treating, preventing, and/or
diagnosing
autoimmune disorders. Many autoimmune disorders result from inappropriate
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recognition of self as foreign material by immune cells. This inappropriate
recognition results in an immune response leading to the destruction of the
host tissue.
Therefore, the administration of polynucleotides and polypeptides of the
invention
that can inhibit an immune response, particularly the proliferation,
differentiation, or
chemotaxis of T-cells, may be an effective therapy in preventing autoimmune
disorders.
Autoimmune diseases or disorders that may be treated, prevented, and/or
diagnosed by polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention include, but are not limited to, one or more of the
following:
autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,
myocarditis,
relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g,
IgA
nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
IS Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's
Disease,
Stiff Man Syndrome, Autoimmune Pulmonary Inflammation, Autism, Guillain-Barre
Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory
eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,
systemic lupus
erhythematosus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities
such
as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin
resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,
rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed
connective
tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's
disease, infertility, glomerulonephritis such as primary glomerulonephritis
and IgA
nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus, and
adrenergic drug resistance (including adrenergic drug resistance with asthma
or cystic
fibrosis), chronic active hepatitis, primary biliary cirrhosis, other
endocrine gland
failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic
dermatitis,
asthma, inflammatory myopathies, and other inflammatory, granulamatous,
degenerative, and atrophic disorders.
Additional autoimmune disorders (that are probable) that may be treated,
prevented, and/or diagnosed with the compositions of the invention include,
but are
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not limited to, rheumatoid arthritis (often characterized, e.g., by immune
complexes in
joints), scleroderma with anti-collagen antibodies (often characterized, e.g.,
by
nucleolar and other nuclear antibodies), mixed connective tissue disease
(often
characterized, e.g., by antibodies to extractable nuclear antigens (e.g.,
ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone
ANA),
pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes, and
intrinsic factor antibodies), idiopathic Addison's disease (often
characterized, e.g., by
humoral and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g.,
by antispermatozoal antibodies), glomerulonephritis (often characterized,
e.g., by
glomerular basement membrane antibodies or immune complexes), bullous
pemphigoid (often characterized, e.g., by IgG and complement in basement
membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often
characterized, e.g., by cell-mediated and humoral islet cell antibodies), and
adrenergic
drug resistance (including adrenergic drug resistance with asthma or cystic
fibrosis)
(often characterized, e.g., by beta-adrenergic receptor antibodies).
Additional autoimmune disorders (that are possible) that may be treated,
prevented, and/or diagnosed with the compositions of the invention include,
but are
not limited to, chronic active hepatitis (often characterized, e.g., by smooth
muscle
antibodies), primary bfliary cirrhosis (often characterized, e.g., by
mitchondrial
antibodies), other endocrine gland failure (often characterized, e.g., by
specific tissue
antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte
antibodies), vasculitis (often characterized, e.g., by Ig and complement in
vessel walls
and/or low serum complement), post-MI (often characterized, e.g., by
myocardial
antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial
antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies
to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to
IgE), asthma
(often characterized, e.g., by IgG and IgM antibodies to IgE), and many other
inflammatory, granulamatous, degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
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prevented, and/or diagnosed using for example, antagonists or agonists,
polypeptides
or polynucleotides, or antibodies of the present invention.
In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention could be used as an agent to
boost
immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
B cell immunodeficiencies that may be ameliorated or treated by
administering the polypeptides or polynucleotides of the invention, and/or
agonists
thereof, include, but are not limited to, severe combined immunodeficiency
(SCID)-X
linked, SC>D-autosomal, adenosine deaminase deficiency (ADA deficiency), X-
linked agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia,
1 S agammaglobulinemia, common variable immunodeficiency (CVI) (acquired),
Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non
X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass.
deficiency (with or without IgA deficiency), antibody deficiency with normal
or
elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa
chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular
dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic
alymophoplasia-aplasia or dysplasia with immunodeficiency, ataxia-
telangiectasia,
short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase
deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and
severe combined immunodeficiency.
T cell deficiencies that may be ameliorated or treated by administering the
polypeptides or polynucleotides of the invention, and/or agonists thereof
include, but
are not limited to, for example, DiGeorge anomaly, thymic hypoplasia, third
and
fourth pharyngeal pouch syndrome, 22q 11.2 deletion, chronic mucocutaneous
candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-
lymphocytopenia,
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immunodeficiency with predominant T cell defect (unspecified), and unspecified
immunodeficiency of cell mediated immunity. In specific embodiments, DiGeorge
anomaly or conditions associated with DiGeorge anomaly are ameliorated or
treated
by, for example, administering the polypeptides or polynucleotides of the
invention,
or antagonists or agonists thereof.
Other immunodeficiencies that may be ameliorated or treated by administering
polypeptides or polynucleotides of the invention, and/or agonists thereof,
include, but
are not limited to, severe combined immunodeficiency (SCID; e.g., X-linked
SCID,
autosomal SCID, and adenosine deaminase deficiency), ataxia-telangiectasia,
Wiskott-Aldrich syndrome, short-limber dwarfism, X-linked lymphoproliferative
syndrome (XLP), Nezelof syndrome (e.g., purine nucleoside phosphorylase
deficiency), MHC Class II deficiency. In specific embodiments, ataxia-
telangiectasia
or conditions associated with ataxia-telangiectasia are ameliorated or treated
by
administering the polypeptides or polynucleotides of the invention, and/or
agonists
thereof.
In a specific preferred embodiment, rheumatoid arthritis is treated,
prevented,
and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention. In another specific preferred
embodiment,
systemic lupus erythemosus is treated, prevented, and/or diagnosed using
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention. In another specific preferred embodiment, idiopathic
thrombocytopenia purpura is treated, prevented, and/or diagnosed using
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention. In another specific preferred embodiment IgA nephropathy is
treated, prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention. In a preferred
embodiment, the
autoimmune diseases and disorders and/or conditions associated with the
diseases and
disorders recited above are treated, prevented, and/or diagnosed using
antibodies
against the protein of the invention.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or other respiratory problems, may also be treated,
prevented, and/or
diagnosed using polypeptides, antibodies, or polynucleotides of the invention,
and/or
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agonists or antagonists thereof. Moreover, these molecules can be used to
treat,
prevent, and/or diagnose anaphylaxis, hypersensitivity to an antigenic
molecule, or
blood group incompatibility.
Moreover, inflammatory conditions may also be treated, diagnosed, and/or
prevented with polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention. Such inflammatory conditions include,
but are
not limited to, for example, respiratory disorders (such as, e.g., asthma and
allergy);
gastrointestinal disorders (such as, e.g., inflammatory bowel disease);
cancers (such
as, e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders
(such as, e.g.,
multiple sclerosis, blood-brain barrier permeability, ischemic brain injury
and/or
stroke, traumatic brain injury, neurodegenerative disorders (such as, e.g.,
Parkinson's
disease and Alzheimer's disease), AIDS-related dementia, and prion disease);
cardiovascular disorders (such as, e.g., atherosclerosis, myocarditis,
cardiovascular
disease, and cardiopulmonary bypass complications); as well as many additional
diseases, conditions, and disorders that are characterized by inflammation
(such as,
e.g., chronic hepatitis (B and C), rheumatoid arthritis, gout, trauma, septic
shock,
pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury,
Grave's
disease, systemic lupus erythematosis, diabetes mellitus (i.e., type 1
diabetes), and
allogenic transplant rejection).
In specific embodiments, polypeptides, antibodies, or polynucleotides of the
invention, and/or agonists or antagonists thereof, are useful to treat,
diagnose, and/or
prevent transplantation rejections, graft-versus-host disease, autoimmune and
inflammatory diseases (e.g., immune complex-induced vasculitis,
glomerulonephritis,
hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis,
experimental
allergic and hyperacute xenograft rejection, rheumatoid arthritis, and
systemic lupus
erythematosus (SLE). Organ rejection occurs by host immune cell destruction of
the
transplanted tissue through an immune response. Similarly, an immune response
is
also involved in GVHD, but, in this case, the foreign transplanted immune
cells
destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the
invention,
and/or agonists or antagonists thereof, that inhibit an immune response,
particularly
the activation, proliferation, differentiation, or chemotaxis of T-cells, may
be an
effective therapy in preventing organ rejection or GVHD.
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Similarly, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may also be used to modulate and/or
diagnose
inflammation. For example, since polypeptides, antibodies, or polynucleotides
of the
invention, and/or agonists or antagonists of the invention may inhibit the
activation,
proliferation and/or differentiation of cells involved in an inflammatory
response,
these molecules can be used to treat, diagnose, or prognose, inflammatory
conditions,
both chronic and acute conditions, including, but not limited to, inflammation
associated with infection (e.g., 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, and resulting from
over
production of cytokines (e.g., TNF or IL-1.).
Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of
the
invention can be used to treat, detect, and/or prevent infectious agents. For
example,
by increasing the immune response, particularly increasing the proliferation
activation
and/or differentiation of B and/or T cells, infectious diseases may be
treated, detected,
and/or prevented. The immune response may be increased by either enhancing an
existing immune response, or by initiating a new immune response.
Alternatively,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may also directly inhibit the infectious agent (refer to
section of
application listing infectious agents, etc), without necessarily eliciting an
immune
response.
Additional preferred embodiments of the invention include, but are not limited
to, the use of polypeptides, antibodies, polynucleotides and/or agonists or
antagonists
in the following applications:
Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig,
pigs,
micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human
primate, and
human, most preferably human) to boost the immune system to produce increased
quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher
affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to
increase an
immune response.
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Administration to an animal (including, but not limited to, those listed
above,
and also including transgenic animals) incapable of producing functional
endogenous
antibody molecules or having an otherwise compromised endogenous immune
system, but which is capable of producing human immunoglobulin molecules by
means of a reconstituted or partially reconstituted immune system from another
animal (see, e.g., published PCT Application Nos. W098/24893, WO/9634096,
WO/9633735, and WO/9110741.
A vaccine adjuvant that enhances immune responsiveness to specific antigen.
An adjuvant to enhance tumor-specific immune responses.
An adjuvant to enhance anti-viral immune responses. Anti-viral immune
responses that may be enhanced using the compositions of the invention as an
adjuvant, include virus and virus associated diseases or symptoms described
herein or
otherwise known in the art. In specific embodiments, the compositions of the
invention are used as an adjuvant to enhance an immune response to a virus,
disease,
or symptom selected from the group consisting of: AIDS, meningitis, Dengue,
EBV,
and hepatitis (e.g., hepatitis B). In another specific embodiment, the
compositions of
the invention are used as an adjuvant to enhance an immune response to a
virus,
disease, or symptom selected from the group consisting o~ HIV/AIDS,
Respiratory
syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and
B,
Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift
Valley
fever, Herpes simplex, and yellow fever.
An adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-
bacterial or anti-fungal immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include bacteria or fungus and
bacteria
or fungus associated diseases or symptoms described herein or otherwise known
in
the art. In specific embodiments, the compositions of the invention are used
as an
adjuvant to enhance an immune response to a bacteria or fungus, disease, or
symptom
selected from the group consisting of: tetanus, Diphtheria, botulism, and
meningitis
type B. In another specific embodiment, the compositions of the invention are
used as
an adjuvant to enhance an immune response to a bacteria or fungus, disease, or
symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium
leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis,
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Streptococcus pneumoniae, Group B streptococcus, Shigella spp.,
Enterotoxigenic
Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgclorferi, and
Plasmodium
(malaria).
An adjuvant to enhance anti-parasitic immune responses. Anti-parasitic
immune responses that may be enhanced using the compositions of the invention
as
an adjuvant, include parasite and parasite associated diseases or symptoms
described
herein or otherwise known in the art. In specific embodiments, the
compositions of
the invention are used as an adjuvant to enhance an immune response to a
parasite. In
another specific embodiment, the compositions of the invention are used as an
adjuvant to enhance an immune response to Plasmodium (malaria).
As a stimulator of B cell responsiveness to pathogens.
As an activator of T cells.
As an agent that elevates the immune status of an individual prior to their
receipt of immunosuppressive therapies.
As an agent to induce higher affinity antibodies.
As an agent to increase serum immunoglobulin concentrations.
As an agent to accelerate recovery of immunocompromised individuals.
As an agent to boost immunoresponsiveness among aged populations.
As an immune system enhancer prior to, during, or after bone marrow
transplant and/or other transplants (e.g., allogeneic or xenogeneic organ
transplantation). With respect to transplantation, compositions of the
invention may
be administered prior to, concomitant with, and/or after transplantation. In a
specific
embodiment, compositions of the invention are administered after
transplantation,
prior to the beginning of recovery of T-cell populations. In another specific
embodiment, compositions of the invention are first administered after
transplantation
after the beginning of recovery of T cell populations, but prior to full
recovery of B
cell populations.
As an agent to boost immunoresponsiveness among individuals having an
acquired loss of B cell function. Conditions resulting in an acquired loss of
B cell
function that may be ameliorated or treated by administering the polypeptides,
antibodies, polynucleotides and/or agonists or antagonists thereof, include,
but are
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not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell
chronic
lymphocytic leukemia (CLL).
As an agent to boost immunoresponsiveness among individuals having a
temporary immune deficiency. Conditions resulting in a temporary immune
deficiency that may be ameliorated or treated by administering the
polypeptides,
antibodies, polynucleotides and/or agonists or antagonists thereof, include,
but are
not limited to, recovery from viral infections (e.g., influenza), conditions
associated
with malnutrition, recovery from infectious mononucleosis, or conditions
associated
with stress, recovery from measles, recovery from blood transfusion, recovery
from
surgery.
As a regulator of antigen presentation by monocytes, dendritic cells, and/or
B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention enhance antigen presentation
or
antagonizes antigen presentation in vitro or in vivo. Moreover, in related
embodiments, said enhancement or antagonization of antigen presentation may be
useful as an anti-tumor treatment or to modulate the immune system.
As an agent to direct an individuals immune system towards development of a
humoral response (i.e. TH2) as opposed to a TH1 cellular response.
As a means to induce tumor proliferation and thus make it more susceptible to
anti-neoplastic agents. For example, multiple myeloma is a slowly dividing
disease
and is thus refractory to virtually all anti-neoplastic regimens. If these
cells were
forced to proliferate more rapidly their susceptibility profile would likely
change.
As a stimulator of B cell production in pathologies such as AIDS, chronic
lymphocyte disorder and/or Common Variable Immunodificiency.
As a therapy for generation and/or regeneration of lymphoid tissues following
surgery, trauma or genetic defect.
As a gene-based therapy for genetically inherited disorders resulting in
immuno-incompetence such as observed among SCm patients.
As an antigen for the generation of antibodies to inhibit or enhance immune
mediated responses against polypeptides of the invention.
As a means of activating T cells.
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As a means of activating monocytes/macrophages to defend against parasitic
diseases that effect monocytes such as Leshmania.
As pretreatment of bone marrow samples prior to transplant. Such treatment
would increase B cell representation and thus accelerate recover.
As a means of regulating secreted cytokines that are elicited by polypeptides
of the invention.
Additionally, polypeptides or polynucleotides of the invention, and/or
agonists
thereof, may be used to treat or prevent IgE-mediated allergic reactions. Such
allergic
reactions include, but are not limited to, asthma, rhinitis, and eczema.
All of the above described applications as they may apply to veterinary
medicine.
Antagonists of the invention include, for example, binding and/or inhibitory
antibodies, antisense nucleic acids, or ribozymes. These would be expected to
reverse
many of the activities of the ligand described above as well as find clinical
or
1 S practical application as:
A means of blocking various aspects of immune responses to foreign agents or
self. Examples include autoimmune disorders such as lupus, and arthritis, as
well as
immunoresponsiveness to skin allergies, inflammation, bowel disease, injury
and
pathogens.
A therapy for preventing the B cell proliferation and Ig secretion associated
with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic
lupus erythramatosus and MS.
An inhibitor of B and/or T cell migration in endothelial cells. This activity
disrupts tissue architecture or cognate responses and is useful, for example
in
disrupting immune responses, and blocking sepsis.
An inhibitor of graft versus host disease or transplant rejection.
A therapy for B cell and/or T cell malignancies such as ALL, Hodgkins
disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas,
multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.
A therapy for chronic hypergammaglobulinemeia evident in such diseases as
monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom's
disease, related idiopathic monoclonalgammopathies, and plasmacytomas.
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A therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
A means of decreasing the involvement of B cells and Ig associated with
Chronic Myelogenous Leukemia.
An immunosuppressive agent(s).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be used to modulate IgE concentrations in vitro or in
vivo.
In another embodiment, administration of polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the invention, may be used
to treat
or prevent IgE-mediated allergic reactions including, but not limited to,
asthma,
rhinitis, and eczema.
The agonists and antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described herein.
The agonists or antagonists may be employed for instance to inhibit
polypeptide chemotaxis and activation of macrophages and their precursors, and
of
neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated
and
CD8 cytotoxic T cells and natural killer cells, in certain auto-immune and
chronic
inflammatory and infective diseases. Examples of autoimmune diseases are
described
herein and include multiple sclerosis, and insulin-dependent diabetes. The
antagonists or agonists may also be employed to treat infectious diseases
including
silicosis, sarcoidosis, idiopathic pulmonary fibrosis by, for example,
preventing the
recruitment and activation of mononuclear phagocytes. They may also be
employed
to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing
eosinophil production and migration. The antagonists or agonists or may also
be
employed for treating atherosclerosis, for example, by preventing monocyte
infiltration in the artery wall.
Antibodies against polypeptides of the invention may be employed to treat
ARDS.
Agonists and/or antagonists of the invention also have uses in stimulating
wound and tissue repair, stimulating angiogenesis, stimulating the repair of
vascular
or lymphatic diseases or disorders. Additionally, agonists and antagonists of
the
invention may be used to stimulate the regeneration of mucosal surfaces.
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In a specific embodiment, polynucleotides or polypeptides, andlor agonists
thereof are used to treat or prevent a disorder characterized by primary or
acquired
immunodeficiency, deficient serum immunoglobulin production, recurrent
infections,
and/or immune system dysfunction. Moreover, polynucleotides or polypeptides,
S and/or agonists thereof may be used to treat or prevent infections of the
joints, bones,
skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,
septic
arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed
herein),
inflammatory disorders, and malignancies, and/or any disease or disorder or
condition
associated with these infections, diseases, disorders and/or malignancies)
including,
but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL,
recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,
hepatitis,
meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis
carnii.
In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention are used to treat, and/or
diagnose an
individual having common variable immunodeficiency disease ("CVID"; also known
as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease.
In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to treat,
diagnose, and/or
prevent (1) cancers or neoplasms and (2) autoimmune cell or tissue-related
cancers or
neoplasms. In a preferred embodiment, polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention conjugated to a toxin
or a
radioactive isotope, as described herein, may be used to treat, diagnose,
and/or
prevent acute myelogeneous leukemia. In a further preferred embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention conjugated to a toxin or a radioactive isotope, as described
herein,
may be used to treat, diagnose, and/or prevent, chronic myelogeneous leukemia,
multiple myeloma, non-Hodgkins lymphoma, andlor Hodgkins disease.
In another specific embodiment, polynucleotides or polypeptides, and/or
agonists or antagonists of the invention may be used to treat, diagnose,
prognose,
andlor prevent selective IgA deficiency, myeloperoxidase deficiency, C2
deficiency,
ataxia-telangiectasia, DiGeorge anomaly, common variable immunodeficiency
(CVI),
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X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), chronic
granulomatous disease (CGD), and Wiskott-Aldrich syndrome.
Examples of autoimmune disorders that can be treated or detected are described
above and also include, but are not limited to: Addison's Disease, hemolytic
anemia,
S antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'
Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmic, Bullous
Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff Man
Syndrome,
Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary
Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,
and
autoimmune inflammatory eye disease.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prognosed, prevented, and/or diagnosed using antibodies against the
polypeptide of
the invention.
As an agent to boost immunoresponsiveness among B cell immunodeficient
individuals, such as, for example, an individual who has undergone a partial
or
complete splenectomy.
Additionally, polynucleotides, polypeptides, and/or antagonists of the
invention may affect apoptosis, and therefore, would be useful in treating a
number of
diseases associated with increased cell survival or the inhibition of
apoptosis. For
example, diseases associated with increased cell survival or the inhibition of
apoptosis
that could be treated or detected by polynucleotides, polypeptides, and/or
antagonists
of the invention, include cancers (such as follicular lymphomas, carcinomas
with p53
mutations, and hormone-dependent tumors, including, but not limited to colon
cancer,
cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma,
lung
cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma,
myxoma,
myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
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glomerulonephritis and rheumatoid arthritis) and viral infections (such as
herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host disease,
acute graft
rejection, and chronic graft rejection. In preferred embodiments,
polynucleotides,
polypeptides, and/or antagonists of the invention are used to inhibit growth,
progression, and/or metastisis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be treated or detected by polynucleotides, polypeptides, and/or
antagonists of
the invention, include, but are not limited to, progression, and/or metastases
of
malignancies and related disorders such as leukemia (including acute leukemias
(e.g.,
acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lyrnphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated or detected
by polynucleotides, polypeptides, and/or antagonists of the invention, include
A>DS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
and
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brain tumor or prior associated disease); autoimmune disorders (such as,
multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-
related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes
(such
S as aplastic anemia), graft v. host disease, ischemic injury (such as that
caused by
myocardial infarction, stroke and reperfusion injury), liver injury (e.g.,
hepatitis
related liver injury, ischemia/reperfusion injury, cholestosis (bile duct
injury) and
liver cancer); toxin-induced liver disease (such as that caused by alcohol),
septic
shock, cachexia and anorexia.
Hyperproliferative diseases and/or disorders that could be detected and/or
treated by polynucleotides, polypeptides, and/or antagonists of the invention,
include,
but are not limited to neoplasms located in the: liver, abdomen, bone, breast,
digestive
system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic,
and
urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected
by
polynucleotides, polypeptides, and/or antagonists of the invention. Examples
of such
hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,
purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's
Disease, histiocytosis, and any other hyperproliferative disease, besides
neoplasia;
located in an organ system listed above.
Hyuerproliferative Disorders
A polynucleotides or polypeptides, or agonists or antagonists of the invention
can be used to treat, prevent,, and/or diagnose hyperproliferative diseases,
disorders,
and/or conditions, including neoplasms. A polynucleotides or polypeptides, or
agonists or antagonists of the present invention may inhibit the proliferation
of the
disorder through direct or indirect interactions. Alternatively, a
polynucleotides or
polypeptides, or agonists or antagonists of the present invention may
proliferate other
cells which can inhibit the hyperproliferative disorder.
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For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing T-cells, hyperproliferative diseases,
disorders, and/or
conditions can be treated, prevented, and/or diagnosed. This immune response
may
be increased by either enhancing an existing immune response, or by initiating
a new
immune response. Alternatively, decreasing an immune response may also be a
method of treating, preventing, and/or diagnosing hyperproliferative diseases,
disorders, and/or conditions, such as a chemotherapeutic agent.
Examples of hyperproliferative diseases, disorders, and/or conditions that can
be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or
agonists or antagonists of the present invention include, but are not limited
to
neoplasms located in the: colon, abdomen, bone, breast, digestive system,
liver,
pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles,
ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative diseases, disorders, and/or conditions can
also be treated, prevented, and/or diagnosed by a polynucleotides or
polypeptides, or
agonists or antagonists of the present invention. Examples of such
hyperproliferative
diseases, disorders, and/or conditions include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or
conditions,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's
Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other
hyperproliferative disease, besides neoplasia, located in an organ system
listed above.
One preferred embodiment utilizes polynucleotides of the present invention to
inhibit aberrant cellular division, by gene therapy using the present
invention, and/or
protein fusions or fragments thereof.
Thus, the present invention provides a method for treating or preventing cell
proliferative diseases, disorders, and/or conditions by inserting into an
abnormally
proliferating cell a polynucleotide of the present invention, wherein said
polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating or
preventing cell-proliferative diseases, disorders, and/or conditions in
individuals
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comprising administration of one or more active gene copies of the present
invention
to an abnormally proliferating cell or cells. In a preferred embodiment,
polynucleotides of the present invention is a DNA construct comprising a
recombinant expression vector effective in expressing a DNA sequence encoding
said
polynucleotides. In another preferred embodiment of the present invention, the
DNA
construct encoding the poynucleotides of the present invention is inserted
into cells to
be treated utilizing a retrovirus, or more preferrably an adenoviral vector
(See G J.
Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by
reference).
In a most preferred embodiment, the viral vector is defective and will not
transform
non-proliferating cells, only proliferating cells. Moreover, in a preferred
embodiment, the polynucleotides of the present invention inserted into
proliferating
cells either alone, or in combination with or fused to other polynucleotides,
can then
be modulated via an external stimulus (i.e. magnetic, specific small molecule,
chemical, or drug administration, etc.), which acts upon the promoter upstream
of said
polynucleotides to induce expression of the encoded protein product. As such
the
beneficial therapeutic affect of the present invention may be expressly
modulated (i.e.
to increase, decrease, or inhibit expression of the present invention) based
upon said
external stimulus.
Polynucleotides of the present invention may be useful in repressing
expression of oncogenic genes or antigens. By "repressing expression of the
oncogenic genes " is intended the suppression of the transcription of the
gene, the
degradation of the gene transcript (pre-message RNA), the inhibition of
splicing, the
destruction of the messenger RNA, the prevention of the post-translational
modifications of the protein, the destruction of the protein, or the
inhibition of the
normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of
the present invention may be administered by any method known to those of
skill in
the art including, but not limited to transfection, electroporation,
microinjection of
cells, or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any
other method described throughout the specification. The polynucleotide of the
present invention may be delivered by known gene delivery systems such as, but
not
limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature
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320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014),
vaccinia virus
system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient
DNA
delivery systems (Pates et al., Nature 313:812 (1985)) known to those skilled
in the
art. These references are exemplary only and are hereby incorporated by
reference.
In order to specifically deliver or transfect cells which are abnormally
proliferating
and spare non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as
described in the art and elsewhere herein) delivery system known to those of
skill in
the art. Since host DNA replication is required for retroviral DNA to
integrate and
the retrovirus will be unable to self replicate due to the lack of the
retrovirus genes
needed for its life cycle. Utilizing such a retroviral delivery system for
polynucleotides of the present invention will target said gene and constructs
to
abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell
proliferative disorder/disease sites in internal organs, body cavities and the
like by use
of imaging devices used to guide an injecting needle directly to the disease
site. The
polynucleotides of the present invention may also be administered to disease
sites at
the time of surgical intervention.
By "cell proliferative disease" is meant any human or animal disease or
disorder, affecting any one or any combination of organs, cavities, or body
parts,
which is characterized by single or multiple local abnormal proliferations of
cells,
groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be
administered as long as it has a biologically inhibiting effect on the
proliferation of
the treated cells. Moreover, it is possible to administer more than one of the
polynucleotide of the present invention simultaneously to the same site. By
"biologically inhibiting" is meant partial or total growth inhibition as well
as
decreases in the rate of proliferation or growth of the cells. The
biologically
inhibitory dose may be determined by assessing the effects of the
polynucleotides of
the present invention on target malignant or abnormally proliferating cell
growth in
tissue culture, tumor growth in animals and cell cultures, or any other method
known
to one of ordinary skill in the art.
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The present invention is further directed to antibody-based therapies which
involve administering of anti-polypeptides and anti-polynucleotide antibodies
to a
mammalian, preferably human, patient for treating, preventing, and/or
diagnosing one
or more of the described diseases, disorders, and/or conditions. Methods for
producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and
monoclonal antibodies are described in detail elsewhere herein. Such
antibodies may
be provided in pharmaceutically acceptable compositions as known in the art or
as
described herein.
A summary of the ways in which the antibodies of the present invention may
be used therapeutically includes binding polynucleotides or polypeptides of
the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without
undue experimentation.
In particular, the antibodies, fragments and derivatives of the present
invention
are useful for treating, preventing, and/or diagnosing a subject having or
developing
cell proliferative and/or differentiation diseases, disorders, and/or
conditions as
described herein. Such treatment comprises administering a single or multiple
doses
of the antibody, or a fragment, derivative, or a conjugate thereof.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors, for example, which serve to increase the number
or
activity of effector cells which interact with the antibodies.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of diseases, disorders, and/or conditions related to polynucleotides
or
polypeptides, including fragements thereof, of the present invention. Such
antibodies,
fragments, or regions, will preferably have an affinity for polynucleotides or
polypeptides, including fragements thereof. Preferred binding affinities
include those
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with a dissociation constant or Kd less than 5X10-6M, 10-~M, 5X10-'M, 10-'M,
5X10
8M, 10-8M, 5 X 10-9M, 10-~M, 5X 10-' °M, 10-' °M, 5 X 10-"M, 10-
"M, 5 X 10~' 2M, 10-
'ZM, 5X10-'3M, 10-'3M, 5X10-'4M, 10-'4M, 5X10-'SM, and 10-'5M.
Moreover, polypeptides of the present invention are useful in inhibiting the
angiogenesis of proliferative cells or tissues, either alone, as a protein
fusion, or in
combination with other polypeptides directly or indirectly, as described
elsewhere
herein. In a most preferred embodiment, said anti-angiogenesis effect may be
achieved indirectly, for example, through the inhibition of hematopoietic,
tumor-
specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J
Natl
Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by
reference).
Antibodies directed to polypeptides or polynucleotides of the present
invention may
also result in inhibition of angiogenesis directly, or indirectly (See Witte
L, et al.,
Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by
reference)).
Polypeptides, including protein fusions, of the present invention, or
fragments
thereof may be useful in inhibiting proliferative cells or tissues through the
induction
of apoptosis. Said polypeptides may act either directly, or indirectly to
induce
apoptosis of proliferative cells and tissues, for example in the activation of
a death-
domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-
1),
TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K,
et.al.,
Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by
reference).
Moreover, in another preferred embodiment of the present invention, said
polypeptides may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or through
stimulating the °
expression of said proteins, either alone or in combination with small
molecule drugs
or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory
proteins
(See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-
33
(1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-
12
(1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby
incorporated by
reference).
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Polypeptides, including protein fusions to, or fragments thereof, of the
present
invention are useful in inhibiting the metastasis of proliferative cells or
tissues.
Inhibition may occur as a direct result of administering polypeptides, or
antibodies
directed to said polypeptides as described elsewere herein, or indirectly,
such as
S activating the expression of proteins known to inhibit metastasis, for
example alpha 4
integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is
hereby
incorporated by reference). Such thereapeutic affects of the present invention
may be
achieved either alone, or in combination with small molecule drugs or
adjuvants.
In another embodiment, the invention provides a method of delivering
compositions containing the polypeptides of the invention (e.g., compositions
containing polypeptides or polypeptide antibodes associated with heterologous
polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted
cells
expressing the polypeptide of the present invention. Polypeptides or
polypeptide
antibodes of the invention may be associated with with heterologous
polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic,
ionic
and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present
invention are
useful in enhancing the immunogenicity and/or antigenicity of proliferating
cells or
tissues, either directly, such as would occur if the polypeptides of the
present
invention 'vaccinated' the immune response to respond to proliferative
antigens and
immunogens, or indirectly, such as in activating the expression of proteins
known to
enhance the immune response (e.g. chemokines), to said antigens and
immunogens.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the invention
may be used to treat, prevent, and/or diagnose cardiovascular diseases,
disorders,
and/or conditions, including peripheral artery disease, such as limb ischemia.
Cardiovascular diseases, disorders, and/or conditions include cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous fistula,
cerebral
arteriovenous malformations, congenital heart defects, pulmonary atresia, and
Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor
triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent
ductus
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arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart
syndrome,
levocardia, tetralogy of fallot, transposition of great vessels, double outlet
right
ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal
defects, such
as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's
Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular diseases, disorders, and/or conditions also include heart
disease, such as arrhythmias, carcinoid heart disease, high cardiac output,
low cardiac
output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm,
cardiac
arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal
dyspnea,
cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy, right ventricular hypertrophy, post-infarction heart rupture,
ventricular
septal rupture, heart valve diseases, myocardial diseases, myocardial
ischemia,
pericardial effusion, pericarditis (including constrictive and tuberculous),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease,
rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and
cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-
type pre-excitation syndrome, Wolff Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias include
paroxysmal tachycardia, supraventricular tachycardia, accelerated
idioventricular
rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial
tachycardia, ectopic
functional tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia,
Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear murmurs, aortic valve prolapse, mural valve prolapse, tricuspid valve
prolapse,
mitral valve insufficiency, mural valve stenosis, pulmonary atresia, pulmonary
valve
insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insufficiency, and tricuspid valve stenosis.
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Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
reperfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema,
aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive
diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular
diseases, disorders,
and/or conditions, diabetic angiopathies, diabetic retinopathy, embolisms,
thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension,
hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-
occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion,
Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia
telangiectasia,
hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose
ulcer,
vasculitis, and venous insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication,
carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion,
Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis
obliterans.
Cerebrovascular diseases, disorders, and/or conditions include carotid artery
diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral
arteriosclerosis, cerebral arteriovenous malformation, cerebral artery
diseases,
cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis,
Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural
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hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
leukomalacia,
vascular headache, cluster headache, migraine, and vertebrobasilar
insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion
injuries, and peripheral limb ischemia. Vasculitis includes aortitis,
arteritis, Behcet's
Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch
purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
Polynucleotides or polypeptides, or agonists or antagonists of the invention,
are especially effective for the treatment of critical limb ischemia and
coronary
disease.
Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides of the invention may be administered as part of a
Therapeutic,
described in more detail below. Methods of delivering polynucleotides of the
invention are described in more detail herein.
Anti-An~io~enesis Activity
The naturally occurnng balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions, such as wound
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healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited. Under conditions of pathological angiogenesis such as that
characterizing
solid tumor growth, these regulatory controls fail. Unregulated angiogenesis
becomes
pathologic and sustains progression of many neoplastic and non-neoplastic
diseases.
A number of serious diseases are dominated by abnormal neovascularization
including solid tumor growth and metastases, arthritis, some types of eye
diseases,
disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et
al.,
Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763
(1995);
Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in
Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-
203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al.,
Science
221:719-725 (1983). In a number of pathological conditions, the process of
angiogenesis contributes to the disease state. For example, significant data
have
accumulated which suggest that the growth of solid tumors is dependent on
angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides for treatment of diseases, disorders, and/or
conditions associated with neovascularization by administration of the
polynucleotides and/or polypeptides of the invention, as well as agonists or
antagonists of the present invention. Malignant and metastatic conditions
which can
be treated with the polynucleotides and polypeptides, or agonists or
antagonists of the
invention include, but are not limited to, malignancies, solid tumors, and
cancers
described herein and otherwise known in the art (for a review of such
disorders, see
Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia
(1985)).Thus, the
present invention provides a method of treating, preventing, and/or diagnosing
an
angiogenesis-related disease and/or disorder, comprising administering to an
individual in need thereof a therapeutically effective amount of a
polynucleotide,
polypeptide, antagonist and/or agonist of the invention. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be utilized in
a variety
of additional methods in order to therapeutically treator prevent a cancer or
tumor.
Cancers which may be treated, prevented, and/or diagnosed with
polynucleotides,
polypeptides, antagonists and/or agonists include, but are not limited to
solid tumors,
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including prostate, lung, breast, ovarian, stomach, pancreas, larynx,
esophagus, testes,
liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney,
bladder, thyroid cancer; primary tumors and metastases; melanomas;
glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal
cancer;
advanced malignancies; and blood born tumors such as leukemias. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be delivered
topically,
in order to treat or prevent cancers such as skin cancer, head and neck
tumors, breast
tumors, and Kaposi's sarcoma.
Within yet other aspects, polynucleotides, polypeptides, antagonists and/or
agonists may be utilized to treat superficial forms of bladder cancer by, for
example,
intravesical administration. Polynucleotides, polypeptides, antagonists and/or
agonists
may be delivered directly into the tumor, or near the tumor site, via
injection or a
catheter. Of course, as the artisan of ordinary skill will appreciate, the
appropriate
mode of administration will vary according to the cancer to be treated. Other
modes
of delivery are discussed herein.
Polynucleotides, polypeptides, antagonists and/or agonists may be useful in
treating, preventing, and/or diagnosing other diseases, disorders, and/or
conditions,
besides cancers, which involve angiogenesis. These diseases, disorders, and/or
conditions include, but are not limited to: benign tumors, for example
hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example, diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed
wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial
angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous
malformations;
ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia;
wound
granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided
for treating, preventing, and/or diagnosing hypertrophic scars and keloids,
comprising
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the step of administering a polynucleotide, polypeptide, antagonist and/or
agonist of
the invention to a hypertrophic scar or keloid.
Within one embodiment of the present invention polynucleotides,
polypeptides, antagonists and/or agonists are directly injected into a
hypertrophic scar
or keloid, in order to prevent the progression of these lesions. This therapy
is of
particular value in the prophylactic treatment of conditions which are known
to result
in the development of hypertrophic scars and keloids (e.g., burns), and is
preferably
initiated after the proliferative phase has had time to progress
(approximately 14 days
after the initial injury), but before hypertrophic scar or keloid development.
As noted
above, the present invention also provides methods for treating, preventing,
and/or
diagnosing neovascular diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic retinopathy,
retrolental fibroplasia and macular degeneration.
Moreover, Ocular diseases, disorders, and/or conditions associated with
neovascularization which can be treated, prevented, and/or diagnosed with the
polynucleotides and polypeptides of the present invention (including agonists
and/or
antagonists) include, but are not limited to: neovascular glaucoma, diabetic
retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of
prematurity
macular degeneration, corneal graft neovascularization, as well as other eye
inflammatory diseases, ocular tumors and diseases associated with choroidal or
iris
neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal.
85:704-710
(1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for
treating or preventing neovascular diseases of the eye such as corneal
neovascularization (including corneal graft neovascularization), comprising
the step
of administering to a patient a therapeutically effective amount of a compound
(as
described above) to the cornea, such that the formation of blood vessels is
inhibited.
Briefly, the cornea is a tissue which normally lacks blood vessels. In certain
pathological conditions however, capillaries may extend into the cornea from
the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it
also becomes clouded, resulting in a decline in the patient's visual acuity.
Visual loss
may become complete if the cornea completely opacitates. A wide variety of
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diseases, disorders, and/or conditions can result in corneal
neovascularization,
including for example, corneal infections (e.g., trachoma, herpes simplex
keratitis,
leishmaniasis and onchocerciasis), immunological processes (e.g., graft
rejection and
Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any
cause),
toxic and nutritional deficiency states, and as a complication of wearing
contact
lenses.
Within particularly preferred embodiments of the invention, may be prepared
for topical administration in saline (combined with any of the preservatives
and
antimicrobial agents commonly used in ocular preparations), and administered
in
eyedrop form. The solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic compositions,
prepared as described above, may also be administered directly to the cornea.
Within
preferred embodiments, the anti-angiogenic composition is prepared with a muco-
adhesive polymer which binds to cornea. Within further embodiments, the anti-
angiogenic factors or anti-angiogenic compositions may be utilized as an
adjunct to
conventional steroid therapy. Topical therapy may also be useful
prophylactically in
corneal lesions which are known to have a high probability of inducing an
angiogenic
response (such as chemical burns). In these instances the treatment, likely in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
Within other embodiments, the compounds described above may be injected
directly into the corneal stroma by an ophthalmologist under microscopic
guidance.
The preferred site of injection may vary with the morphology of the individual
lesion,
but the goal of the administration would be to place the composition at the
advancing
front of the vasculature (i.e., interspersed between the blood vessels and the
normal
cornea). In most cases this would involve perilimbic corneal injection to
"protect" the
cornea from the advancing blood vessels. This method may also be utilized
shortly
after a corneal insult in order to prophylactically prevent corneal
neovascularization.
In this situation the material could be injected in the perilimbic cornea
interspersed
between the corneal lesion and its undesired potential limbic blood supply.
Such
methods may also be utilized in a similar fashion to prevent capillary
invasion of
transplanted corneas. In a sustained-release form injections might only be
required 2-
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3 times per year. A steroid could also be added to the injection solution to
reduce
inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for
treating or preventing neovascular glaucoma, comprising the step of
administering to
a patient a therapeutically effective amount of a polynucleotide, polypeptide,
antagonist and/or agonist to the eye, such that the formation of blood vessels
is
inhibited. In one embodiment, the compound may be administered topically to
the
eye in order to treat or prevent early forms of neovascular glaucoma. Within
other
embodiments, the compound may be implanted by injection into the region of the
anterior chamber angle. Within other embodiments, the compound may also be
placed in any location such that the compound is continuously released into
the
aqueous humor. Within another aspect of the present invention, methods are
provided
for treating or preventing proliferative diabetic retinopathy, comprising the
step of
administering to a patient a therapeutically effective amount of a
polynucleotide,
1 S polypeptide, antagonist and/or agonist to the eyes, such that the
formation of blood
vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in order to increase the local concentration of the polynucleotide,
polypeptide, antagonist and/or agonist in the retina. Preferably, this
treatment should
be initiated prior to the acquisition of severe disease requiring
photocoagulation.
Within another aspect of the present invention, methods are provided for
treating or preventing retrolental fibroplasia, comprising the step of
administering to a
patient a therapeutically effective amount of a polynucleotide, polypeptide,
antagonist
and/or agonist to the eye, such that the formation of blood vessels is
inhibited. The
compound may be administered topically, via intravitreous injection and/or via
intraocular implants.
Additionally, diseases, disorders, and/or conditions which can be treated,
prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists
and/or
agonists include, but are not limited to, hemangioma, arthritis, psoriasis,
angiofibroma, atherosclerotic plaques, delayed wound healing, granulations,
hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber
syndrome,
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pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, diseases, disorders, and/or conditions and/or states, which can be
treated, prevented, and/or diagnosed with the the polynucleotides,
polypeptides,
agonists and/or agonists include, but are not limited to, solid tumors, blood
born
tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors,
for
example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for
example,
diabetic retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft
rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and
uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations,
hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma,
vascular
adhesions, myocardial angiogenesis, coronary collaterals, cerebral
collaterals,
arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber
Syndrome,
plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma
1 S fibromuscular dysplasia, wound granulation, Crohn's disease,
atherosclerosis, birth
control agent by preventing vascularization required for embryo implantation
controlling menstruation, diseases that have angiogenesis as a pathologic
consequence
such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter
pylori),
Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control method, an amount of the compound
sufficient to block embryo implantation is administered before or after
intercourse and
fertilization have occurred, thus providing an effective method of birth
control,
possibly a "morning after" method. Polynucleotides, polypeptides, agonists
and/or
agonists may also be used in controlling menstruation or administered as
either a
peritoneal lavage fluid or for peritoneal implantation in the treatment of
endometriosis.
Polynucleotides, polypeptides, agonists and/or agonists of the present
invention may be incorporated into surgical sutures in order to prevent stitch
granulomas.
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a
wide variety of surgical procedures. For example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
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to coat or spray an area prior to removal of a tumor, in order to isolate
normal
surrounding tissues from malignant tissue, and/or to prevent the spread of
disease to
surrounding tissues. Within other aspects of the present invention,
compositions (e.g.,
in the form of a spray) may be delivered via endoscopic procedures in order to
coat
tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects
of the
present invention, surgical meshes which have been coated with anti-
angiogenic
compositions of the present invention may be utilized in any procedure wherein
a
surgical mesh might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition may be
utilized
during abdominal cancer resection surgery (e.g., subsequent to colon
resection) in
order to provide support to the structure, and to release an amount of the
anti-
angiogenic factor.
Within further aspects of the present invention, methods are provided for
treating tumor excision sites, comprising administering a polynucleotide,
polypeptide,
agonist and/or agonist to the resection margins of a tumor subsequent to
excision,
such that the local recurrence of cancer and the formation of new blood
vessels at the
site is inhibited. Within one embodiment of the invention, the anti-angiogenic
compound is administered directly to the tumor excision site (e.g., applied by
swabbing, brushing or otherwise coating the resection margins of the tumor
with the
anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be
incorporated into known surgical pastes prior to administration. Within
particularly
preferred embodiments of the invention, the anti-angiogenic compounds are
applied
after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, polynucleotides, polypeptides,
agonists and/or agonists may be administered to the resection margin of a wide
variety of tumors, including for example, breast, colon, brain and hepatic
tumors. For
example, within one embodiment of the invention, anti-angiogenic compounds may
be administered to the site of a neurological tumor subsequent to excision,
such that
the formation of new blood vessels at the site are inhibited.
The polynucleotides, polypeptides, agonists and/or agonists of the present
invention may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
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Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the
lighter "d
group" transition metals.
Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal species may form transition metal complexes. Suitable complexes of the
above-mentioned transition metal species include oxo transition metal
complexes.
Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include metavanadate and orthovanadate complexes such as, for example,
ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
Representative examples of tungsten and molybdenum complexes also include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide complexes. Suitable tungstate complexes include ammonium tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
A wide variety of other anti-angiogenic factors may also be utilized within
the
context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells),
(Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide
Peptidoglycan
Complex (SP- PG) (the function of this compound may be enhanced by the
presence
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of steroids such as estrogen, and tamoxifen citrate); Staurosporine;
modulators of
matrix metabolism, including for example, proline analogs, cishydroxyproline,
d,L-
3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile
fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.
267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480,
1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin
(Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-
serum;
alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987);
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole;
and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of
apoptosis
that could be treated, prevented, and/or diagnosed by the polynucleotides or
polypeptides and/or antagonists or agonists of the invention, include cancers
(such as
follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent
tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic
cancer,
melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast
cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune
diseases,
disorders, and/or conditions (such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes viruses, pox
viruses and
adenoviruses), inflammation, graft v. host disease, acute graft rejection, and
chronic
graft rej ection. In preferred embodiments, the polynucleotides or
polypeptides, and/or
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agonists or antagonists of the invention are used to inhibit growth,
progression, and/or
metasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be treated, prevented or diagnosed by the polynucleotides or
polypeptides, or
S agonists or antagonists of the invention, include, but are not limited to,
progression,
and/or metastases of malignancies and related disorders such as leukemia
(including
acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic)
leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas
(e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited
to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lyrnphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented,
and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, include AIDS; neurodegenerative diseases,
disorders,
and/or conditions (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic
lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain
tumor or
prior associated disease); autoimmune diseases, disorders, and/or conditions
(such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis,
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Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus
and
immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic
syndromes (such as aplastic anemia), graft v. host disease, ischemic injury
(such as
that caused by myocardial infarction, stroke and reperfusion injury), liver
injury (e.g.,
hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile
duct injury)
and liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic
shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing the polynucleotides or polypeptides, and/or
agonists
or antagonists of the invention, for therapeutic purposes, for example, to
stimulate
epithelial cell proliferation and basal keratinocytes for the purpose of wound
healing,
and to stimulate hair follicle production and healing of dermal wounds.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
invention,
may be clinically useful in stimulating wound healing including surgical
wounds,
excisional wounds, deep wounds involving damage of the dermis and epidermis,
eye
tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers,
dermal
ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting
from heat
exposure or chemicals, and other abnormal wound healing conditions such as
uremia,
malnutrition, vitamin deficiencies and complications associted with systemic
treatment with steroids, radiation therapy and antineoplastic drugs and
antimetabolites. Polynucleotides or polypeptides, and/or agonists or
antagonists of
the invention, could be used to promote dermal reestablishment subsequent to
dermal
loss
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could be used to increase the adherence of skin grafts to a wound
bed and
to stimulate re-epithelialization from the wound bed. The following are a non-
exhaustive list of grafts that polynucleotides or polypeptides, agonists or
antagonists
of the invention, could be used to increase adherence to a wound bed:
autografts,
artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular
grafts, Blair-
Brown grafts, bone graft, brephoplastic grafts, cubs graft, delayed graft,
dermic graft,
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epidermic graft, fascia graft, full thickness graft, heterologous graft,
xenograft,
homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal
graft, Ollier-
Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft,
split skin
graft, thick split graft. The polynucleotides or polypeptides, and/or agonists
or
antagonists of the invention, can be used to promote skin strength and to
improve the
appearance of aged skin.
It is believed that the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, will also produce changes in hepatocyte
proliferation,
and epithelial cell proliferation in the lung, breast, pancreas, stomach,
small intesting,
and large intestine. The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could promote proliferation of epithelial cells
such as
sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing
goblet
cells, and other epithelial cells and their progenitors contained within the
skin, lung,
liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or
agonists
or antagonists of the invention, may promote proliferation of endothelial
cells,
keratinocytes, and basal keratinocytes.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could also be used to reduce the side effects of gut toxicity that
result from
radiation, chemotherapy treatments or viral infections. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may have a
cytoprotective effect on the small intestine mucosa. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may also
stimulate
healing of mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could further be used in full regeneration of skin in full and
partial
thickness skin defects, including burns, (i.e., repopulation of hair
follicles, sweat
glands, and sebaceous glands), treatment of other skin defects such as
psoriasis. The
polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could
be used to treat epidermolysis bullosa, a defect in adherence of the epidermis
to the
underlying dermis which results in frequent, open and painful blisters by
accelerating
reepithelialization of these lesions. The polynucleotides or polypeptides,
and/or
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agonists or antagonists of the invention, could also be used to treat gastric
and
doudenal ulcers and help heal by scar formation of the mucosal lining and
regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis,
are
diseases which result in destruction of the mucosal surface of the small or
large
intestine, respectively. Thus, the polynucleotides or polypeptides, and/or
agonists or
antagonists of the invention, could be used to promote the resurfacing of the
mucosal
surface to aid more rapid healing and to prevent progression of inflammatory
bowel
disease. Treatment with the polynucleotides or polypeptides, and/or agonists
or
antagonists of the invention, is expected to have a significant effect on the
production
of mucus throughout the gastrointestinal tract and could be used to protect
the
intestinal mucosa from injurious substances that are ingested or following
surgery.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention,
could be used to treat diseases associate with the under expression of the
polynucleotides of the invention.
Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists
of
the invention, could be used to prevent and heal damage to the lungs due to
various
pathological states. A growth factor such as the polynucleotides or
polypeptides,
and/or agonists or antagonists of the invention, which could stimulate
proliferation
and differentiation and promote the repair of alveoli and brochiolar
epithelium to
prevent or treat acute or chronic lung damage. For example, emphysema, which
results in the progressive loss of aveoli, and inhalation injuries, i.e.,
resulting from
smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium
and
alveoli could be effectively treated, prevented, and/or diagnosed using the
polynucleotides or polypeptides, and/or agonists or antagonists of the
invention.
Also, the polynucleotides or polypeptides, and/or agonists or antagonists of
the
invention, could be used to stimulate the proliferation of and differentiation
of type II
pneumocytes, which may help treat or prevent disease such as hyaline membrane
diseases, such as infant respiratory distress syndrome and bronchopulmonary
displasia, in premature infants.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could stimulate the proliferation and differentiation of
hepatocytes and,
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thus, could be used to alleviate or treat liver diseases and pathologies such
as
fulminant liver failure caused by cirrhosis, liver damage caused by viral
hepatitis and
toxic substances (i.e., acetaminophen, carbon tetraholoride and other
hepatotoxins
known in the art).
In addition, the polynucleotides or polypeptides, and/or agonists or
antagonists
of the invention, could be used treat or prevent the onset of diabetes
mellitus. In
patients with newly diagnosed Types I and II diabetes, where some islet cell
function
remains, the polynucleotides or polypeptides, and/or agonists or antagonists
of the
invention, could be used to maintain the islet function so as to alleviate,
delay or
prevent permanent manifestation of the disease. Also, the polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, could be used
as an
auxiliary in islet cell transplantation to improve or promote islet cell
function.
Neurological Diseases
Nervous system diseases, disorders, and/or conditions, which can be treated,
prevented, and/or diagnosed with the compositions of the invention (e.g.,
polypeptides, polynucleotides, and/or agonists or antagonists), include, but
are not
limited to, nervous system injuries, and diseases, disorders, and/or
conditions which
result in either a disconnection of axons, a diminution or degeneration of
neurons, or
demyelination. Nervous system lesions which may be treated, prevented, and/or
diagnosed in a patient (including human and non-human mammalian patients)
according to the invention, include but are not limited to, the following
lesions of
either the central (including spinal cord, brain) or peripheral nervous
systems: (I)
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; (2) traumatic lesions, including lesions caused by
physical
injury or associated with surgery, for example, lesions which sever a portion
of the
nervous system, or compression injuries; (3) malignant lesions, in which a
portion of
the nervous system is destroyed or injured by malignant tissue which is either
a
nervous system associated malignancy or a malignancy derived from non-nervous
system tissue; (4) infectious lesions, in which a portion of the nervous
system is
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destroyed or 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; (5) 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 associated with Parkinson's
disease,
Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis
(ALS); (6)
lesions associated with nutritional diseases, disorders, and/or conditions, in
which a
portion of the nervous system is destroyed or injured by a nutritional
disorder or
disorder of metabolism including but not limited to, vitamin B12 deficiency,
folic
acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-
Bignami
disease (primary degeneration of the corpus callosum), and alcoholic
cerebellar
degeneration; (7) neurological lesions associated with systemic diseases
including,
but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic
lupus
erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic
substances
including alcohol, lead, or particular neurotoxins; and (9) 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,
progressive
multifocal leukoencephalopathy, and central pontine myelinolysis.
In a preferred embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to protect neural cells from the
damaging effects
of cerebral hypoxia. According to this embodiment, the compositions of the
invention are used to treat, prevent, and/or diagnose neural cell injury
associated with
cerebral hypoxia. In one aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are used to
treat, prevent,
and/or diagnose neural cell injury associated with cerebral ischemia. In
another
aspect of this embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or diagnose
neural cell
injury associated with cerebral infarction. In another aspect of this
embodiment, the
polypeptides, polynucleotides, or agonists or antagonists of the invention are
used to
treat, prevent, and/or diagnose or prevent neural cell injury associated with
a stroke.
In a further aspect of this embodiment, the polypeptides, polynucleotides, or
agonists
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or antagonists of the invention are used to treat, prevent, and/or diagnose
neural cell
injury associated with a heart attack.
The compositions of the invention which are useful for treating or preventing
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, compositions of the invention which elicit any of the following
effects may
be useful according to the invention: (1) increased survival time of neurons
in culture;
(2) increased sprouting of neurons in culture or in vivo; (3) increased
production of a
neuron-associated molecule in culture or in vivo, e.g., choline
acetyltransferase or
acetylcholinesterase with respect to motor neurons; or (4) 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
routinely be measured using a method set forth herein or otherwise known in
the art,
such as, for example, the method set forth in Arakawa et al. (J. Neurosci.
10:3507-3515 (1990)); increased sprouting of neurons may be detected by
methods
known in the art, such as, for example, the methods set forth in Pestronk et
al. (Exp.
Neurol. 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));
increased production of neuron-associated molecules may be measured by
bioassay,
enzymatic assay, antibody binding, Northern blot assay, etc., using techniques
known
in the art and 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 diseases, disorders, and/or conditions
that may be treated, prevented, and/or diagnosed according to the invention
include,
but are not limited to, diseases, disorders, and/or conditions 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 diseases, disorders, and/or conditions 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-
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Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Further, polypeptides or polynucleotides of the invention may play a role in
neuronal survival; synapse formation; conductance; neural differentiation,
etc. Thus,
compositions of the invention (including polynucleotides, polypeptides, and
agonists
or antagonists) may be used to diagnose and/or treat or prevent diseases or
disorders
associated with these roles, including, but not limited to, learning and/or
cognition
disorders. The compositions of the invention may also be useful in the
treatment or
prevention of neurodegenerative disease states and/or behavioural disorders.
Such
neurodegenerative disease states and/or behavioral disorders include, but are
not
limited to, Alzheimers Disease, Parkinsons Disease, Huntingtons Disease,
Tourette
Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder,
panic disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception. In
addition,
compositions of the invention may also play a role in the treatment,
prevention and/or
detection of developmental disorders associated with the developing embryo, or
sexually-linked disorders.
Additionally, polypeptides, polynucleotides and/or agonists or antagonists of
the invention, may be useful in protecting neural cells from diseases, damage,
disorders, or injury, associated with cerebrovascular disorders including, but
not
limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid
stenosis, or
Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral
anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,
cerebral artery
diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis,
sinus
thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or
subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral
ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or
vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct),
leukomalacia,
periventricular, and vascular headache (e.g., cluster headache or migraines).
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, for therapeutic purposes, for example,
to
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stimulate neurological cell proliferation and/or differentiation. Therefore,
polynucleotides, polypeptides, agonists and/or antagonists of the invention
may be
used to treat and/or detect neurologic diseases. Moreover, polynucleotides or
polypeptides, or agonists or antagonists of the invention, can be used as a
marker or
detector of a particular nervous system disease or disorder.
Examples of neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include brain diseases, such as metabolic brain diseases which includes
phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase
deficiency,
pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain
edema, brain neoplasms such as cerebellar neoplasms which include
infratentorial
neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar
diseases such as cerebellar ataxia which include spinocerebellar degeneration
such as
ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-
Joseph
Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as
infratentorial
neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis,
globoid cell
leukodystrophy, metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include cerebrovascular disorders (such as carotid artery diseases which
include
carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral
amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformations, cerebral artery diseases, cerebral embolism and
thrombosis such as carotid artery thrombosis, sinus thrombosis and
Wallenberg's
Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and
subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as
transient
cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar
insufficiency,
vascular dementia such as multi-infarct dementia, periventricular
leukomalacia,
vascular headache such as cluster headache and migraine.
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Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include dementia such as AIDS Dementia Complex, presenile dementia such as
Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as
Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such
as
mufti-infarct dementia, encephalitis which include encephalitis periaxialis,
viral
encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis
Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic
syndrome,
Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy such as
generalized
epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy
which
includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as
complex
partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-
traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua, and
Hallervorden-
Spatz Syndrome.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include hydrocephalus such as Dandy-Walker Syndrome and normal pressure
hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral
malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri
pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral
toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central
nervous
system infections such as AIDS Dementia Complex, Brain Abscess, subdural
empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine
Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and
cerebral
malaria.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include meningitis such as arachnoiditis, aseptic meningtitis such as viral
meningtitis
which includes lymphocytic choriomeningitis, Bacterial meningtitis which
includes
Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such
as
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Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal
tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural
effusion,
meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as
transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes
bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as
Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-
Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include central nervous system neoplasms such as brain neoplasms that include
cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle
neoplasms
such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial
neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural
neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral
sceloris which includes adrenoleukodystrophy, encephalitis periaxialis,
globoid cell
leukodystrophy, diffuse cerebral sclerosis such as metachromatic
leukodystrophy,
allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
progressive
multifocal leukoencephalopathy, multiple sclerosis, central pontine
myelinolysis,
transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue
Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord
diseases
such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular
atrophy
such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord
neoplasms
such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff Man Syndrome,
mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's
Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff
Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-
Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis
such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal
syndrome,
phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett
Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome,
nervous system abnormalities such as holoprosencephaly, neural tube defects
such as
anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,
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encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina
bifida cystica and spina bifida occulta.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include hereditary motor and sensory neuropathies which include Charcot-Marie
Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic
paraplegia,
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such
as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations
(such
as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde
amnesia,
apraxia, neurogenic bladder, cataplexy, communicative disorders such as
hearing
disorders that includes deafness, partial hearing loss, loudness recruitment
and
tinnitus, language disorders such as aphasia which include agraphia, anomia,
broca
aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language
development disorders, speech disorders such as aphasia which includes anomia,
1 S broca aphasia and Wernicke Aphasia, articulation disorders, communicative
disorders
such as speech disorders which include dysarthria, echolalia, mutism and
stuttering,
voice disorders such as aphonia and hoarseness, decerebrate state, delirium,
fasciculation, hallucinations, meningism, movement disorders such as angelman
syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia,
myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle
rigidity such
as stiff man syndrome, muscle spasticity, paralysis such as facial paralysis
which
includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such
as
diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external
ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic
Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia,
respiratory
paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders
such as
ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color
vision
defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders
such as
hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism,
spasm such as trismus, unconsciousness such as coma, persistent vegetative
state and
syncope and vertigo, neuromuscular diseases such as amyotonia congenita,
amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron
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disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie
Disease
and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy,
Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy,
Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic
Paraparesis
and Stiff Man Syndrome, peripheral nervous system diseases such as acrodynia,
amyloid neuropathies, autonomic nervous system diseases such as Adie's
Syndrome,
Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, Reflex
Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as
Acoustic Nerve Diseases such as Acoustic Neuroma which includes
Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-
Rosenthal Syndrome, ocular motility disorders which includes amblyopia,
nystagmus,
oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Homer's
Syndrome, Chronic Progressive External Ophthalmoplegia which includes Kearns
Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve
Paralysis,
Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic
Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,
Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating
Diseases
such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as
diabetic foot.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include nerve compression syndromes such as carpal tunnel syndrome, tarsal
tunnel
syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve
compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia,
facial
neuralgia and trigeminal neuralgia, neuritis such as experimental allergic
neuritis,
optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as
polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-
Marie
Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic
Paraplegia
and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies
which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome,
Sciatica, Gustatory Sweating and Tetany).
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Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present
invention can be used to treat, prevent, and/or diagnose infectious agents.
For
example, by increasing the immune response, particularly increasing the
proliferation
and differentiation of B and/or T cells, infectious diseases may be treated,
prevented,
and/or diagnosed. The immune response may be increased by either enhancing an
existing immune response, or by initiating a new immune response.
Alternatively,
polypeptide or polynucleotide and/or agonist or antagonist of the present
invention
may also directly inhibit the infectious agent, without necessarily eliciting
an immune
response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide
or
polypeptide and/or agonist or antagonist of the present invention. Examples of
viruses, include, but are not limited to Examples of viruses, include, but are
not
limited to the following DNA and RNA viruses and viral families: Arbovirus,
Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,
Caliciviridae,
Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae
(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes
Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma
virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as
Smallpox or
Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus),
and Togaviridae (e.g., Rubivirus). Viruses falling within these families can
cause a
variety of diseases or symptoms, including, but not limited to: arthritis,
bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),
Japanese B
encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma,
chickenpox,
hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold,
Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g.,
Kaposi's, warts),
and viremia. polynucleotides or polypeptides, or agonists or antagonists of
the
invention, can be used to treat, prevent, and/or diagnose any of these
symptoms or
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diseases. In specific embodiments, polynucleotides, polypeptides, or agonists
or
antagonists of the invention are used to treat, prevent, and/or diagnose:
meningitis,
Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific
embodiment polynucleotides, polypeptides, or agonists or antagonists of the
invention
are used to treat patients nonresponsive to one or more other commercially
available
hepatitis vaccines. In a further specific embodiment polynucleotides,
polypeptides, or
agonists or antagonists of the invention are used to treat, prevent, and/or
diagnose
AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can be treated, prevented, and/or diagnosed by a polynucleotide or
polypeptide
and/or agonist or antagonist of the present invention include, but not limited
to,
include, but not limited to, the following Gram-Negative and Gram-positive
bacteria
and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium,
Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae
(e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,
Borrelia
(e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter,
Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,
Enterotoxigenic
E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella,
Salmonella
(e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia),
Erysipelothrix,
Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,
Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter,
Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections
(e.g.,
Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B),
Pasteurella),
Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,
Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g.,
Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal
families can cause the following diseases or symptoms, including, but not
limited to:
bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,
uveitis),
gingivitis, opportunistic infections (e.g., AIDS related infections),
paronychia,
prosthesis-related infections, Reiter's Disease, respiratory tract infections,
such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease,
Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,
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meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria,
Leprosy,
Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases
(e.g.,
cellulitis, dermatocycoses), toxemia, urinary tract infections, wound
infections.
S Polynucleotides or polypeptides, agonists or antagonists of the invention,
can be used
to treat, prevent, and/or diagnose any of these symptoms or diseases. In
specific
embodiments, polynucleotides, polypeptides, agonists or antagonists of the
invention
are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism,
and/or
meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated,
prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist
or
antagonist of the present invention include, but not limited to, the following
families
or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis,
Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,
Theileriasis,
Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,
Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium
ovate). These parasites can cause a variety of diseases or symptoms,
including, but
not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease
(e.g.,
dysentery, giardiasis), liver disease, lung disease, opportunistic infections
(e.g., AIDS
related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides
or
polypeptides, or agonists or antagonists of the invention, can be used
totreat, prevent,
and/or diagnose any of these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the invention are
used to
treat, prevent, and/or diagnose malaria.
Preferably, treatment or prevention using a polypeptide or polynucleotide
and/or agonist or antagonist of the present invention could either be by
administering
an effective amount of a polypeptide to the patient, or by removing cells from
the
patient, supplying the cells with a polynucleotide of the present invention,
and
returning the engineered cells to the patient (ex vivo therapy). Moreover, the
polypeptide or polynucleotide of the present invention can be used as an
antigen in a
vaccine to raise an immune response against infectious disease.
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Regeneration
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention can be used to differentiate, proliferate, and attract cells,
leading to the
regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of
tissues
could be used to repair, replace, or protect tissue damaged by congenital
defects,
trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery, including
cosmetic plastic
surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
or cardiac), vasculature (including vascular and lymphatics), nervous,
hematopoietic,
and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration
occurs without or decreased scarring. Regeneration also may include
angiogenesis.
Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention may increase regeneration of tissues difficult to heal. For
example,
increased tendon/ligament regeneration would quicken recovery time after
damage.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention
could also be used prophylactically in an effort to avoid damage. Specific
diseases
that could be treated, prevented, and/or diagnosed include of tendinitis,
carpal tunnel
syndrome, and other tendon or ligament defects. A further example of tissue
regeneration of non-healing wounds includes pressure ulcers, ulcers associated
with
vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide or polypeptide and/or agonist or antagonist of the present
invention to
proliferate and differentiate nerve cells. Diseases that could be treated,
prevented,
and/or diagnosed using this method include central and peripheral nervous
system
diseases, neuropathies, or mechanical and traumatic diseases, disorders,
and/or
conditions (e.g., spinal cord disorders, head trauma, cerebrovascular disease,
and
stoke). Specifically, diseases associated with peripheral nerve injuries,
peripheral
neuropathy (e.g., resulting from chemotherapy or other medical therapies),
localized
neuropathies, and central nervous system diseases (e.g., Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-
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Drager syndrome), could all be treated, prevented, and/or diagnosed using the
polynucleotide or polypeptide and/or agonist or antagonist of the present
invention.
Chemotaxis
S A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes
cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils,
epithelial and/or endothelial cells) to a particular site in the body, such as
inflammation, infection, or site of hyperproliferation. The mobilized cells
can then
fight off and/or heal the particular trauma or abnormality.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may increase chemotaxic activity of particular cells. These
chemotactic
molecules can then be used to treat, prevent, andlor diagnose inflammation,
infection,
hyperproliferative diseases, disorders, and/or conditions, or any immune
system
disorder by increasing the number of cells targeted to a particular location
in the body.
For example, chemotaxic molecules can be used to treat, prevent, and/or
diagnose
wounds and other trauma to tissues by attracting immune cells to the injured
location.
Chemotactic molecules of the present invention can also attract fibroblasts,
which can
be used to treat, prevent, and/or diagnose wounds.
It is also contemplated that a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention may inhibit chemotactic activity. These
molecules
could also be used totreat, prevent, and/or diagnose diseases, disorders,
and/or
conditions. .Thus, a polynucleotide or polypeptide and/or agonist or
antagonist of the
present invention could be used as an inhibitor of chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules
that bind to the polypeptide or for molecules to which the polypeptide binds.
The
binding of the polypeptide and the molecule may activate (agonist), increase,
inhibit
(antagonist), or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleotides, proteins (e.g.,
receptors),or
small molecules.
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Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand,
a structural
or functional mimetic. (See, Coligan et al., Current Protocols in Immunology
1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the
natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide, either as a secreted protein or on the
cell
membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli.
Cells expressing the polypeptide (or cell membrane containing the expressed
polypeptide) are then preferably contacted with a test compound potentially
containing the molecule to observe binding, stimulation, or inhibition of
activity of
either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the
polypeptide, wherein binding is detected by a label, or in an assay involving
competition with a labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The
antibody can measure polypeptide level or activity by either binding, directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
Additionally, the receptor to which a polypeptide of the invention binds can
be
identified by numerous methods known to those of skill in the art, for
example, ligand
panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1 (2),
Chapter 5, (1991)). For example, expression cloning is employed wherein
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polyadenylated RNA is prepared from a cell responsive to the polypeptides, for
example, NIH3T3 cells which are known to contain multiple receptors for the
FGF
family proteins, and SC-3 cells, and a cDNA library created from this RNA is
divided
into pools and used to transfect COS cells or other cells that are not
responsive to the
polypeptides. Transfected cells which are grown on glass slides are exposed to
the
polypeptide of the present invention, after they have been labelled. The
polypeptides
can be labeled by a variety of means including iodination or inclusion of a
recognition
site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-
radiographic analysis. Positive pools are identified and sub-pools are
prepared and re-
transfected using an iterative sub-pooling and re-screening process,
eventually
yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffinity linked with cell membrane or extract preparations that
express
the receptor molecule. Cross-linked material is resolved by PAGE analysis and
exposed to X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and subjected to
protein
microsequencing. The amino acid sequence obtained from microsequencing would
be used to design a set of degenerate oligonucleotide probes to screen a cDNA
library
to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA shuffling") may be
employed to modulate the activities of polypeptides of the invention thereby
effectively generating agonists and antagonists of polypeptides of the
invention. See
generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and
5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33
(1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques
24(2):308-13 (1998) (each of these patents and publications are hereby
incorporated
by reference). In one embodiment, alteration of polynucleotides and
corresponding
polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments into a desired
polynucleotide
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sequence of the invention molecule by homologous, or site-specific,
recombination.
In another embodiment, polynucleotides and corresponding polypeptides of the
invention may be alterred by being subjected to random mutagenesis by error-
prone
PCR, random nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections, parts, domains,
fragments, etc., of the polypeptides of the invention may be recombined with
one or
more components, motifs, sections, parts, domains, fragments, etc. of one or
more
heterologous molecules. In preferred embodiments, the heterologous molecules
are
family members. In further preferred embodiments, the heterologous molecule is
a
growth factor such as, for example, platelet-derived growth factor (PDGF),
insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha,
epidermal
growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic
protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B,
decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors
(GDFs),
nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-betas, and
glial-
derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the
polypeptides of the invention. Biologically active fragments are those
exhibiting
activity similar, but not necessarily identical, to an activity of the
polypeptide. The
biological activity of the fragments may include an improved desired activity,
or a
decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to
identify those which modulate the action of the polypeptide of the present
invention.
An example of such an assay comprises combining a mammalian fibroblast cell, a
the
polypeptide of the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell would
normally
proliferate. A control assay may be performed in the absence of the compound
to be
screened and compared to the amount of fibroblast proliferation in the
presence of the
compound to determine if the compound stimulates proliferation by determining
the
uptake of 3[H] thymidine in each case. The amount of fibroblast cell
proliferation is
measured by liquid scintillation chromatography which measures the
incorporation of
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3[H] thymidine. Both agonist and antagonist compounds may be identified by
this
procedure.
In another method, a mammalian cell or membrane preparation expressing a
receptor for a polypeptide of the present invention is incubated with a
labeled
polypeptide of the present invention in the presence of the compound. The
ability of
the compound to enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system following
interaction of a compound to be screened and the receptor is measured and the
ability
of the compound to bind to the receptor and elicit a second messenger response
is
measured to determine if the compound is a potential agonist or antagonist.
Such
second messenger systems include but are not limited to, cAMP guanylate
cyclase,
ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers.
The molecules discovered using these assays can be used to treat, prevent,
and/or
diagnose disease or to bring about a particular result in a patient (e.g.,
blood vessel
growth) by activating or inhibiting the polypeptide/molecule. Moreover, the
assays
can discover agents which may inhibit or enhance the production of the
polypeptides
of the invention from suitably manipulated cells or tissues. Therefore, the
invention
includes a method of identifying compounds which bind to the polypeptides of
the
invention comprising the steps o~ (a) incubating a candidate binding compound
with
the polypeptide; and (b) determining if binding has occurred. Moreover, the
invention includes a method of identifying agonists/antagonists comprising the
steps
o~ (a) incubating a candidate compound with the polypeptide, (b) assaying a
biological activity , and (b) determining if a biological activity of the
polypeptide has
been altered.
Also, one could identify molecules bind a polypeptide of the invention
experimentally by using the beta-pleated sheet regions contained in the
polypeptide
sequence of the protein. Accordingly, specific embodiments of the invention
are
directed to polynucleotides encoding polypeptides which comprise, or
alternatively
consist of, the amino acid sequence of each beta pleated sheet regions in a
disclosed
polypeptide sequence. Additional embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or alternatively consist
of,
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any combination or all of contained in the polypeptide sequences of the
invention.
Additional preferred embodiments of the invention are directed to polypeptides
which
comprise, or alternatively consist of, the amino acid sequence of each of the
beta
pleated sheet regions in one of the polypeptide sequences of the invention.
Additional
embodiments of the invention are directed to polypeptides which comprise, or
alternatively consist of, any combination or all of the beta pleated sheet
regions in one
of the polypeptide sequences of the invention.
Targeted Delivery
In another embodiment, the invention provides a method of delivering
compositions to targeted cells expressing a receptor for a polypeptide of the
invention,
or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be
associated with heterologous polypeptides, heterologous nucleic acids, toxins,
or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In
one
embodiment, the invention provides a method for the specific delivery of
compositions of the invention to cells by administering polypeptides of the
invention
(including antibodies) that are associated with heterologous polypeptides or
nucleic
acids. In one example, the invention provides a method for delivering a
therapeutic
protein into the targeted cell. In another example, the invention provides a
method for
delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or
double
stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or
replicate
episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention (e.g., polypeptides of the invention or antibodies of the
invention) in
association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic
effector systems, radioisotopes, holotoxins, modified toxins, catalytic
subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell
that under defined conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not limited to,
radioisotopes
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known in the art, compounds such as, for example, antibodies (or complement
fixing
containing portions thereof) that bind an inherent or induced endogenous
cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin,
abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed
antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is
meant a
non-toxic compound that is converted by an enzyme, normally present in the
cell, into
a cytotoxic compound. Cytotoxic prodrugs that may be used according to the
methods of the invention include, but are not limited to, glutamyl derivatives
of
benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or
mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives
of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention,
or
the polynucleotides encoding these polypeptides, to screen for molecules which
modify the activities of the polypeptides of the present invention. Such a
method
would include contacting the polypeptide of the present invention with a
selected
compounds) suspected of having antagonist or agonist activity, and assaying
the
activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by
using the polypeptides of the present invention, or binding fragments thereof,
in any
of a variety of drug screening techniques. The polypeptide or fragment
employed in
such a test may be affixed to a solid support, expressed on a cell surface,
free in
solution, 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. One may measure, for example, the
formulation
of complexes between the agent being tested and a polypeptide of the present
invention.
Thus, the present invention provides methods of screening for drugs or any
other agents which affect activities mediated by the polypeptides of the
present
invention. These methods comprise contacting such an agent with a polypeptide
of the
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present invention or a fragment thereof and assaying for the presence of a
complex
between the agent and the polypeptide or a fragment thereof, by methods well
known
in the art. In such a competitive binding assay, the agents to screen are
typically
labeled. Following incubation, free agent is separated from that present in
bound
S form, and the amount of free or uncomplexed label is a measure of the
ability of a
particular agent to bind to the polypeptides of the present invention.
Another technique for drug screening provides high throughput screening for
compounds having suitable binding affinity to the polypeptides of the present
invention, and is described in great detail in European Patent Application
84/03564,
published on September 13, 1984, which is incorporated herein by reference
herein.
Briefly stated, large numbers of different small peptide test compounds are
synthesized on a solid substrate, such as plastic pins or some other surface.
The
peptide test compounds are reacted with polypeptides of the present invention
and
washed. Bound polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in the
aforementioned
drug screening techniques. In addition, non-neutralizing antibodies may be
used to
capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing antibodies capable of binding polypeptides of the
present
invention specifically compete with a test compound for binding to the
polypeptides
or fragments thereof. In this manner, the antibodies are used to detect the
presence of
any peptide which shares one or more antigenic epitopes with a polypeptide of
the
invention.
Polvpeptides of the Invention Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying
polypeptides and nonpolypeptides that bind polypeptides of the invention, and
the
polypeptide of the invention binding molecules identified thereby. These
binding
molecules are useful, for example, as agonists and antagonists of the
polypeptides of
the invention. Such agonists and antagonists can be used, in accordance with
the
invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:
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a. contacting a polypeptide of the invention with a plurality of molecules;
and
b. identifying a molecule that binds the polypeptide of the invention.
The step of contacting the polypeptide of the invention with the plurality of
molecules may be effected in a number of ways. For example, one may
contemplate
immobilizing the polypeptide of the invention on a solid support and bringing
a
solution of the plurality of molecules in contact with the immobilized
polypeptide of
the invention. Such a procedure would be akin to an affinity chromatographic
process,
with the affinity matrix being comprised of the immobilized polypeptide of the
invention. The molecules having a selective affinity for the polypeptide of
the
invention can then be purified by affinity selection. The nature of the solid
support,
process for attachment of the polypeptide of the invention to the solid
support,
solvent, and conditions of the affinity isolation or selection are largely
conventional
and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into
1 S substantially separate fractions comprising a subset of or individual
polypeptides. For
instance, one can separate the plurality of polypeptides by gel
electrophoresis, column
chromatography, or like method known to those of ordinary skill for the
separation of
polypeptides. The individual polypeptides can also be produced by a
transformed host
cell in such a way as to be expressed on or about its outer surface (e.g., a
recombinant
phage). Individual isolates can then be "probed" by the polypeptide of the
invention,
optionally in the presence of an inducer should one be required for
expression, to
determine if any selective affinity interaction takes place between the
polypeptide of
the invention and the individual clone. Prior to contacting the polypeptide of
the
invention with each fraction comprising individual polypeptides, the
polypeptides
could first be transferred to a solid support for additional convenience. Such
a solid
support may simply be a piece of filter membrane, such as one made of
nitrocellulose
or nylon. In this manner, positive clones could be identified from a
collection of
transformed host cells of an expression library, which harbor a DNA construct
encoding a polypeptide having a selective affinity for a polypeptide of the
invention.
Furthermore, the amino acid sequence of the polypeptide having a selective
affinity
for the polypeptide of the invention can be determined directly by
conventional means
or the coding sequence of the DNA encoding the polypeptide can frequently be
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determined more conveniently. The primary sequence can then be deduced from
the
corresponding DNA sequence. If the amino acid sequence is to be determined
from
the polypeptide itself, one may use microsequencing techniques. The sequencing
technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound
polypeptide of the invention, or alterntatively, unbound polypeptides, from a
mixture
of the polypeptide of the invention and the plurality of polypeptides prior to
attempting to determine or to detect the presence of a selective affinity
interaction.
Such a wash step may be particularly desirable when the polypeptide of the
invention
or the plurality of polypeptides is bound to a solid support.
The plurality of molecules provided according to this method may be provided
by way of diversity libraries, such as random or combinatorial peptide or
nonpeptide
libraries which can be screened for molecules that specifically bind to a
polypeptide
of the invention. Many libraries are known in the art that can be used, e.g.,
chemically
synthesized libraries, recombinant (e.g., phage display libraries), and in
vitro
translation-based libraries. Examples of chemically synthesized libraries are
described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991,
Nature
354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology
12:709-710;Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
Ohlmeyer
et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994,
Proc. Natl.
Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et
al.,
1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO
93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-
5383.
Examples of phage display libraries are described in Scott and Smith, 1990,
Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.
B., et
al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth.
152:149-157;
Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated
Aug.
18, 1994.
In vitro translation-based libraries include but are not limited to those
described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and
Mattheakis
et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.
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By way of examples of nonpeptide libraries, a benzodiazepine library (see
e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be
adapted for
use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-
9371)
can also be used. Another example of a library that can be used, in which the
amide
functionalities in peptides have been permethylated to generate a chemically
transformed combinatorial library, is described by Ostresh et al. (1994, Proc.
Natl.
Acad. Sci. USA 91:11138-11142).
The variety of non-peptide libraries that are useful in the present invention
is
great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list
benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-
mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes,
xanthines,
aminimides, and oxazolones as among the chemical species that form the basis
of
various libraries.
Non-peptide libraries can be classified broadly into two types: decorated
monomers and oligomers. Decorated monomer libraries employ a relatively simple
scaffold structure upon which a variety functional groups is added. Often the
scaffold
will be a molecule with a known useful pharmacological activity. For example,
the
scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are
assembled together in ways that create new shapes that depend on the order of
the
monomers. Among the monomer units that have been used are carbamates,
pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the
side
chain is attached to the alpha amino group rather than the alpha carbon, form
the basis
of another version of non-peptide oligomer libraries. The first non-peptide
oligomer
libraries utilized a single type of monomer and thus contained a repeating
backbone.
Recent libraries have utilized more than one monomer, giving the libraries
added
flexibility.
Screening the libraries can be accomplished by any of a variety of commonly
known methods. See, e.g., the following references, which disclose screening
of
peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218;
Scott
and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques
13:422-
427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et
al.,
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1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al.,
1992,
Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-
6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat.
No.
5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo,
1993,
Science 263:671-673; and CT Publication No. WO 94/18318.
In a specific embodiment, screening to identify a molecule that binds a
polypeptide of the invention can be carried out by contacting the library
members
with a polypeptide of the invention immobilized on a solid phase and
harvesting those
library members that bind to the polypeptide of the invention. Examples of
such
screening methods, termed "panning" techniques are described by way of example
in
Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques
13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting
proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al.,
1991,
Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules
that
specifically bind to a polypeptide of the invention.
Where the polypeptide of the invention binding molecule is a polypeptide, the
polypeptide can be conveniently selected from any peptide library, including
random
peptide libraries, combinatorial peptide libraries, or biased peptide
libraries. The term
"biased" is used herein to mean that the method of generating the library is
manipulated so as to restrict one or more parameters that govern the diversity
of the
resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides
in
which the probability of finding a particular amino acid at a given position
of the
peptide is the same for all 20 amino acids. A bias can be introduced into the
library,
however, by specifying, for example, that a lysine occur every fifth amino
acid or that
positions 4, 8, and 9 of a decapeptide library be fixed to include only
arginine.
Clearly, many types of biases can be contemplated, and the present invention
is not
restricted to any particular bias. Furthermore, the present invention
contemplates
specific types of peptide libraries, such as phage displayed peptide libraries
and those
that utilize a DNA construct comprising a lambda phage vector with a DNA
insert.
As mentioned above, in the case of a polypeptide of the invention binding
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molecule that is a polypeptide, the polypeptide may have about 6 to less than
about 60
amino acid residues, preferably about 6 to about 10 amino acid residues, and
most
preferably, about 6 to about 22 amino acids. In another embodiment, a
polypeptide of
the invention binding polypeptide has in the range of 15-100 amino acids, or
20-50
amino acids.
The selected polypeptide of the invention binding polypeptide can be obtained
by chemical synthesis or recombinant expression.
Antisense And Ribozyme (Antagonists)
In specific embodiments, antagonists according to the present invention are
nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the
complementary strand thereof, and/or to nucleotide sequences contained a
deposited
clone. In one embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is separately
administered
(see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides
as
Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
Antisense technology can be used to control gene expression through antisense
DNA
or RNA, or through triple-helix formation. Antisense techniques are discussed
for
example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix
formation is discussed in, for instance, Lee et al., Nucleic Acids Research,
6:3073
(1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science,
251:1300
(1991). The methods are based on binding of a polynucleotide to a
complementary
DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit
the growth of the non-lymphocytic leukemia cell line HL-60 and other cell
lines was
previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These
experiments were performed in vitro by incubating cells with the
oligoribonucleotide.
A similar procedure for in vivo use is described in WO 91/15580. Briefly, a
pair of
oligonucleotides for a given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is flanked by an
EcoRl
site on the 5 end and a HindIII site on the 3 end. Next, the pair of
oligonucleotides is
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heated at 90°C for one minute and then annealed in 2X ligation buffer
(20mM TRIS
HCl pH 7.5, IOmM MgCl2, IOMM dithiothreitol (DTT) and 0.2 mM ATP) and then
ligated to the EcoRl/Hind III site of the retroviral vector PMV7 (WO
91/15580).
For example, the S' coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide
is designed to be complementary to a region of the gene involved in
transcription
thereby preventing transcription and the production of the receptor. The
antisense
RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of
the
mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced
intracellularly by transcription from an exogenous sequence. For example, a
vector or
a portion thereof, is transcribed, producing an antisense nucleic acid (RNA)
of the
invention. Such a vector would contain a sequence encoding the antisense
nucleic
1 S acid of the invention. Such a vector can remain episomal or become
chromosomally
integrated, as long as it can be transcribed to produce the desired antisense
RNA.
Such vectors can be constructed by recombinant DNA technology methods standard
in the art. Vectors can be plasmid, viral, or others known in the art, used
for
replication and expression in vertebrate cells. Expression of the sequence
encoding a
polypeptide of the invention, or fragments thereof, can be by any promoter
known in
the art to act in vertebrate, preferably human cells. Such promoters can be
inducible
or constitutive. Such promoters include, but are not limited to, the SV40
early
promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et
al.,
Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc.
Natl.
Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a gene of
interest.
However, absolute complementarity, although preferred, is not required. A
sequence
"complementary to at least a portion of an RNA," referred to herein, means a
sequence having sufficient complementarity to be able to hybridize with the
RNA,
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forming a stable duplex; in the case of double stranded antisense nucleic
acids of the
invention, a single strand of the duplex DNA may thus be tested, or triplex
formation
may be assayed. The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid Generally, the
larger the
hybridizing nucleic acid, the more base mismatches with a RNA sequence of the
invention it may contain and still form a stable duplex (or triplex as the
case may be).
One skilled in the art can ascertain a tolerable degree of mismatch by use of
standard
procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the message, e.g.,
the 5' untranslated sequence up to and including the AUG initiation codon,
should
work most efficiently at inhibiting translation. However, sequences
complementary
to the 3' untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature,
372:333-335 (1994). Thus, oligonucleotides complementary to either the 5' - or
3'
non- translated, non-coding regions of a polynucleotide sequence of the
invention
could be used in an antisense approach to inhibit translation of endogenous
mRNA.
Oligonucleotides complementary to the 5' untranslated region of the mRNA
should
include the complement of the AUG start codon. Antisense oligonucleotides
complementary to mRNA coding regions are less efficient inhibitors of
translation but
could be used in accordance with the invention. Whether designed to hybridize
to the
5' -, 3' - or coding region of mRNA, antisense nucleic acids should be at
least six
nucleotides in length, and are preferably oligonucleotides ranging from 6 to
about 50
nucleotides in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety, or
phosphate backbone, for example, to improve stability of the molecule,
hybridization,
etc. 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., Proc. Natl. Acad. Sci. U.S.A. 86:6553-
6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT
Publication
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NO: W088/09810, published December 15, 1988) or the blood-brain barrier (see,
e.g., PCT Publication NO: W089/10134, published April 25, 1988), hybridization-
triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976
(1988))
or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To
this end,
the oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including, but not limited to, 5-
fluorouracil,
5-bromouracil, S-chlorouracil, S-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
S-methylcytosine, N6-adenine, 7-methylguanine, 5-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.
The antisense oligonucleotide may also comprise at least one modified sugar
moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one modified phosphate backbone selected from the group including, but not
limited
to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
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hybrids with complementary RNA in which, contrary to the usual b-units, the
strands
run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641
(1987)).
The oligonucleotide is a 2-0-methylribonucleotide (moue et al., Nucl. Acids
Res.,
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (moue et al., FEBS Lett.
215:327-330 (1987)).
Polynucleotides of the invention may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such as are
commercially available from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides may be synthesized by the method of Stein et
al.
(Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports (Sarin et al., Proc.
Natl.
Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.
While antisense nucleotides complementary to the coding region sequence of
the invention could be used, those complementary to the transcribed
untranslated
region are most preferred.
Potential antagonists according to the invention also include catalytic RNA,
or
a ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes
that
cleave mRNA at site specific recognition sequences can be used to destroy
mRNAs
corresponding to the polynucleotides of the invention, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated
by flanking regions that form complementary base pairs with the target mRNA.
The
sole requirement is that the target mRNA have the following sequence of two
bases:
5' -UG-3' . The construction and production of hammerhead ribozymes is well
known in the art and is described more fully in Haseloff and Gerlach, Nature,
334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage
sites within each nucleotide sequence disclosed in the sequence listing.
Preferably,
the ribozyme is engineered so that the cleavage recognition site is located
near the 5'
end of the mRNA corresponding to the polynucleotides of the invention; i.e.,
to
increase efficiency and minimize the intracellular accumulation of non-
functional
mRNA transcripts.
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As in the antisense approach, the ribozymes of the invention can be composed
of modified oligonucleotides (e.g. for improved stability, targeting, etc.)
and should
be delivered to cells which express the polynucleotides of the invention in
vivo.
DNA constructs encoding the ribozyme may be introduced into the cell in the
same
manner as described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct "encoding" the
ribozyme under the control of a strong constitutive promoter, such as, for
example,
pol III or pol II promoter, so that transfected cells will produce sufficient
quantities of
the ribozyme to destroy endogenous messages and inhibit translation. Since
ribozymes unlike antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth
and proliferation effects of the polypeptides of the present invention on
neoplastic
cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore,
retard or
prevent abnormal cellular growth and proliferation, for example, in tumor
formation
or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular
diseases, and prevent the proliferation of epithelial lens cells after
extracapsular
cataract surgery. Prevention of the mitogenic activity of the polypeptides of
the
present invention may also be desirous in cases such as restenosis after
balloon
angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar
tissue during wound healing.
The antagonist/agonist may also be employed to treat, prevent, and/or
diagnose the diseases described herein.
Thus, the invention provides a method of treating or preventing diseases,
disorders, and/or conditions, including but not limited to the diseases,
disorders,
and/or conditions listed throughout this application, associated with
overexpression of
a polynucleotide of the present invention by administering to a patient (a) an
antisense
molecule directed to the polynucleotide of the present invention, and/or (b) a
ribozyme directed to the polynucleotide of the present invention.
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invention, and/or (b) a ribozyme directed to the polynucleotide of the present
invention
Other Activities
The polypeptide of the present invention, as a result of the ability to
stimulate
vascular endothelial cell growth, may be employed in treatment for stimulating
re
vascularization of ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions. These
polypeptide
may also be employed to stimulate angiogenesis and limb regeneration, as
discussed
above.
The polypeptide may also be employed for treating wounds due to injuries,
burns, post-operative tissue repair, and ulcers since they are mitogenic to
various cells
of different origins, such as fibroblast cells and skeletal muscle cells, and
therefore,
facilitate the repair or replacement of damaged or diseased tissue.
The polypeptide of the present invention may also be employed stimulate
neuronal growth and to treat, prevent, and/or diagnose neuronal damage which
occurs
in certain neuronal disorders or neuro-degenerative conditions such as
Alzheimer's
disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the
invention may have the ability to stimulate chondrocyte growth, therefore,
they may
be employed to enhance bone and periodontal regeneration and aid in tissue
transplants or bone grafts.
The polypeptide of the present invention may be also be employed to prevent
skin aging due to sunburn by stimulating keratinocyte growth.
The polypeptide of the invention may also be employed for preventing hair
loss, since FGF family members activate hair-forming cells and promotes
melanocyte
growth. Along the same lines, the polypeptides of the present invention may be
employed to stimulate growth and differentiation of hematopoietic cells and
bone
marrow cells when used in combination with other cytokines.
The polypeptide of the invention may also be employed to maintain organs
before transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing
tissue of mesodermal origin to differentiate in early embryos.
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The polypeptide or polynucleotides and/or agonist or antagonists of the
present invention may also increase or decrease the differentiation or
proliferation of
embryonic stem cells, besides, as discussed above, hematopoietic lineage.
The polypeptide or polynucleotides and/or agonist or antagonists of the
present invention may also be used to modulate mammalian characteristics, such
as
body height, weight, hair color, eye color, skin, percentage of adipose
tissue,
pigmentation, size, and shape (e.g., cosmetic surgery). Similarly,
polypeptides or
polynucleotides and/or agonist or antagonists of the present invention may be
used to
modulate mammalian metabolism affecting catabolism, anabolism, processing,
utilization, and storage of energy.
Polypeptide or polynucleotides and/or agonist or antagonists of the present
invention may be used to change a mammal's mental state or physical state by
influencing biorhythms, caricadic rhythms, depression (including depressive
diseases,
disorders, and/or conditions), tendency for violence, tolerance for pain,
reproductive
capabilities (preferably by Activin or Inhibin-like activity), hormonal or
endocrine
levels, appetite, libido, memory, stress, or other cognitive qualities.
Polypeptide or polynucleotides and/or agonist or antagonists of the present
invention may also be used as a food additive or preservative, such as to
increase or
decrease storage capabilities, fat content, lipid, protein, carbohydrate,
vitamins,
minerals, cofactors or other nutritional components.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical to a sequence of at least about 50 contiguous nucleotides in the
nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of
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the Clone Sequence and ending with the nucleotide at about the position of the
3'
Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of
the Start Codon and ending with the nucleotide at about the position of the 3'
Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
Similarly preferred is a nucleic acid molecule wherein said sequence of
contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X
in the
range of positions beginning with the nucleotide at about the position of the
5'
Nucleotide of the First Amino Acid of the Signal Peptide and ending with the
nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as
defined for SEQ 117 NO:X in Table 1.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 150
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide-
sequence which is at least 95% identical to a sequence of at least about 500
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide sequence which is at least 95% identical to the nucleotide sequence
of SEQ
ID NO:X beginning with the nucleotide at about the position of the 5'
Nucleotide of
the First Amino Acid of the Signal Peptide and ending with the nucleotide at
about
the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID
NO:X
in Table 1.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence of SEQ ID NO:X.
Also preferred is an isolated nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule, wherein said
nucleic
acid molecule which hybridizes does not hybridize under stringent
hybridization
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conditions to a nucleic acid molecule having a nucleotide sequence consisting
of only
A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which
comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1,
S which DNA molecule is contained in the material deposited with the American
Type
Culture Collection and given the ATCC Deposit Number shown in Table 1 for said
cDNA Clone Identifier.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in the nucleotide sequence of a human cDNA clone identified by a
cDNA
Clone Identifier in Table l, which DNA molecule is contained in the deposit
given the
ATCC Deposit Number shown in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of
at least SO contiguous nucleotides is included in the nucleotide sequence of
the
complete open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to sequence of at least 150
contiguous
nucleotides in the nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to sequence
of at
least 500 contiguous nucleotides in the nucleotide sequence encoded by said
human
cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is a method for detecting in a biological
sample a nucleic acid molecule comprising a nucleotide sequence which is at
least
95% identical to a sequence of at least SO contiguous nucleotides in a
sequence
selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table l; and a nucleotide sequence
encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
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1; which method comprises a step of comparing a nucleotide sequence of at
least one
nucleic acid molecule in said sample with a sequence selected from said group
and
determining whether the sequence of said nucleic acid molecule in said sample
is at
least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a nucleic acid molecule comprising said sequence
selected from said group. Similarly, also preferred is the above method
wherein said
step of comparing sequences is performed by comparing the nucleotide sequence
determined from a nucleic acid molecule in said sample with said sequence
selected
from said group. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
A further preferred embodiment is a method for identifying the species, tissue
or cell type of a biological sample which method comprises a step of detecting
nucleic
acid molecules in said sample, if any, comprising a nucleotide sequence that
is at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from the group consisting of a nucleotide sequence of SEQ m NO:X
wherein X is any integer as defined in Table 1; and a nucleotide sequence
encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
The method for identifying the species, tissue or cell type of a biological
sample can comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide sequences, wherein
at least
one sequence in said panel is at least 95% identical to a sequence of at least
50
contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein identified in Table l, which method comprises a step of detecting in a
biological sample obtained from said subject nucleic acid molecules, if any,
comprising a nucleotide sequence that is at least 95% identical to a sequence
of at
least 50 contiguous nucleotides in a sequence selected from the group
consisting of: a
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nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in
Table 1;
and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA
Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at
least two nucleotide sequences, wherein at least one sequence in said panel is
at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from said group.
Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise
a panel of at least two nucleotide sequences, wherein at least one sequence in
said
panel is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a
sequence selected from the group consisting of: a nucleotide sequence of SEQ
ID
NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about 10 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1.
Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in the amino acid sequence of SEQ ID NO:Y in the range of
positions beginning with the residue at about the position of the First Amino
Acid of
the Secreted Portion and ending with the residue at about the Last Amino Acid
of the
Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
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Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to a sequence of at least about 100 contiguous
amino
acids in the amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to the complete amino acid sequence of SEQ ID
NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 90% identical to a sequence of at least about 10 contiguous
amino
acids in the complete amino acid sequence of a secreted protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is a polypeptide wherein said sequence of contiguous amino
acids is included in the amino acid sequence of a secreted portion of the
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in
the amino acid sequence of the secreted portion of the protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table l and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to the amino acid sequence of the secreted portion of
the protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1.
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Further preferred is an isolated antibody which binds specifically to a
polypeptide comprising an amino acid sequence that is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table 1; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
Further preferred is a method for detecting in a biological sample a
polypeptide comprising an amino acid sequence which is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table l; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1; which method comprises a step of comparing an amino acid sequence of at
least
one polypeptide molecule in said sample with a sequence selected from said
group
and determining whether the sequence of said polypeptide molecule in said
sample is
at least 90% identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino
acid sequence of at least one polypeptide molecule in said sample with a
sequence
selected from said group comprises determining the extent of specific binding
of
polypeptides in said sample to an antibody which binds specifically to a
polypeptide
comprising an amino acid sequence that is at least 90% identical to a sequence
of at
least 10 contiguous amino acids in a sequence selected from the group
consisting of:
an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1; and a complete amino acid sequence of a protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is the above method wherein said step of comparing sequences
is performed by comparing the amino acid sequence determined from a
polypeptide
molecule in said sample with said sequence selected from said group.
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Also preferred is a method for identifying the species, tissue or cell type of
a
biological sample which method comprises a step of detecting polypeptide
molecules
in said sample, if any, comprising an amino acid sequence that is at least 90%
identical to a sequence of at least 10 contiguous amino acids in a sequence
selected
S from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein
Y is
any integer as defined in Table 1; and a complete amino acid sequence of a
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is the above method for identifying the species, tissue or cell
type of a biological sample, which method comprises a step of detecting
polypeptide
molecules comprising an amino acid sequence in a panel of at least two amino
acid
sequences, wherein at least one sequence in said panel is at least 90%
identical to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
above
group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein identified in Table 1, which method comprises a step of detecting in a
biological sample obtained from said subject polypeptide molecules comprising
an
amino acid sequence in a panel of at least two amino acid sequences, wherein
at least
one sequence in said panel is at least 90% identical to a sequence of at least
10
contiguous amino acids in a sequence selected from the group consisting of: an
amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
In any of these methods, the step of detecting said polypeptide molecules
includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence encoding a
polypeptide wherein said polypeptide comprises an amino acid sequence that is
at
least 90% identical to a sequence of at least 10 contiguous amino acids in a
sequence
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240
selected from the group consisting o~ an amino acid sequence of SEQ ID NO:Y
wherein Y is any integer as defined in Table 1; and a complete amino acid
sequence
of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone
Identifier in Table 1 and contained in the deposit with the ATCC Deposit
Number
shown for said cDNA clone in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide
sequence encoding a polypeptide has been optimized for expression of said
polypeptide in a prokaryotic host.
Also preferred is an isolated nucleic acid molecule, wherein said polypeptide
comprises an amino acid sequence selected from the group consisting of: an
amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Further preferred is a method of making a recombinant vector comprising
inserting any of the above isolated nucleic acid molecule into a vector. Also
preferred
is the recombinant vector produced by this method. Also preferred is a method
of
making a recombinant host cell comprising introducing the vector into a host
cell, as
well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising
culturing this recombinant host cell under conditions such that said
polypeptide is
expressed and recovering said polypeptide. Also preferred is this method of
making
an isolated polypeptide, wherein said recombinant host cell is a eukaryotic
cell and
said polypeptide is a secreted portion of a human secreted protein comprising
an
amino acid sequence selected from the group consisting of an amino acid
sequence of
SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid
of
the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table
1 and
said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y
is
defined in Table 1; and an amino acid sequence of a secreted portion of a
protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The isolated polypeptide produced by this method is also
preferred.
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Also preferred is a method of treatment of an individual in need of an
increased level of a secreted protein activity, which method comprises
administering
to such an individual a pharmaceutical composition comprising an amount of an
isolated polypeptide, polynucleotide, or antibody of the claimed invention
effective to
increase the level of said protein activity in said individual.
The above-recited applications have uses in a wide variety of hosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig, camel,
horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog,
cat, non-
human primate, and human. In specific embodiments, the host is a mouse,
rabbit,
goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred
embodiments, the host is a mammal. In most preferred embodiments, the host is
a
human.
In specific embodiments of the invention, for each "Contig ID" listed in the
fourth column of Table 5, preferably excluded are one or more polynucleotides
comprising, or alternatively consisting of, a nucleotide sequence referenced
in the
fifth column of Table 5 and described by the general formula of a-b, whereas a
and b
are uniquely determined for the corresponding SEQ ID NO:X referred to in
column 3
of Table 5. Further specific embodiments are directed to polynucleotide
sequences
excluding one, two, three, four, or more of the specific polynucleotide
sequences
referred to in the fifth column of Table 5. In no way is this listing meant to
encompass
all of the sequences which may be excluded by the general formula, it is just
a
representative example. All references available through these accessions are
hereby
incorporated by reference in their entirety.
TABLE 5
Gene cDNA CloneNT Contig Public Accession Numbers
ID
No. ID SEQ
ID
NO:
X
1 HDPAP35 11 1014385 AA127696, AI608902, AA121533,
AI819376,
AI690414, AI799320, AA037078,
AW207276,
AI190009, AI189908, AI733919,
AA292201,
AI186293, C18658, AI424335,
AI678841, 863655,
AI275846, AI192451, AI361211,
H89848, 827271,
AA399416, N26384, N42253, D63181,
H61437,
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H90695, T83206, 878533, 878465,
826741,
863550, 826975, AA373164, AI208396,
AI202996, H62437, AI568166,
828443, 825415,
830906, N32809, 827470, 863656,
AI826502,
863505, 830860, AA368815, and
AF177937.
1 HDPAP35 26 906806 AA292201, AI733919, AA399416,
and
AF177937.
1 HDPAP35 27 902330 AA127696, AI608902, AA121533,
AI819376,
AI424335, AA037078, AI690414,
AI799320,
AI678841, AI275846, AW207276,
AI190009,
AI189908, RG3655, C18658, AI186293,
AI192451,
AI361211, 827271, H89848, T83206,
N26384,
N42253, D63181, H61437, H90695,
878465,
878533, 826741, 863550, 826975,
AA373164,
AI208396, 828443, H62437, AI568166,
AI826502,
827470, 863656, 825415, AI202996,
830906,
N32809, 863505, and 830860.
1 HDPAP35 28 887675 AI733919, AA292201, AA399416,
AI202996,
AA373164, AA368815, and AF177937.
2 HAMFC93 12 900586 AI245433, 819672, T80223, F05628,
F06854,
F08386, 244059, 818499, and
AF055030.
2 HAMFC93 29 906819 AA522810, AA255643, AA425656,
AI588920,
AI650351, AW299625, AW338844,
AI916819,
AI763055, AI081567, AI081569,
AI478792,
AI056363, AI360130, W96031,
H29070,
AA192342, AI083572, AI081344,
AI089950,
AI572861, AI423467, AI401499,
AI261544,
AA806097, AI129885, AI003809,
AA427763,
AI768935, C06006, N80333, AW271475,
W90464,
846410, W67631, AA910461, AI682057,
W96045,
T75273, AI522122, W94664, AI277323,
AI580318,
AI687583, AA035340, AI366519,
H19055,
AI277502, AI924428, AI753937,
N71788,
AI674998, AA515661, 816164,
815316, 819672,
T31979, 816165, W94808, AW084413,
866672,
243164, 239246, AI696837, H19345,
815425,
AI890272, F06351, AA324890,
AA633574,
AI200277, AA987848, 244059,
N35552,
AI245433, AA293579, F06854,
815424, T80223,
W19999, 844459, AI290371, AI280283,
T05756,
C01791, F03118, AA992270, AI004847,
F12961,
AI979311, F02641, 854961, X97515,
F10559,
865758, 818499, AI468682, 839178,
AA331471,
T64415, 867391, F08386, H28967,
F04611,
AI868715, AI300215, AI468615,
F05628, 863245,
865759, F01890, AA626472, AI633337,
842216,
N71948, F03261, AI942227, AI424608,
AI354266,
863298, AA508475, AA333487,
854772, 821358,
AA659684, C03175, AI033331,
N56429,
AI123688, AI033570, N99683,
N91342, AI311616,
AA978060, and AF055030.
2 HAMFC93 30 904749 W96031, H29070, AI572861, AI245433,
AW338844, AI763055, AI650351,
AI360130,
846410, W90464, AI401499, W67631,
W96045,
T75273, W94664, AI423467, AI056363,
AI089950,
AI924428, H19055, AA515661,
819672, C06006,
W94808, AW271475, H19345, AI753937,
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AI277502, AI687583, AA324890,
816165,
816164, 244059, 844459, AI366519,
815424,
F06854, 243164, F06351, 815425,
T80223,
W 19999, AI290371, AI280283,
T05756, F03118,
F12961, 866672, AA293579, 865758,
818499,
AA331471, F08386, AI696837,
F04611, F05628,
C01791, F01890, AI468682, F10559,
839178,
F03261, H28967, AI868715, 854961,
854772,
821358, C03175, AA806097, AI033331,
AW084413, AI277323, AA333487,
AI300215,
AI682057, AI979311, AA910461,
AI633337,
AA992270, AI004847, AI890272,
AA035340,
AI468615, AA192342, AA987848,
T64415,
X97515, AA633574, AI424608,
AI311616,
AA508475, AI033570, AL119399,
AL079794,
AL047675, AI623941, AL037030,
AI439443,
AL037558, AW021717, AL036634,
AL042382,
AW020397, AI567582, 836271,
AI446373,
AI890907, AA572758, AL037454,
AW162194,
AA641818, AL135022, AI923837,
AW160376,
AL037649, AL048656, AA580663,
AI538885,
AL036901, AI913452, AI345562,
AI499652,
AI866770, AI349628, AI609128,
AI818977,
AI609594, AW 161156, AL046200,
AI525653,
AI538850, AL118781, AW071362,
AW301300,
AI285826, AI433590, AI343037,
AL045163,
AW 151136, AI491775, AW075084,
AI310925,
AI859991, AI340603, AI355779,
AI307505,
AI500061, AI307543, AI334884,
AL041150,
AL036631, AI340659, AL119863,
AW268083,
AW 161579, AI345005, AI633062,
AI311892,
AI307736, AA420722, AI537677,
AI349266,
AW 163554, AI670009, AI932949,
AI433157,
AI917963, AI961589, AL119457,
AI288285,
AI702073, AL040241, AI690748,
AL119836,
AI244249, AW 104056, AL036705,
AL135517,
AW 163834, AI471282, AI473451,
AI345688,
AI872423, AL036274, AW051088,
AI343059,
AI538764, AI633125, AA579232,
AI698391,
AI002285, AI538564, AW268122,
AI349245,
AI915291, AI349933, AW 152182,
AL039086,
AW023338, AW 162189, AW020693,
AI582932,
AW020095, AW238688, AL047100,
AI301046,
AI889189, AI521560, AI349246,
AI345253,
AI366992, AW059828, AI343091,
AI624993,
AI866469, AI345677, AI801325,
AL038605,
AI537191, AI267162, AI340519,
AI884318,
AI287233, AI523806, AI439350,
AI972074,
AL047422, AI635467, AL079963,
AI868204,
AI348854, AI349256, W74529,
AI954504,
AI312152, AW269097, AL121270,
AI366968,
AA579618, AA070889, AL047344,
AL079728,
AI312399, AL039858, AI284517,
AI348897,
AI345251, AL118752, AW071412,
AI590423,
AF055030, AF210052, AF067790,
I48978,
AF090943, AF183393, A77033,
A77035,
AF111849, I89947, AL080159,
E07108, U78525,
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Y07905, A65341, Y11587, AL110197,
AL137488,
AL122110, AF069506, AL137271,
AL137550,
AL133113, A08916, AL137529,
Y16645,
AL049466, AF126247, AL133640,
A08913,
A08910, AL080074, A08909, AF090900,
AL117457, AL050116, AL096744,
AL137533,
AR038854, I48979, AL023657,
AL137480,
A58524, A58523, AF078844, Y11254,
AL137478,
AF087943, AR059958, AL117649,
AF026124,
AL133665, AF106657, X79812,
X70685,
AL049314, X72889, I33392, A93350,
AL133067,
AF100931, E05822, AL049382,
X80340,
AL050172, AL122100, AL137294,
AL110222,
AF061943, I89931, A08908, X84990,
D16301,
A08912, S78214, AL133560, I49625,
AL050277,
AR013797, AF090934, X87582,
AF031147,
AL080148, AF026816, AL080140,
AL137557,
AF111112, AL137463, AL117648,
AR034830,
I96214, AF079763, AF090903,
A18777,
AL122050, 237987, AF061795,
AF151685,
I66342, S68736, AF185576, AL080163,
AFI 13691,
AF104032, AL137479, A08907,
AB016226,
AF113019, U77594, X53587, AR000496,
U39656,
I03321, AL049452, AJ242859,
AF177401,
AF090901, U35846, A08911, AL080124,
AF113677, AF097996, AL049938,
AR038969,
AL117435, AF111851, U58996,
AF032666,
Y14314, A45787, AF039138, AF039137,
AF139986, X65873, AJ012755,
AL122123,
AR011880, AL049430, AL133075,
AL050108,
AL080137, A03736, S76508, AF081197,
AF081195, I89934, E01573, E02319,
U49434,
AL137429, L19437, S36676, U67958,
282022,
AL117460, AL133557, AL110221,
AL050149,
AL050138, U42766, U91329, AF106862,
AF057300, AF057299, X81464,
A21103,
AF017437, AL137526, AL049300,
AF113699,
AL133016, AL050092, AL133606,
AF079765,
AF061573, U51587, AF091084,
AF113690,
AL049283, AL110196, AJ000937,
AL133080,
AF132676, AL133081, AF061836,
AB007812,
X92070, AF090886, AJ003118,
AL122093,
AL137521, X83508, AL110218,
AF118070,
E04233, AF146568, U55017, AL137538,
Y09972,
AF113676, AL049324, AL122118,
I68732,
U88966, U75932, AR020905, X82434,
AF113689,
AL117583, X52128, AL117585,
U68233, I92592,
AL137574, AF158248, U00763,
AL133010,
AL137476, I00734, E12747, 561953,
AB019565,
E03348, AF118090, E00617, E00717,
E00778,
AF162270, AL137656, AF061981,
AL133568,
U80742, AL137527, AL096751,
AL050393,
L04504, E15569, A93016, AF119337,
I42402,
AF176651, AJ238278, M86826,
E01314, S79832,
U72621, AL137641, AF022363,
I89944, and
AL137283.
~DPMS12 13 91192~ AI302789, AA811403, AA280098,
~ AI859802,
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AI399882, AI889993, AI955126,
AI566118,
AA281112, AI890958, AI218594,
AW304705,
AI923781, AI571059, A1298752,
AA588151,
AW194947, AI520733, AW072661,
AI858683,
AA569130, C16026, AA445939,
AA034484,
AA037785, AA281535, AA281210,
AI922456,
N51740, AI453683, AA452036,
AA640052,
AA494120, AI559965, AI536050,
W32322,
AI122851, AI377559, W32652,
N77069,
AA716267, AA034483, AA280236,
AA280031,
AW089437, AI597785, AI474880,
AA873639,
H65003, D61846, AA642010, AW270089,
AW363819, AA174150, AB020686,
and
AL035701.
3 HDPMS12 31 907113 AL035701, andAB020686.
3 HDPMS12 32 907128 AI302789, AA280098, AA281112,
AA569130,
AA445939, AA640052, AA494120,
N77069,
AA280236, AI474880, AI597785,
H65003,
C16026, D61846, AW194947, AI566118,
AI955126, AI536050, AB020686,
and AL035701.
3 HDPMS12 33 907127 AI566118, AI955126, AW194947,
AW304705,
AI298752, AW072661, C16026,
AI399882,
AA281535, AI520733, AI536050,
AA034484,
W32652, AA037785, AA034483,
AA280031,
AI859802, AA811403, AW089437,
AI889993,
D61846, AI218594, AI890958,
AI122851,
AI923781, AA588151, AW363819,
AI571059,
AI474880, AA494120, AA569130,
H65003,
AI858683, AB020686, and AL035701.
3 HDPMS12 34 907126 AW270089,AB020686, andAL035701.
3 HDPMS12 35 763259 AA811403, AI859802, AI858683,
AI889993,
AI218594, AI890958, AI923781,
AI922456,
AI571059, AA588151, AI453683,
AI399882,
AA452036, AI520733, N51740,
AA034484,
AA037785, AA281210, AI559965,
W32322,
AI122851, AI377559, AA642010,
AA716267,
AA873639, H65004, AW363819,
AA174150,
AL035701, and AB020686.
4 HKAAV61 14 1035509 AI821806, AI821058, AI821110,
AA670417,
AI623110, AA651619, AI821856,
AW072286,
AA635690, AI815232, AW273382,
AW 190718,
AI433157, AI702073, AI613038,
AL119863,
AL045500, AI567846, AI500061,
AI249497,
AI866106, AW 129659, AW026882,
AI969655,
AI696626, AA872614, AI811344,
AI868204,
AI890507, AW 169234, AI537677,
F27438,
AW020693, AI283914, AI572934,
AI452560,
AI802542, AI640873, AI619502,
AI612913,
AW131282, AL040169, AI539771,
AI648509,
AI536685, AW173449, AI884469,
AL041150,
AI872545, AW162189, AW238688,
AI564719,
AI567971, AL036802, AI926790,
AI917252,
AI249323, AA908294, AL110306,
AI699865,
AI917963, AI310575, AI929108,
AI288305,
AW262042, AI868931, AW161156,
AI340533,
AL036146, AW081255, AI866770,
AL134830,
AI637584, AW169671, AW151136,
AI590227,
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AW104724, W45127, AA135879,
AI567940,
AW300889, AI498067, AI802240,
AI494201,
AL119791, AI950892, AI351588,
AI888621,
AI536638, AI445025, AW 198090,
AI004207,
AI312428, AI871697, AI269205,
AI538259,
AI619426, AI340603, AW020095,
AI625079,
AI440239, AI635461, AI439452,
AI670009,
AI445432, AI890907, AI635942,
AW087160,
AL120700, AI687728, AI567128,
AI432969,
299428, AL041734, AW 163823,
AI340519,
N33175, AI684053, AA420722,
AI345608,
AI475371, AW149311, AW166903,
AW075667,
AI539780, AI923989, AI828682,
AW 148408,
AW080327, AI818683, AI634345,
AW051088,
832821, AI921248, AI609409,
AI633125,
AI687127, AI274508, AI934011,
AI698391,
AI431327, AI537273, AI610690,
AI251221,
AI697137, AI915291, AI682971,
AI469532,
AL138457, AW152182, AW163834,
AL039086,
AI580190, AW080379, AI582932,
AW087207,
AI889189, AI521560, AW 191844,
AA572758,
AI473536, AI345471, AI524671,
AI799470,
AI866751, AW087445, AL037454,
AI340627,
AI887396, AI859991, AW 169132,
AI581033,
AI349645, AI587114, AW 148363,
AI620284,
AI288285, W74529, AW300782,
AL048323,
AI343091, AI285448, AL048340,
AA806720,
AW 160905, AW 130922, AI270055,
AW078895,
AI858827, AW002362, AW191003,
AL120854,
AI918435, AW 162194, AI863382,
AI872423,
AL036673, AI434741, AW262565,
AI862144,
AI932949, AL036274, AI445990,
AI554821,
AI702068, AW 161579, AW 129106,
AI824746,
AL046944, AI611738, AI198402,
AI560679,
AI433976, AL036361, AL047344,
AI624206,
AI306613, AI621209, AI499986,
AI521040,
AI345543, AI630928, AW 103878,
AL043975,
AI590830, AW302988, AW083573,
AI345745,
AA641818, AI538885, AL042382,
AI365256,
AI687362, AI254731, AI801325,
AI310582,
AI500523, AI636456, AI318569,
AI445992,
AI866801, AI269862, AI886753,
AI564749,
AI352497, AI828583, AI609331,
AW074869,
AI610307, AI271786, I89947,
AL122095, I48978,
I33392, AL080159, AF180525,
I48979, AL133613,
Y07905, AL137557, AL137294,
AL122050,
A08916, AR038854, A08910, A08909,
AL117457,
AL050024, AL133640, A08913,
AF113694,
AL050116, Y09972, X70685, AL117583,
AL096744, AL080148, U42766,
AL137271,
Y16645, AF097996, AL133075,
U72620,
AF017437, AL049283, 237987,
AL122093,
S78214, AJ000937, AF177401,
AL117435,
E02221, AL137480, AL122110,
I89931,
AF090943, X87582, AF090900,
AL110225,
AR011880, I49625, AL137459,
A12297,
AL050277, AL133557, A93350,
AF125949,
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A08912, AL133031, AF090903,
Y14314,
AL050149, AL133113, AF106862,
AF032666,
AL137476, AL133560, AF113019,
X82434,
Y11587, AL133080, X83508, AL110280,
X98834,
AF118070, A77033, A77035, E02349,
AF111851,
AL122045, A45787, AF104032,
A21103,
AF067790, AF090934, Y11254,
AF087943,
AF113699, AL133016, AF146568,
E05822,
A58524, A58523, A65341, 282022,
I46765,
AF183393, AF159148, AF111849,
AL117440,
AL133568, AF090901, AL050138,
AL050393,
U35846, A03736, AF113677, AL049466,
AF162270, AL049314, I03321,
AL050146,
AL133565, AL137463, AL049382,
AJ238278,
I09499, AF091084, AF113690,
AL049430,
AF210052, AL137538, AF017152,
AL136842,
AJ012755, 563521, AF132676,
AF061836,
AL117460, AL110221, AF090886,
AL137533,
U68387, U78525, AL137479, L19437,
AL137560,
L30117, X84990, U00763, AL137550,
AF185576,
AL122118, AL110228, AL137478,
I42402,
AC002467, AR020905, Y10936,
AF057300,
E03348, AL080074, AF113689,
AF057299,
AF118064, AL133093, A08908,
AF079763,
AR059958, AF142672, AF158248,
AF061981,
AF090896, AL122123, AF111112,
S61953,
ALI 10196, AR000496, U39656,
E07108, 568736,
X93495, AL117432, AL122121,
AF061943,
I00734, AB019565, AR013797,
AL137283,
AF118094, AL049938, AL110197,
AL049452,
AF026124, AL023657, E00617,
E00717, E00778,
AL050108, AL133665, AL137488,
AL137527,
AF081197, AF081195, AF113013,
AF026816,
A57389, AF067728, I26207, U80742,
AL080137,
AL117394, AF030513, AB008792,
X96540,
AB008791, X06146, AR038969,
AL110296,
AF153205, I30339, I30334, AL133606,
I29004,
A93016, Y10655, AL122049, and
I09360.
4 HKAAV61 37 897639 AI821806, AW 190718, AW273382,
AW 173449,
AI821058, AI004207, AI821110,
AI279844,
AA670417, AI623110, AA651619,
AI351588,
AI821856, AW072286, AA135879,
U66684,
W45127, AA872614, AA635690,
AA230178,
AI198402, W45086, AI866106,
AA228314,
AI572934, AL122095, and AL133613.
HDPCJ43 15 963210 AA700160, AI872123, AW055047,
AW338216,
AI393645, AA740794, AW373953,
AI241468,
AI017842, AL045616, W72806,
AA969034,
H17561, AA906689, D11553, AA961283,
T32867,
AL045615, D57538, H00373, T59678,
824874,
AA630157, AA319359, T98106,
T98026,
AA907502, W72805, T59814, AA328115,
W52508, AA577514, AA094924,
and AL137432.
5 HDPCJ43 38 907076 AW392670, AL119496, AL119363,
AW372827,
AL119319, U46346, AL119335,
AL119391,
AL119484, AL119497, AL119443,
AW363220,
CA 02389722 2002-04-30
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AW384394, AL119439, AL134902,
U46347,
U46351, U46350, U46349, AL119444,
AL119457,
AL119399, AL119324, 299396,
AL134518,
AL119355, AL119483, AL119522,
U46341,
AL119341, AI142132, AL119396,
AL134528,
U46345, AL 134524, AL042970,
AL 119418,
AL042614, AL042980, AL037205,
AL134538,
AL042965, AL042544, AL042850,
AL042995,
AL042542, AL042975, AL042450,
AL079683,
AL119320, AL043029, AL042551,
AL043037,
AL042428, AL043039, AL043019,
AL042984,
AL043003, AL119304, AL119464,
AB026436,
AR054110, A81671, AR060234,
AR066494, and
AR069079.
HDPCJ43 39 902173 AA700160, AI872123, AW338216,
AW055047,
AA740794, AI393645, AW373953,
AI241468,
AL045616, W72806, H17561, AI017842,
AA969034, AA906689, D11553,
AA961283,
T32867, T59678, D57538, H00373,
AA907502,
AA630157, 824874, T98106, T98026,
AA319359,
W72805, T59814, AA328115, W52508,
AA577514, AA094924, AI926593,
AI866465,
AA504514, AI648567, AI434242,
AI242736,
AL045413, AI096771, AI471429,
AI440238,
AI554821, AW151136, AI539771,
AI537677,
AI494201, AI500659, AI815232,
AI801325,
AI500523, AI538850, AI582932,
AI923989,
AI284517, AI872423, AI500706,
AI445237,
AI491776, AW 151138, AI521560,
AI889189,
AI500662, AW 172723, AI284509,
AI440263,
AI889168, AI866573, AI633493,
AI434256,
AI866469, AI805769, AI888661,
AI284513,
AI888118, AI432644, AI859991,
AI436429,
AI355779, AI889147, AI371228,
AI440252,
AL047422, AI888523, AI866786,
AI610557,
AI860003, AI687166, AI887499,
AI539781,
AI887775, AI590043, AL042377,
AI559957,
AI289791, AI582912, AI539800,
AI538885,
AI927233, AI333104, AI500714,
AI285439,
AI623736, AI581033, AI274759,
AI491710,
AI431307, AI358701, AI804505,
AI567971,
AI431316, AI828574, AW 151979,
AI867068,
AI539707, AI702065, AI798303,
AI885949,
AW089557, AI285419, AI521571,
AI469775,
AI866581, AI433157, AI567953,
AI815150,
AI446495, AI690946, AI925402,
AI801152,
AI539260, AW 163834, AI537187,
AA814782,
AW074057, AL047398, AI225248,
AI561170,
AI698352, AL045619, AW 104641,
AL045626,
AI610616, AL042365, AI687168,
AI890907,
AI866691, AL040459, AI439762,
AI434240,
AI371251, AI866510, AW 151131,
AA808311,
AW 151974, AI923046, AW080402,
AI445620,
AI049850, AI273179, AI371243,
AI952433,
AW089275, AW129310, AI371229,
AL039287,
AL042981, AI866461, AW081343,
AW 151132,
AI474646, AI560545, AI885920,
AI866503,
CA 02389722 2002-04-30
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AW079432, AL048403, AA878808,
AI473451,
AW089009, AA488429, AI285417,
AI815239,
AI621341, AI344935, AI431238,
AW191003,
AI433611, AI476086, AW025279,
AI249497,
AA761557, AI560679, AI611816,
AI805774,
AI828583, AL120700, AW020381,
AI866458,
AL121328, AI433976, AL048375,
AW089424,
AI432666, AI282249, AW 197139,
AA808175,
AI567944, AI571575, AL039390,
AI049856,
A1366900, AI888621, AI887785,
AA641818,
AI266558, AW079768, AI366910,
AI889191,
AI636619, AI440260, AI436438,
AL042533,
AW083804, AL135065, AL045500,
AI623799,
AI049851, AI620284, AI431323,
AL040241,
AI049859, AI275175, AI493559,
AL042694,
AI539863, AA715307, AI524654,
AA809974,
AI633125, AI499463, AI355008,
AI818353,
AI610362, AI697243, AA911767,
AI340519,
AW023338, AL079960, AA835947,
AI340603,
AL137432, AJ238278, AF090934,
AL110280,
I89947, AF185576, I48978, AL133080,
U30290,
E07108, AL137521, AL133070,
AF113013,
A08910, A08909, AL137480, A08908,
I17544,
AJ010277, AL117626, I68732,
AL137526,
AL049430, AL117583, A08913,
M19658,
AL137258, A08912, A08911, AL133067,
AL133093, X87582, AF109155,
AL133049,
AL133665, AL122118, AL137550,
576508,
AL122123, AF028823, AL050024,
AL117416,
AF151109, 577771, AF106657,
AL080124,
A18777, AF067790, AS7389, I33392,
AF132676,
AL133640, AF061836, AL122098,
AF177401,
I32738, X72387, AF002985, A08907,
AF113694,
AL133084, AF090943, A65341,
AL122050,
E12580, AF112208, AF043493,
297214, I00734,
AF111 I 12, I41145, I89931,
AL137478, AL122104,
X62580, AL137538, AF185614,
AL117457,
AF124728, E00617, E00717, E00778,
AL117394,
E02221, X72889, E12747, I49625,
AL122110,
X66862, AL049283, E05822, A77033,
A77035,
AL049426, S79832, AF022363,
AR0388S4,
AL133560, X63410, AR013797,
AL122049,
AL137459, X84990, A93350, AF090903,
I89934,
A08916, AF119337, L13297, AR012379,
U67958,
AL117585, AL133075, AF146568,
AL133072,
X63574, AL110171, X79812, 282022,
AL110197,
AB007812, AF090896, AL137488,
AF090901,
AR034821, U42766, M92439, I48979,
AL049347,
AL137281, U62807, AL080074,
U37359,
AF194030, AL096728, AL0501SS,
AF137367,
AL137548, AR068753, 561953,
AF078844,
Y10655, A76335, 272491, AL122111,
AL080158,
AF026124, AF017152, 237987,
AL117578,
AF090900, A07647, AL096744,
AL133113,
U35846, J05277, AF000145, AF008439,
AL035458, AL137300, AL080118,
AB019565,
AL136884, Y10080, AR019470,
AF118090,
CA 02389722 2002-04-30
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M85164, U95114, AL122100, I09499,
A45787,
X86693, AL137660, AL110225,
S53987,
AL050138, AF079765, AJ006417,
AL050277,
578214, A30330, A30331, L04849,
AL133104,
AF113690, AF126247, X56039,
AF097996,
Y10936, AL137560, D89079, AL133081,
AL050172, I26207, X55446, X06146,
U42031,
AL137711, AF205861, U68387,
AL080156,
AL122093, AL137527, AF002672,
AJ012755,
AF104032, X83508, AF081195,
AF113691,
AC002467, D55641, AL137429,
AL049324,
AL049300, AL110196, AF030165,
AL049423,
AL122106, AF176651, AF017790,
AL133077,
U80742, AL080137, AL137705,
AL049452,
AL133010, AL137557, AL133565,
L04504,
E07361, AL137627, A92311, AF113019,
AL133637, AF113677, AL049938,
X70685,
AF111851, A49139, AF183393,
M96857, U00686,
AL049466, AF040751, AF030513,
AL137641,
A03736, AL122121, U89295, and
A18788.
HDPCJ43 40 853790 AL045615 and AL137432.
6 HKACM93 41 907085 AA326415, AW392670, U46341,
AL119497,
AW372827, AL119324, AL119457,
ALI 19396,
AW384394, AW363220, AL119319,
AL119391,
AL119363, ALl 19443, 299396,
U46351,
AL119484, AL119341, AL119355,
U46350,
U46347, U46349, AL119483, AL134518,
AL119444, U46346, AL119335,
AL119496,
AL119399, AL134920, AL134533,
ALI 19439,
AL119522, AL037205, AL042433,
AL134525,
AL134524, U46345, AL042614,
AL134538,
AL042965, AL042975, AL134531,
AL119418,
AL042544, AI142132, AL042450,
AL042551,
AL042970, AL043029, AL042542,
AL043019,
AL042984, AL043003, AL119464,
AL119488,
AB026436, AR054110, A81671,
AR069079,
AR060234, and AR066494.
6 HKACM93 42 906154 W91896, AW003420, AI952478,
W92036,
AW014836, AA588510, AI625123,
AA233897,
AA552314, AI394000, AI694853,
800094,
AA234032, AI391587, H14912,
H14619, and
AI940122.
6 HKACM93 43 906150 AA223404.
7 HHEMM74 17 941955 AI912020, AI738591, AI673200,
AW 195629,
AW207103, AI914327, AI016102,
AI948562,
AA515654, AI858984, AI021976,
AA824295,
AW003109, AI288261, AI332790,
AA447206,
AI863407, AW271426, AI673222,
AW274813,
AI093417, AW 197033, AA846300,
AI989749,
AA917651, AI933079, AI198249,
AA149282,
AW337461, AA029228, N89854,
AA476264,
AI650694, AA805734, 228929,
AL042753,
AI862408, AW 137892, AW250017,
AL138455,
H43959, AI679782, AA029227,
AL043009,
AA078301, AI400383, AL042853,
AL138265,
AI971136, AL043718, AL079812,
AI284640,
AI133164, AF039185, AW301350,
AI805607,
CA 02389722 2002-04-30
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AW303196, AI334443, AI499938,
H01583,
AL037683, H01483, AL041690,
AA587604,
AL046409, AW274349, AL048626,
AL045053,
AA631507, AL040921, AI963720,
AI307608,
AL119691, AI431303, AA581903,
AI270117,
AW021583, AW088846, AW072923,
AW 193265,
AA610491, AW419262, AI613280,
AA129446,
AA594725, AA723017, AL039996,
AI254316,
AI281881, AL042377, AI350211,
AW188484,
AA490183, AI708009, AI610920,
AI862409,
AA563926, AI305766, AA584082,
AW406447,
AI192631, AL046205, AW270270,
AI367497,
AI537506, AW245747, AI457397,
AA577906,
AI076616, AW276827, AW265385,
AI345654,
AI281903, AI097429, AL134972,
AI754955,
AI821271, AI537955, AL120687,
AI924251,
AL044940, AI732865, AI370074,
AA547979,
AI625244, AW276586, AI473943,
AI246796,
AI345681, AI345675, AW438643,
AI434695,
AI110770, AW406755, AA525190,
AW327868,
F36273, AI471481, AA665330,
AI375710,
AI305547, AA587256, AW163293,
AI564185,
AA682912, AA877817, AI358571,
AI890348,
H86301, AI354426, AW023672,
AA572713,
AI357551, AA720702, AI688846,
AI345518,
AI370094, AI745325, AL048925,
AA652764,
AI732186, AI341664, AI270559,
AA584167,
AW083402, AW 193432, AI064864,
AI249997,
AL044858, AA077579, AW410400,
AA515829,
AW268300, AL119649, AI053790,
AA974503,
AL042420, AW276817, AI200051,
AA491814,
843152, 849809, AI149478, AA594145,
AA846876, AA584195, AA491284,
AW 148792,
AI148277, AA598586, AA521323,
AA666332,
AW265393, AI379719, AL039958,
AI312309,
AI35S206, AI801591, AI568678,
AI538852,
AW029038, AA468022, AI623898,
AI539563,
AW 162489, AIOS3672, AC004084,
ABO11110,
AC005696, AC008372, AC000025,
AC005015,
AC009516, 285986, AF053356,
AC006480,
AC004963, AL023284, AC004686,
AC005081,
AF196779, AL022721, L44140,
AJ003147,
AC005839, AC007055, 293017,
AC004832,
AC004638, AL049759, AC002350,
AC005695,
AC006285, AC005291, AC005821,
AC005037,
AC005670, AC006040, AC005520,
299716,
U91326, AC002565, AC004019,
AC005940,
AC006241, AC006965, AC003037,
AC006449,
297054, AL035587, AC007193,
AC002316,
AL121603, AC007227, AC006960,
AC005911,
AC005914, AL021878, AL049776,
AL022163,
AC006211, AC005527, AL035458,
AC005837,
AC007546, AC005500, AL050318,
AL022238,
AL031283, AC005231, AL049795,
AC004453,
U95742, AC005620, AC007298,
AL023553,
AF129756, U80017, AC004821,
AL096701,
269666, AL031587, AC002549,
AL133485,
CA 02389722 2002-04-30
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AL133448, AC005488, AC007666,
AC005519,
AC006512, 283844, AC005778,
AC005971,
AL020993, AC007731, AC005295,
AL096776,
AC006251, AC005086, AL033521,
AC005562,
AF001549, AC007676, AC008115,
AC009263,
AL139054, U62293, AC006057,
AC005529,
AC002476, AC005261, AL022476,
AL031311,
AL031680, AL078463, AC000052,
AC006312,
AC007216, AC005754, U85195,
AC002470,
AC012331, AL034420, AC005011,
AC002308,
AC005280, AL009181, AC003029,
AC002563,
AC005412, AL008716, AL109984,
AL031602,
AC007919, AC004813, AC006487,
AC005225,
AF 134726, AC004967, AL022316,
AC002400,
L78833, AE000658, 286090, 294721,
AC008079,
AC004383, AC007226, AC007993,
AC004032,
AC006501, AC006013, AL022315,
AF111168,
AC020663, 293241, AL022318,
AC004263,
AC007384, AC006111, AC005772,
AC002059,
AL049569, U62317, AF196969,
AC004534,
AL031229, AC006511, AC007277,
AL020997,
AL049780, AC007242, AL031662,
AC005829,
U47924, AC003957, AL049869,
AC005071,
AC004966, AC002365, AL008583,
AC004526,
AC006205, U91328, AL096774,
AL035659,
AL008718, AC005740, AL031767,
AC006112,
AF001548, AP000031, AC005484,
AC002310,
AC003688, AC005057, AC005667,
AC007308,
AC002094, AC005480, AL009179,
AC010072,
AC006120, AP000045, AC005703,
AC016831,
AL049643, AC005553, AC005531,
AC005180,
282097, AC002301, AC004865,
AC006344,
AC007011, AC008101, AL132712,
AC002472,
AC003003, AC005755, AC007021,
AL031657,
AC003043, AC006115, AF207550,
AL023807,
AC018633, AL109758, AL049872,
AC005089,
AL008582, AC005900, AC006479,
AL022311,
AP000555, AC005004, AL035411,
AL031846,
AC006441, AC004814, AP000557,
AC003048,
AL109985, AC004883, 299495,
AL121653,
AC004812, U91321, AC007686,
AC000353,
AC000035, AC007057, AL080243,
AC016025,
AF165142, AC006538, and AC006581.
7 HHEMM74 44 906815 AA078301, H01583, H01483, AA857812,
AI917891, AW044118, AI335387,
AA598605,
AA832175, AA831638, AW128958,
AI354602,
AA078020, AA484892, AI583259,
AI249997,
AI620992, AC004084, AC007193,
AC005696,
AP000359, AL008582, AC008372,
284480,
AC005519, AF053356, AC005520,
AC004383,
AC005015, 299716, AJ003147,
AC004686,
L78833, AC000052, AC002369,
283844,
AC007226, AL034429, AL022322,
AC002563,
AC005081, 293023, AC002091,
U07562,
AC002470, AP001054, AP000553,
AC002350,
AC005102, AC007225, AL023284,
AC005722,
AL023807, AC004659, AL031680,
AC005913,
CA 02389722 2002-04-30
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AC007546, AC006430, AL034379,
AC006480,
AC004895, AC006241, AC005071,
AC002365,
AC005527, AL133163, AC005914,
AC004841,
AC004685, AC009516, AP000046,
AP000114,
AC005829, AF091512, AL139054,
AL121603,
L44140, AL021154, AC005004,
AC004000,
AC005911, AL049759, AC006312,
AC007993,
AC007308, AC004019, 293017,
AL050307,
AC007114, AL049745, AL049776,
U62293,
AC004002, AC003037, AC004638,
AC016025,
AL035683, AC005666, AL133448,
AC000353,
AL031985, 298051, AL022326,
AC005386,
AC007637, AC006544, AC007676,
297054,
AC002300, AF165926, AC005726,
AC007151,
AC004531, AC005086, AL049780,
AL022163,
AL031577, AC007055, AC005907,
AC005057,
AC006077, AC003070, AP000356,
AC002565,
AC005089, AC005209, AC006014,
AC00651 I,
298044, AL031295, AP000248,
AC004408,
AC007228, AL031602, 293930,
AC005280,
AC002316, AF196971, AC005919,
295116,
AC004797, AL035415, AF045555,
AL096791,
AC011311, AF109907, AC004813,
AC004263,
AL020993, AC005225, AC002477,
285986,
284467, AC020663, U91327, AP000115,
AC007666, AL021918, AF196779,
AF196969,
AC002492, M89651, AL023575,
AC006064,
AC005695, 285996, AL031657,
AC006512,
AL024498, AC005778, U91321,
AL031281,
AC005049, 283846, AC003103,
AC012384,
AC003051, AC008101, AL049869,
AC006509,
AC005067, AC006449, AC006538,
AL109758,
298200, AC007845, AC008044,
AC006088,
AC005632, AC005011, AC004765,
AL049553,
295115, U91323, AC009247, AJ246003,
AF196972, AL080243, AC007066,
AL133245,
AC004526, AC005920, AC005250,
AC007388,
AC004386, AP000496, AL132992,
AL033521,
AC007999, AC002310, AC005531,
AL109628,
AL031681, AC002400, U91326,
AC006071,
AC005105, AC007919, AC005261,
284469,
AC003108, AP000513, 284466,
L77570,
AC005180, AC002312, AC004948,
AC004890,
AC005363, AC005837, AL109798,
295114,
AC005288, AC004491, U47924,
AC005486,
AC003101, AC005387, AL020995,
AC006023,
AC005255, AL050318, 298742,
295152,
AC002550, AC004967, AC005231,
AC003665,
AL078477, AC006486, AL022476,
AC004832,
AC005971, AC007358, U95740,
AL050348,
AF047825, AP000211, and AA402180.
7 HHEMM74 45 902458 AI912020, AI738591, AI673200,
AW195629,
AW207103, AI914327, AI016102,
AI948562,
AA515654, AI858984, AI021976,
AA824295,
AW003109, AI288261, AA029227,
AI332790,
AA447206, AI863407, AW271426,
AI673222,
AW274813, AI093417, AW 197033,
AA846300,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
254
AI989749, AA917651, AI933079,
AI198249,
AA149282, AW337461, AA029228,
N89854,
AA476264, AI650694, AA805734,
AA149376,
228929, 853155, AI862408, AW374891,
AW 137892, AA010232, AW250017,
H43959,
AI400383, AA077229, AI971136,
AA077669,
AI862409, AW405369, AW386712,
AW386708,
843152, 849809, H22270, AA077893,
AA889934,
AW373785, F00253, M91506, AF139794,
AA077980, AI282751, AA010157,
AW393714,
F01048, AI349772, AW166645,
AW071349,
AI815383, AW080838, AL119049,
AI906328,
AL047042, AI907070, AI149592,
AA077544,
AI207510, AI907061, AI868831,
AI624859,
AW132121, AI220734, AI349645,
AI345111,
AI909662, AI340582, AI344182,
AI343112,
AL121270, AI345860, AW268253,
AI349614,
AL045500, AI500553, AW162071,
AL120854,
AL036396, AI682106, AI590482,
AA528822,
AL036802, AI580190, AI684265,
AI251485,
AI349598, AI436456, AL046849,
AI334902,
AI064830, AI525064, AI687376,
AI909666,
AI690751, AL135661, AI799305,
AA613907,
AI907056, AI433976, AW 117882,
AL036759,
AI433157, AI863014, AI920968,
AW303152,
AI500077, AL036146, AI583316,
AI309401,
AI813914, AI547175, AW168591,
AL119791,
AI027531, AA603930, AI345744,
AA528491,
AI969601, AL047763, AL120736,
AL119748,
AI524991, AI560012, AI569870,
AI521012,
AI687728, AI349933, AI679724,
AI469532,
AI687415, AI608667, AW302965,
AW238730,
AI568870, AL040169, AI475371,
AW 103371,
AI934036, AI567351, AA640779,
AI702406,
AI969567, AL121365, AL036274,
AI538716,
AI343059, AI753683, AI567632,
AL036980,
AI673256, AW089572, AI873731,
AI440426,
AI349256, AW071417, AI285735,
AA585422,
AI312152, AL036240, AI499393,
AI349937,
AA938383, AW301409, AI818683,
AI866780,
AI497733, AI625079, AW074993,
AI889203,
AI255071, AI682743, AL038605,
AI349004,
AI282655, AW274192, AI609592,
AI250293,
AI345735, AI800453, AI631107,
AW235035,
AI366549, AI635942, AI690835,
AI557728,
AI686926, AW195957, AI636456,
AI678302,
AI307466, AI281779, AA528529,
AI697137,
AI671679, AW087445, AI635461,
AI366991,
AI439087, AA572758, AI275175,
AW169653,
AI564719, AI340519, AI866608,
AI597918,
AI445432, AW068845, AI633419,
AL038778,
AI636445, AI612913, AI590128,
AA528585,
AI699857, AI568854, AI620284,
AI886532,
AI556971, AI498579, AL040243,
AI696846,
AI919058, AI610307, AI857296,
AI446606,
AI758437, AI800433, AA523030,
AI540832,
AI613017, ABO11110, AC004084,
I48979,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
255
AL133640, AF090934, 578214,
AF113691,
256822, L31396, AP090900, L31397,
AF 118064,
AL049938, AL050393, Y11587,
AF118070,
AF078844, AL133016, AF090943,
AL050146,
AF113013, A08916, AL117457,
AF125949,
AL110196, AL137527, A93016,
AL117460,
AF090903, AJ242859, AL122050,
AL133606,
AF090901, AF104032, AL080060,
AL049452,
AF113694, I89947, AL110221,
S68736, AF113690,
AF113676, AR059958, AF113689,
AL050149,
X84990, AF106862, Y16645, I89931,
A08913,
U42766, AF090896, AB019565,
AL096744,
AL133075, AF113677, AL050108,
AF113019,
AL122093, AL050116, AL049466,
AL049314,
AL049430, E03348, AC004093,
AL050277,
AC005992, AC007390, AL080137,
AL133565,
AF017152, AL080124, AL133557,
AC006336,
AL137283, AF113699, X63574,
AL137557,
AL133093, AR011880, AC002467,
AF158248,
I48978, AL122121, AL117394,
AF111851,
Y11254, AF097996, AF144093,
AL133080,
AC006965, E07361, AL122123,
U91329,
AJ000937, AL137459, AL121603,
AF146568,
AL110225, X82434, AF177401,
AF091084,
AC004690, AC006371, U00763,
AF079765,
E05822, AF125948, AF091512,
AL137550,
AF017437, AL050138, I49625,
U95739,
AL049776, AL133560, E07108,
AC005225,
A65341, AC007172, E02349, AC010077,
AL117585, AC007298, AL117583,
AL049300,
AJ238278, 561953, AC000111,
AF061943,
A08910, AL049382, AF183393,
AL049464,
AL050024, AC004686, AC008394,
A08912,
AL034374, AL117435, AL122098,
X70685,
AC005488, AL137271, AC007043,
AC005886,
282022, A58524, A58523, AC005291,
AL080127,
AL035587, AL022165, AL137463,
AF067728,
AC006944, AL133113, AL133445,
A03736,
AC002464, A08909, AL137538,
284814,
AL122110, I33392, AL022147,
A77033, A77035,
AL096776, AL031346, X93495,
AF118094,
U80742, X72889, AC006840, A12297,
and
AL049283.
7 HHEMM74 46 895682 AA029227, AA077579, AA149376,
AA077544,
AI912020, AA010232, AI738591,
AC004084, and
ABO11110.
8 HSYAZ50 18 1027673 AW02862 and H58607.
8 HSYAZ50 47 852318 AW028622.
8 HSYAZ50 48 902235 AW028622, AA595740, H58608,
H58607,
AC002039, AC002045, and AC007200.
8 HSYAZ50 49 882732 AL079444, AI972850, AI968041,
AW245683,
AW006960, AW027253, AA651725,
AA557203,
AI346500, AA628058, AA888978,
AA609162,
AA025053, AW069699, AA722679,
AI755144,
W93595, AI083933, AI185644,
N39238,
AA393616, AI081868, N95391,
AL079445,
N26655, AI027313, AA934594,
AA983627,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
256
AAG94280, AI35G126, AA74G230,
AI493015,
WG8062, AW383214, AI281236,
AI278112,
AA186911, WG8063, W73332, N51027,
AA459559, AA405910, AA263095,
AI268103,
AI052280, AI753963, H49465,
N51042, W353G8,
AI091959, AI192708, N51035,
AA778366,
N56710, N73444, W95769, 836307,
AA179574,
C03383, H18870, AI818424, AW245326,
W95726,
AI079721, AI203076, H18871,
N29702, H49464,
AA296459, H03252, AA505296,
H85293,
AA772581, AA405724, AA627796,
AA032241,
AW351501, N27044, AA296458,
AA758070,
AA654129, AA731674, AA835855,
AA599399,
AI240246, AA180170, AA630588,
AA564639,
AI708735, AW275924, W25227,
AA151422,
AA179342, 849238, AI378971,
AW090338,
AA180169, W73353, AI190100,
W93491,
AW445146, H03251, AA362124,
AA303019,
AA025052, AA151421, AI187864,
AA356383,
AA033649, N46682, 825955, C04087,
AA872633,
AA689558, H49937, AA593719,
W32932, H49940,
AW029622, AW076097, AI871706,
AA503103,
AA430581, AW057769, AI956128,
AI921229,
H48152, AW085888, AI568021,
AW161023,
AW006763, AW006996, AA937417,
AW075529,
AA579703, and AI270G30.
9 HDPAQ55 19 1002722 AA584014, AW265468, AW 148821,
AI702049,
AW438539, AA584034, AW410844,
AL121039,
AI064968, AL021394, AC004084,
AC005484,
AC005971, AC004841, D87675,
AL096791,
AL008582, AC005746, AC005755,
AC002544,
AL133448, AC005081, AL121658,
AL031589,
AC005288, AC004228, AL031311,
AC007993,
AF047825, AL031577, AC005839,
AC004983,
AF053356, AC005089, AF134726,
AL022476,
AL009181, AL031670, AL109952,
AF109907,
AC006211, AC004814, AC007676,
AC005324,
AC004770, AP000692, AL031427,
AL009179,
298048, AL109628, AL121603,
AC004125,
AC005940, AC004797, AC004167,
AC005274,
AC005412, AC004033, AC005291,
AC005696,
AC009516, AC005399, AC002310,
AC002996,
AC006544, AL022322, AL049869,
AC002477,
AC004796, AC005730, AC004890,
U47924,
AC012099, AC002395, AC020663,
AC005837,
AC004491, AC007546, U95742,
AC002470,
AL034548, AC006241, AL031591,
AF001550,
ACOb8372, AL020997, AL031005,
AC005736,
283844, 299716, AC005632, AL031295,
AP001053, AL133353, AL049776,
AC004659,
AC006449, 283840, AC004821,
AC006111,
AC005562, AC002314, AC004098,
AC004638,
297056, AC005082, AC005057,
AC005049,
AC005911, AC004386, AF111168,
AC005071,
AP000553, AL022238, AC01131
l, AL132712,
AL121769, AC004263,'AC005520,
AC000066,
AC005048, AC005920, AL035587,
AC004815,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
257
AL023807, 285986, AL049759,
AL049872,
AL096701, AF196970, AL109984,
AC005697,
AC002126, AC005409, AC003982,
284480,
AC007066, AC004253, AC005519,
AC005821,
AP000211, AP000065, AC007999,
AL049709,
AC005225, AC005037, AC005041,
AC004531,
AC007055, AP000555, AL022318,
U07563,
AL121653, AL021546, AC005332,
AL133163,
AF196779, AL022721, AL008718,
AC002400,
AC004760, AC007114, AL032821,
AC007371,
AF111169, AF165926, AL109627,
298949,
AF001548, AL022326, AC006312,
AC003043,
and AC002352.
9 HDPAQ55 50 885594 AL021394.
9 HDPAQ55 51 891373 AL134990, AI871361, AI192081,
AI457414,
AI393551, AW131821, AA704524,
AI435337,
AI571078, AA029986, AA351075,
AA702823,
T87870, AA342740, AW369850,
AA884585,
AA833651, AA350232, AA086340,
813094,
AW103794, T97188, AA285358,
AW203960,
AA086196, AA313216, AL119324,
AL134524,
AA311780, AW370641, AL119319,
AW363220,
AW392670, AW384394, AL119457,
U46349,
AL119443, AL119363, AL119444,
U46346,
AW372827, AL119464, F03544,
AL119399,
AL119391, AL119484, AL119355,
U46350,
299396, AL119483, U46347, AL042544,
AL119497, U46351, AL134920,
AL119439,
U46341, AL119341, AL119496,
AL119418,
U46345, AL119335, AL134528,
AI142134,
AL119522, AL134538, AL037205,
AI142131,
AI561106, AL134518, AL119396,
D87440,
AC007308, and AC002470.
9 HDPAQ55 52 693713 AA584014, AA584034, AI344906,
N35135,
AW020612, T34066, AI921744,
AA125758,
AA380695, T10218, AA493348,
897635,
AI281622, AA525156, AA533653,
AW273201,
AW273146, AI572680, N49298,
AA410788,
AW 169469, AI309059, AI872229,
AW 192002,
AI821892, AA736488, AA228778,
AA342029,
AI521019, AA644090, AA362670,
AA485289,
N52441, AA564591, AA947352,
AL021394,
AC003043, AL096791, AL049694,
AF001549,
AL133448, AP000356, AC002369,
AP000503,
AC007201, AL022163, AP000133,
AP000211,
AC004812, AC005519, AL109627,
AC002425,
AL031311, AC006285, AC007637,
292542,
AL133163, AF134726, AL121603,
AL096701,
AC005015, AF129756, AC006101,
AC005004,
AC005288, AL022326, AC004020,
AC006512,
AC004821, AC006057, AC005914,
AC002544,
AC002996, AC005695, AL023575,
AC004084,
U91326, AC004477, 297056, AP000030,
U85195,
AL022320, 282176, AC007055,
AC005632,
AC005102, AC002395, AC005971,
AC005225,
293017, AC004686, AC004876,
AE000658,
AC005920, AJ246003, AP000501,
U80017,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
258
AL132712, AC005332, AC004685,
AC002477,
AC005291, AC002350, AC005921,
AF053356,
AC003029, 283844, AC007298,
AC005031,
AC002565, AC005399, AC005081,
285986,
AL035086, AC003684, AL049830,
AL031602,
AC004797, AC005037, 285987,
AC005839,
U47924, AL031680, M89651, AC016025,
AJ003147, AC007536, AL139054,
U6372.1,
AC004687, AC002551, AD000092,
AC007216,
AL022322, U52112, AC003663,
AC004975,
AC008044, AF196779, AC006111,
AC006515,
AC005082, AC005046, AL022238,
AC005049,
AP000553, AF196969, AC005484,
AC007546,
AC005837, AP000692, AC004883,
297630,
AL021707, AC007229, AL035400,
AP000502,
AL034400, AC002310, AC005800,
AC005755,
297054, AF205588, AL022316,
L78810,
AL009181, AP000359, AC007387,
AC002039,
AC005412, AC006441, AC005722,
AF196971,
AL031281, AC002316, AL031588,
AC007686,
AL034420, AC004659, AL035405,
AF064861,
AC005231, L44140, AC002301,
AC004167,
AC004834, AF 111169, AC006088,
AL024498,
AC005207, AC006064, AL133353,
AC005089,
AF111167, AF200465, AL109984,
AC007371,
298941, AC004079, AC004967,
AC004841,
AL050318, AC006468, AL049776,
U62293,
AC000353, AC008085, AL031767,
AP000952,
AL020997, AC005480, AC007040,
AC003101,
AL121655, AC006261, AF139813,
AL031685,
AC008372, AC006597, AC006530,
AC006141,
AC006011, AL049759, AC003109,
AL022165,
AC005088, AL049576, AC007308,
U91322,
AC004000, AC005702, U95739,
283822,
AP000513, AC002404, AL023803,
AC002470,
AL009183, AF038458, AC005696,
AL079340,
AL031427, AC004859, AP000045,
AP000113,
AC005409, AC002375, 299716,
AL035460,
AL031846, AF050154, AL049795,
AC005520,
AC005666, AC006014, AC007227,
AP000115,
AC004678, AB023048, AC005620,
AC005065,
AC005828, AC004491, AC007537,
AC004996,
AC005300, AL096703, AC006006,
AC004228,
AL133245, AL121652, AL049872,
AL034423,
AC004983, AL008721, AF045555,
and
AC006270.
HKAFT66 20 946512 AA436785, AI804932, AA310516,
AA873013,
AA251417, AI798761, AA250867,
AI826225,
AI811785, AI539808, AL039086,
AI554821,
AI784252, AW105601, AW054931,
AL119863,
AI280747, AI611738, AW 193134,
AW 118518,
AI610362, AL042628, AL036980,
AI312428,
AA833760, AI829327, AI433384,
AI589267,
AI269862, AI564723, AI624548,
AW302992,
AI802542, AI569583, AW 169653,
AI800453,
AI439717, AI567612, AI611348,
AI869367,
AW081797, AL038605, AI570781,
AW051258,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
259
AI802240, AI274728, AW071417,
AI571909,
AW 152469, AI612885, AW022682,
AW 148320,
AI569579, AI252023, AI608936,
AW075413,
AI500077, AI318280, AI636585,
AI251434,
AI862144, AI890806, AI625094,
AI955906,
AI309401, AI431424, AI343112,
AI612913,
AW268253, AW301300, AI349598,
AI554344,
AL036664, AW075207, AI345735,
AI678357,
AI475394, AI633419, AI348897,
AI500659,
AI247193, AI648684, AI313320,
AI627988,
AI251221, AI630928, AI340627,
AAG40779,
AI340603, AI587143, AI312146,
AI312339,
AA572758, AI889168, AI345258,
AI348854,
AW 193000, AW020693, AI457369,
AL040243,
AI445115, AI620866, AW081242,
AI610645,
AI801325, AI866798, AI932794,
AI689248,
AI340582, AA225339, AA427700,
AI270707,
AI955467, AI681985, AI684265,
AW074993,
AI349614, AI590415, AI811353,
AI354283,
AI564247, AI302910, AI955917,
AI349256,
AI312152, AI174394, H89138,
AW075084,
AI567351, AI800433, AI950664,
AL036288,
AI634224, AI349937, AW089572,
AI334884,
AL039132, AI670009, AI349957,
AI307708,
AI312325, AW269097, AI609409,
AI591420,
AW 190042, AI572892, AL045266,
AL121286,
AI916419, AI307520, AI925156,
AI445237,
AI306613, AI434256, AA012905,
AI917123,
AL134999, AW075351, AI874166,
AL036736,
AL036901, AW 151138, AW268302,
AW023590,
AI306705, AI436456, AI608667,
W33163,
AI282281, AW 170635, AW073994,
AW268122,
AI349645, AW074869, AI537677,
AW169634,
AI308032, AI343059, AL036631,
AW268072,
AW403717, AI758735, AL036396,
AI349933,
AI921176, AI524671, AI439601,
AI554245,
AI564736, AW084194, AI497733,
AI288285,
AI934036, AI963224, AL038445,
AI698391,
AI273094, AA508692, AA857311,
AI343030,
AW080079, AI680388, AW071380,
AW080279,
AL042551, AW081449, AI336585,
AI500706,
AI307543, AI349787, AI453413,
AW301505,
AI689420, AI270055, AW 149227,
AI889189,
AI500662, AL040241, AL121496,
AI620639,
AI366992, AI566670, AI538085,
AI917252,
AI452560, AW089310, AW268220,
AI281837,
AI334450, AI653836, AL134259,
AI918655,
AI420521, AI624056, AW068845,
AI621209,
AL036403, AI434020, AL036945,
AI521012,
AI683099, AI682725, AW088903,
AL038504,
AI537515, AL045500, AL119791,
AW088899,
AW059713, AW087838, I48979,
I48978,
AR011880, AF017437, I89947,
L31396, L31397,
AF113013, AL117460, X72889,
AF090934,
I33392, A65341, A08916, A08913,
I89931,
AL049452, A08910, I49625, A08909,
AF090896,
AL050146, Y11587, A77033, A77035,
AL137560,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
260
AF118070, AL117435, I03321,
AL049464,
AJ238278, AJ242859, AF113694,
AF017152,
AL137463, U35846, AF090943,
AF118094,
AF113691, AF079765, AL137550,
AL117583,
X93495, AL133080, Y11254, AF111851,
AL133075, AF125948, AF146568,
AL133113,
U00763, 561953, A58524, A58523,
AJ000937,
X84990, AL133557, AF158248,
AL122093,
AL133606, X96540, X70685, AL050149,
AL133016, AL110225, AL117394,
AL117457,
AL122098, AL050116, AL080124,
U72620,
AL110221, AF113676, AL137527,
AFl 13690,
AF113019, AF113699, AL049314,
AL137538,
E07108, AF177401, S68736, AL050393,
AF078844, AF091084, X82434,
AF113677,
AF097996, AL080159, 282022,
AL137459,
U80742, AB019565, AL122123,
AL137557,
U42766, AL137648, AL050277,
AL117585,
AF090903, AF125949, AL050108,
A12297,
AL133565, X63574, AL122121,
AL049466,
AF118064, AF090900, AL133640,
E02349,
A03736, E03348, AF113689, AL050024,
AL110196, AL122050, AF087943,
AJ012755,
AL110197, S78214, AF090901,
AL080060,
U67958, AF183393, AL133560,
U91329,
AF106862, AF104032, AF061943,
AL122110,
Y16645, AL049938, AL133093,
AL049430,
AL049382, AL137271, AR059958,
AL080127,
AL080137, AL050138, AL137521,
E07361,
A93016, AL122049, Y09972, AL049300,
X65873,
A08912, AF067728, AL096744,
A93350,
AL137283, I09360, X98834, E05822,
I42402,
AF026124, AL049283, E15569,
AL133072,
AL133014, AF026816, I26207,
AF210052,
AL133077, I17767, AL133568,
AF119337,
U58996, Y07905, AL137556, A45787,
A90832,
AF111112, AR000496, U39656,
Y14314,
AL110280, E08263, E08264, M30514,
I00734,
AL133104, AF003737, E00617,
E00717, E00778,
AF057300, AF057299, X87582,
AL137523,
AL133098, AF079763, AF153205,
AF185576,
AL117440, A07647, AR038969,
AR038854,
AC006371, AF106827, AL050172,
AF162270,
AL137526, 272491, U96683, AL137476,
U68387,
AL137300, AL133067, AL080074,
L19437,
E08631, AL080148, AJ006417,
E04233, L30117,
U49908, AF061573, AL137292,
U78525,
AL137480, AF008439, X92070,
AR013797,
AL122111, AF111849, AL137533,
237987,
AF100931, AL133081, I66342,
AL137294,
AL122118, AF177767, AL137273,
AL050092,
AL117432, E02221, X62580, X81464,
AF051325,
AF081197, AL137429, L13297,
AF132676,
AF061836, and I41145.
HKAFT66 53 889258 AA436785, AI804932, AA310516,
AA251417,
AA873013, AI798761, AA250867,
AI280747,
AI826225, AI811785, AI539808,
AL039086,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
261
AI554821, AI784252, AW105601,
AW054931,
AL119863, AI611738, AW 193134,
AW 118518,
AI610362, AL042628, AL036980,
AI312428,
AA833760, AI829327, AI318280,
AI433384,
AIS89267, AI269862, AI564723,
AI624548,
AW302992, AI802542, AI569583,
AW169653,
AI800453, AI439717, AI567612,
AI611348,
AI869367, AW081797, AL038605,
AI570781,
AW051258, AI802240, AI274728,
AW071417,
AI571909, AW152469, AI612885,
AW022682,
AW 148320, AI636585, AI569579,
AI252023,
AI608936, AW075413, AI500077,
AI247193,
AI251434, AI862144, AI475394,
AI890806,
AI625094, AI955906, AI309401,
AI554344,
AI612913, AL040243, AI431424,
AI343112,
AW268253, AW301300, AI349598,
AL036664,
AW075207, AI925156, AI345735,
AI678357,
AI564247, AI633419, AI348897,
AI587143,
AI500659, AI630928, AI648684,
AI313320,
AI627988, AI251221, AW 193000,
AI340627,
AA640779, AI340603, AI312146,
AI312339,
AA572758, AI889168, AI345258,
AI348854,
AI457369, AW020693, AI445115,
AW075351,
' AI620866, AI270707, AW081242,
AI610645,
AI801325, AI866798, AI932794,
AI689248,
AI340582, AI955917, AA225339,
AA427700,
AI684265, AI955467, AI590415,
AI681985,
AW074993, AI349614, AI282281,
AI811353,
AI354283, AI302910, AA508692,
AI349256,
AI634224, AI312152, AI174394,
H89138,
AW075084, AI567351, AL039132,
AI800433,
AI950664, AL036288, AI349937,
AW089572,
AI334884, AI670009, AI349957,
AI307708,
AI312325, AI609409, AW269097,
AI591420,
AW190042, AI572892, AL045266,
AL121286,
AI916419, AI307520, AI445237,
AI306613,
AI434256, AI917123, AL036901,
AA012905,
AL134999, AI874166, AL036736,
AW 151138,
AW268302, AL121496, AI436456,
AI608667,
AW023590, AI758735, AI306705,
W33163,
AI680388, AW169634, AW170635,
AW073994,
AI887247, AW268122, AI349645,
AW074869,
AI537677, AI308032, AI343059,
AL036631,
AW268072, AW301505, AW403717,
AL036396,
AI349933, AI564736, AI921176,
AI524671,
AI439601, AW084194, AI554245,
AI934036,
AI497733, AI963224, AI288285,
AA857311,
AI273094, AI452560, AW080079,
AL038445,
AI698391, AI538085, AW080279,
AI343030,
AI420521, AW081449, AL042551,
AI281837,
AI336585, AI500706, AI307543,
AI349787,
AI270055, AI918655, AI689420,
AW 149227,
AI889189, AI500662, AI621209,
AI566670,
AL040241, AI620639, AL134259,
AI366992,
AW089310, AI917252, AI434020,
AW268220,
AI653836, AI334450, AI624056,
AI682725,
AW068845, AI683099, AL036403,
AL036945,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
262
AI521012, A1632033, AW088903,
AI923357,
AL038504, AI537515, AL045500,
AL119791,
AW088899, AI440426, I48979,
I48978, AR011880,
AF017437, 189947, L31396, L31397,
AF113013,
AL117460, X72889, AF090934,
I33392, A65341,
A08916, A08913, I89931, AL049452,
A08910,
I49625, A08909, AF090896, AL050146,
Y11587,
A77033, A77035, AL137560, AF118070,
AL117435, I03321, AL049464,
AJ238278,
AJ242859, AF113694, AF017152,
AL137463,
U35846, AF090943, AF118094,
AF113691,
AL137550, AL117583, X93495,
AL133080,
Y11254, AF111851, U00763, AL133075,
AF125948, AF146568, 561953,
A58524, A58523,
X84990, AL133557, AF158248,
AL122093,
AL133606, X96540, X70685, AL050149,
AL133016, AL110225, AL117394,
AL117457,
AL122098, AL050116, AL080124,
U72620,
AL110221, AF113676, AL137527,
AF113690,
AF113019, AF113699, AL049314,
AL137538,
E07108, AF177401, 568736, AL050393,
AF078844, AF091084, X82434,
AF113677,
AF097996, AL080159, 282022,
AL137459,
U80742, AL133113, AB019565,
AL122123,
AL137557, AL137648, U42766,
AL050277,
AL117585, AF090903, AF125949,
AL050108,
A12297, AL133565, AF079765,
X63574,
AL122121, AL049466, AF118064,
AF090900,
AL133640, E02349, A03736, E03348,
AF113689,
AL050024, AL110196, AJ000937,
AL122050,
AF087943, AJ012755, AL110197,
S78214,
AF090901, AL080060, U67958,
AF183393,
AL133560, U91329, AF106862,
AF104032,
AF061943, AL122110, Y16645,
AL049938,
AL133093, AL049430, AL049382,
AL137271,
AR059958, AL080127, AL080137,
AL050138,
AL137521, E07361, A93016, AL122049,
AL049300, X65873, A08912, AF067728,
AL096744, A93350, AL137283,
I09360, X98834,
I42402, Y09972, AF026124, AL049283,
E15569,
AL133014, AF026816, I26207,
AL133077,
AL133072, E05822, AF119337,
U58996,
AL137556, A45787, A90832, E08263,
E08264,
M30514, I00734, AF111112, AL110280,
AR000496, U39656, I17767, Y14314,
E00617,
E00717, E00778, AF057300, AF057299,
AF210052, AL133098, AF079763,
AL133568,
AF003737, AL117440, Y07905,
AL133104,
AF153205, AR038969, AC006371,
A07647,
U96683, AL050172, U68387, AF162270,
272491,
AF185576, X87582, AL133067,
L19437,
AL137523, E08631, AL080148,
AL137476,
AR038854, AL137526, AL080074,
E04233,
AF061573, AL137292, U49908,
L30117,
AF008439, AF106827, U78525,
AF111849,
AL137300, AF100931, AJ006417,
AL050092,
AL137533, AL137480, X81464,
AF051325,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
263
166342, X92070, AR013797, AL122111,
237987,
E02221, I41145, AL137273, AL080086,
AF061981, AF081197, X53587,
AC006112,
AL133081, AL137488, AL137294,
AL117432,
AC006222, and AL122118.
HKAFT66 54 904790 AI804932, AA310516, AA873013,
AA251417,
AI798761, AA250867, and AA436785.
11 HLWAZ70 21 996196 AL121021, AA478639, AI740941,
AA935638,
AI925600, AI804088, AI990095,
AI767129,
AI761835, AA807158, AI990664,
AA281985,
AI263809, AI561047, AL121020,
AA482585,
AI015241, AI452488, AW 130146,
AW 183325,
AI285906, AA252067, AI151521,
N27193,
AA464616, AA621472, AI253619,
AI276734,
AA984520, AI445847, T08918,
AA115579,
H06365, AA912329, H06366, H24140,
AI215547,
AW029608, AI914672, AA721213,
AI474224,
N39939, AA927470, H22959, AA377375,
N43772,
815255, 242938, T94383, AA115553,
AA626206,
AA252049, 239057, T30110, 244683,
AA483097,
F03697, AW022470, AW160534,
AA857847,
AW090492, AA693340, AI439062,
AA629977,
AW090451, AW080076, AI682759,
AI476694,
AF032666, AC004227, and AL133558.
11 HLWAZ70 55 853918 AA419393, AL121020, AA626206,
H06365,
M78773, AA478749, T08918, AA479969,
H24140,
AA377375, 242938, T94471, 815255,
AA252049,
T30110, and 244683.
11 HLWAZ70 56 842503 AA281985, AA115579, AI253619,
T08918,
H24140, N39939, AL121020, 815255,
N43772,
T30110, AL121021, 244683, AA478639,
H06365,
AA252049, 242938, AA935638,
AI740941,
AI925600, AI804088, and AI990095.
11 HLWAZ70 57 837204 AL121021, AA478639, AI740941,
AA935638,
AI925600, AI804088, AI990095,
AI767129,
AI761835, AA807158, AI990664,
AI263809,
AA482585, AI015241, AI561047,
AI452488,
N27193, AW 130146, AW 183325,
AI285906,
AA252067, AI151521, AA464616,
AA621472,
H06366, AI276734, AA984520,
AI445847,
AA912329, AI215547, AW029608,
AA721213,
AI914672, AI474224, AA927470,
H22959,
N43772, T94383, AA115553, 239057,
AA483097,
F03697, AW022470, AI253619,
and AA115579.
12 HLWBG83 22 965821 AI829724, AI694428, N22679,
AI762907,
AI806332, AI380380, AI523992,
AI628644,
AA741042, AI913030, AW303336,
AI299752,
AA748396, AI307811, AW262837,
AI700492,
AW 137241, N34570, AA309892,
AI001938,
AA227556, AA004818, AI265995,
AA485060,
AA085344, AI222772, AI202579,
AA291154,
AI024937, AI002097, F37438,
AA317927,
AI285855, F06707, M79121, AI884776,
H27017,
AA349070, AA127427, AI298473,
H92628,
AA777242, AA016085, 818356,
843342, H92411,
AI341409, 807069, AA765453,
AA082872,
AW001270, N31159, 239721, AI247009,
891161,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
264
AI699824, 842560, 836921, 817620,
AI241808,
AA888480, AA004971, W23194,
AW296173,
AA324057, H44419, 807026, F31857,
801579,
AI867998, AA485153, and T88976.
12 HLWBG83 59 902341 AI829724, AI694428, N22679,
AI762907,
AI806332, AI380380, AI523992,
AI628644,
AA741042, AI913030, AW303336,
AI299752,
AA748396, AI307811, AW262837,
AI700492,
AW 137241, N34570, AA309892,
AI001938,
AA227556, AA004818, AI265995,
AA485060,
AA085344, AI222772, AI202579,
AA291154,
AI024937, AI002097, F37438,
AA317927,
AI285855, AI884776, F06707,
M79121, H27017,
AA349070, AA127427, AI298473,
H92628,
AA777242, AA016085, 843342,
H92411, 818356,
AI341409, 807069, AA082872,
AA765453,
N31159, AW001270, 239721, AI247009,
891161,
AI699824, 842560, 836921, AI241808,
817620,
AA004971, AA888480, W23194,
AW296173,
AA324057, H44419, 807026, F31857,
801579,
AI867998, AA485153, T88976,
AI432644,
AI623302, AW 128846, AI431337,
AW081103,
AI432653, AI431345, AI432649,
AI431351,
AI431354, AI431243, AI432674,
AI432673,
AI432664, AI431346, AI431357,
AI431254,
AI432658, AI432676, AI432665,
AI432654,
AI431241, AI431353, AW 128900,
AI431347,
AI431230, AI431248, AI431328,
AI432655,
AI431310, AI431312, AI431330,
AI432672,
AI432651, AI432647, AI432677,
AI432661,
AI432675, AI492519, AI431255,
AI432662,
AI431340, AI432650, AW 128897,
AI431307,
AI431316, AI791349, AI492520,
AI431236,
AI431751, AI492509, AI492510,
AI432643,
AI432657, AI431308, AI431247,
AW 128884,
AW129223,Y17793, andAF064854.
12 HLWBG83 61 893445 AI205344, T79516, H40127, 853453,
AA308554,
871046, AW003570, T52637, W42479,
T85050,
H03071, AI742770, AW295666,
874283,
AI867731, AW 166915, AI081512,
AI826447,
AW082101, AI887870, AI091580,
AA010979,
AA557279, AI936402, 853454,
AW338583,
W42544, AA377100, AC000400,
and D17093.
13 HRABS65 23 884575 AI018571, T95378, 816525, and
AA730275.
15 HLWAX42 25 1047527 T39369, T40536, T46954, T46980,
T47016,
T48748, T48931, T48932, T49294,
T49295,
T49594, T49595, T52101, T53608,
T53609,
T53780, T55348, T58695, T86946,
T87029,
T89007, 809231, 809343, 809737,
810973,
T80772, T81450, T96992, T97106,
800500,
800601, 800900, 802529, 816480,
816677,
816678, 816780, 821233, 821732,
821924,
822008, 822855, 822938, 823043,
823734,
823784, 825327, 826523, 827583,
827616,
827682, 828197, 828645, 830775,
831271,
831669, 831800, 831852, 831877,
832578,
833658, 833772, 833887, 833888,
834690,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
265
834795, 835968, 836334, R36G16,
836617,
837905, 853822, 853871, 863207,
863254,
863766, 865618, RG5795, 865892,
866039,
866040, 866669, RG7000, 867146,
867250,
867255, 867389, 867494, RG7874,
RG7875,
868144, 868699, 869167, 870512,
870525,
870609, 870625, 874369, 874371,
874603,
875872, 875881, 877664, 877665,
877724,
877831, 877956, 878057, 878329,
878796,
878932, 879031, 879098, R79G45,
879731,
880248, 880360, 880766, 881471,
881936,
881937, 882345, 882759, H00188,
H00932,
H00977, H01187, H01193, H01283,
H01290,
H01310, H01715, H01741, H02301,
H02411,
H02755, H02962, H03055, H03455,
H03539,
H04260, H12600, H12650, H13646,
H13647,
H18383, H20702, H21182, H21846,
H23647,
H23849, H40161, H40182, H40199,
H40200,
H42464, H46515, H46534, 892416,
892415,
894434, 896235, 896290, 898784,
899823,
899891, H49863, H50046, H50439,
H51478,
H53537, H53536, H54070, H54071,
H59583,
H59584, H60972, H61545, H61600,
H61601,
H62263, H63662, H63941, H66992,
H66993,
H68996, H70950, H73711, H74226,
H78317,
H78318, H80281, H80378, H81049,
H81056,
H81051, H81144, H81146, H81153,
H81514,
H89486, H89632, H94918, H95146,
H95182,
N58483, N74660, N78072, N94503,
W00998,
W04151, W23953, W31947, W39660,
W84789,
W84836, W86279, W86323, W93671,
N91085,
AA004267, AA004406, AA029995,
AA029105,
AA043911, AA046219, AA082581,
AA085579,
AA088178, AA088608, AA122102,
AA131924,
AA148932, AA148933, AA148465,
AA148466,
AA148534, AA156623, AA156601,
AA156838,
AA234575, AA234636, H94864,
AA595787,
AA568860, AA747591, AA962143,
D78668,
D78695, D78703, D78757, D78764,
D78775,
D78780, D78806, D78811, D78839,
D78838,
D78832, D79043, D79107, D79189,
N57914,
N63734, N70340, AA005377, C17370,
C17492,
C21504, C16923, C16968, C17009,
C17044,
C17064, C17938, C17954, C18029,
C18327,
C18615, C18791, C18911, C19002,
C19062,
C19086, AA131925, C17677, C17797,
AA443241,
AA599478, AA653499, AA456487,
AA705356,
AA708613, AA709381, AI014957,
AI032539,
AI033574, AI057251, AI075154,
AI086754,
AI088338, AI088494, AI090290,
AI093496,
800047, 809738, AA694405, AA699451,
AA702947, AI271743, AI373324,
AI492481,
AI126573, AI128118, AI129553,
AI150568,
AI151343, AI160937, AI168432,
AI168453,
AI189909, AI200046, AI200363,
AI220869,
AI276116, AI342605, AI702300,
AI768458,
AI953638, AW265710, AW 193364,
AW444884,
CA 02389722 2002-04-30
WO 01/34768 PCT/US00/30039
266
and AW611716.
15 HLWAX42 63 905459 AI110886, AA156838, AI953638,
C17797,
AI457612, AI492481, H51478,
AI743340,
AA156601, AI129553, AI276116,
AI768458,
AA595787, AI088338, N58483,
AA709381,
C18029, AI566069, W04151, AI342605,
AI805980,
N57914, AA131924, AI742050,
H94864,
AI086754, AI075154, AA088178,
W39660,
N70340, AA148932, 881936, AI057251,
D78838,
AA043911, 869918, C18911, N91085,
D78775,
H80378, C16968, AA004406, H21846,
AA443241,
H21182, D78832, 870625, W86323,
AA131925,
H23849, AI014957, N74660, AA708613,
D63290,
C17677, AI393674, H20702, AA005377,
866040,
AA122102, H66992, AA004267,
H81514,
AA088608, AI160937, H59583,
800047, H94918,
AA962143, H60972, 892415, D79043,
D78780,
878329, H42464, AI220869, 894434,
AI090290,
H23647, AA705356, N63734, AI168432,
880360,
867494, T87029, C18615, C17009,
D78695,
867255, 866039, T86946, H53536,
D78757,
H18383, 863766, H12650, H01310,
H50439,
898784, 863254, 821924, C18791,
AA368244,
H01283, AA369083, AI168453,
899891, H81153,
AI200046, 882398, 898785, H80281,
H81146,
867250, 892416, H40182, D63217,
D78806,
825327, 809738, T47017, AA148933,
AA368675,
T96992, H70950, H61600, H01290,
H50046,
W93671, H01187, H03455, D78764,
H00932,
T47016, AA367587, C19062, W86279,
AA653499,
836334, H01193, 831852, H00188,
867389,
810973, H53537, 831800, H00240,
W31947,
H78317, 880766, H61601, H46515,
H46534,
H03054, H03055, T53780, 826523,
816677,
D78668, 882345, H81051, 809737,
H40199,
C17492, 802140, H12600, H59584,
831669,
867874, 821776, H40161, 865892,
H00977,
836616, AA367783, H81056, 881937,
AA327133,
C16923, D78811, T89007, 899823,
876046,
H54070, 823784, C17044, 875881,
821732,
H89486, AA369734, 800900, 877956,
AA085579,
876055, AI373324, 827616, 833887,
878932,
828645, 809343, 896235, H01715,
H66993,
C17064, 865795, 875872, 823734,
835968,
AA082581, H49863, D63268, H54071,
H01741,
809231, C19002, 827583, H03539,
AI702300,
832578, 821233, D78839, H78318,
H61545,
H13646, 827682, AI494132, T48748,
W31324,
863207, H13647, AA046219, 828197,
802529,
W00998, 853822, 816780, 882759,
867875,
870525, C19086, 880248, H68996,
816678,
825628, 816480, 833888, 836617,
AA368730,
T46980, AW 197984, T55348, T80772,
T81450,
822008, 830775, 853871, 866669,
874603,
879031, H02301, H40200, H62263,
and C21504.
15 HLWAX42 64 905721 AI953638, C17797, AI457612,
AI743340,
AI129553, AI276116, AA709381,
AI566069,
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267
W04151, AI342605, AI805980,
AA131924,
N57914, H94864, N70340, D78838,
N91085,
D78775, C16968, AA443241, W86323,
AA131925,
C17677, D63290, AA005377, AA004267,
H81514,
800047, H94918, H60972, D79043,
894434,
878329, AI220869, 880360, N63734,
AI090290,
867494, T87029, C18615, 867255,
H12650,
H53536, D78757, T86946, 898784,
H01310,
863254, C18791, AA368244, H81153,
899891,
AA369083, 882398, 867250, 898785,
D63217,
825327, AA368675, T47017, H70950,
809738,
H61600, W93671, D78764, W86279,
AA653499,
H00240, H53537, H78317, H03054,
H61601,
826523, T53780, C17492, 809737,
H12600,
H40199, 882345, 831669, 802140,
H00977,
821776, H40161, AA367783, AA327133,
H81056,
899823, D78811, C16923, 876046,
823784,
821732, H54070, 875881, C17044,
800900,
877956, AA369734, 876055, 827616,
833887,
878932, 828645, H01715, C17064,
H66993,
875872, 823734, AA082581, H54071,
C19002,
832578, D78839, H78318, H61545,
AI702300,
T48748, AI014957, 863207, 802529,
816780,
882759, 880248, H68996, 816480,
833888,
AA368730, AA369082, AA367501,
AI110886,
AI742050, 830775, 835968, AA367587,
827583,
AA156601, 879031, AW197984,
AI160937,
866669, AA367502, AA088178,
T80772,
AI088338, AI200046, 853871,
870525, 809231,
896235, AI768458, T89007, N58483,
T81450,
H62263, T55348, AA962143, and
AA369013.
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Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Examples
Example 1: Isolation of a Selected cDNA Clone From the Deposited Sample
Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector.
Table 1 identifies the vectors used to construct the cDNA library from which
each
clone was isolated. In many cases, the vector used to construct the library is
a phage
vector from which a plasmid has been excised. The table immediately below
correlates the related plasmid for each phage vector used in constructing the
cDNA
library. For example, where a particular clone is identified in Table 1 as
being
isolated in the vector "Lambda Zap," the corresponding deposited clone is in
"pBluescript."
Vector Used to Construct Library Correspondin~De~osited
Plasmid
Lambda Zap pBluescript (pBS)
Uni-Zap XR pBluescript (pBS)
Zap Express pBK
lafmid BA plafmid BA
pSportl pSportl
pCMVSport 2.0 pCMVSport 2.0
pCMVSport 3.0 pCMVSport 3.0
pCR~2.1 pCR~2.1
Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap
XR (U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic
Acids Res.
16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res.
17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992))
are
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commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey
Pines Road, La Jolla, CA, 92037. pBS contains an ampicillin resistance gene
and
pBK contains a neomycin resistance gene. Both can be transformed into E. coli
strain
XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK-, KS+
and KS. The S and K refers to the orientation of the polylinker to the T7 and
T3
primer sequences which flank the polylinker region ("S" is for SacI and "K" is
for
KpnI which are the first sites on each respective end of the linker). "+" or "-
" refer to
the orientation of the fl origin of replication ("ori"), such that in one
orientation,
single stranded rescue initiated from the fl on generates sense strand DNA and
in the
other, antisense.
Vectors pSportl, pCMVSport 2.0 and pCMVSport 3.0, were obtained from
Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport
vectors
contain an ampicillin resistance gene and may be transformed into E. coli
strain
DH10B, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 (1993).) Vector lafinid BA (Bento Soares, Columbia
University,
NY) contains an ampicillin resistance gene and can be transformed into E. coli
strain
XL-1 Blue. Vector pCR~2.1, which is available from Invitrogen, 1600 Faraday
Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed into E. coli strain DH10B, available from Life Technologies. (See,
for
instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et
al.,
Bio/Technology 9: (1991).) Preferably, a polynucleotide of the present
invention
does not comprise the phage vector sequences identified for the particular
clone in
Table 1, as well as the corresponding plasmid vector sequences designated
above.
The deposited material in the sample assigned the ATCC Deposit Number
cited in Table 1 for any given cDNA clone also may contain one or more
additional
plasmids, each comprising a cDNA clone different from that given clone. Thus,
deposits sharing the same ATCC Deposit Number contain at least a plasmid for
each
cDNA clone identified in Table 1. Typically, each ATCC deposit sample cited in
Table 1 comprises a mixture of approximately equal amounts (by weight) of
about 50
plasmid DNAs, each containing a different cDNA clone; but such a deposit
sample
may include plasmids for more or less than SO cDNA clones, up to about 500
cDNA
clones.
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Two approaches can be used to isolate a particular clone from the deposited
sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is
directly
isolated by screening the clones using a polynucleotide probe corresponding to
SEQ
ID NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported.
The oligonucleotide is labeled, for instance, with 32P-y-ATP using T4
polynucleotide
kinase and purified according to routine methods. (E.g., Maniatis et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY
(1982).)
The plasmid mixture is transformed into a suitable host, as indicated above
(such as
XL-1 Blue (Stratagene)) using techniques known to those of skill in the art,
such as
those provided by the vector supplier or in related publications or patents
cited above.
The transformants are plated on 1.5% agar plates (containing the appropriate
selection
agent, e.g., ampicillin) to a density of about 150 transformants (colonies)
per plate.
1 S These plates are screened using Nylon membranes according to routine
methods for
bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A
Laboratory
Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to
1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the
SEQ m NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5' NT and
the 3' NT of the clone defined in Table 1 ) are synthesized and used to
amplify the
desired cDNA using the deposited cDNA plasmid as a template. The polymerase
chain reaction is carned out under routine conditions, for instance, in 25 u1
of reaction
mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture
is
1.5-S mM MgClz, 0.01 % (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25
pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation at 94 degree C for 1 min; annealing at 55 degree C for 1 min;
elongation
at 72 degree C for 1 min) are performed with a Perkin-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and
the DNA band with expected molecular weight is excised and purified. The PCR
product is verified to be the selected sequence by subcloning and sequencing
the
DNA product.
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Several methods are available for the identification of the S' or 3' non-
coding
portions of a gene which may not be present in the deposited clone. These
methods
include but are not limited to, filter probing, clone enrichment using
specific probes,
and protocols similar or identical to 5' and 3' "RACE" protocols which are
well
known in the art. For instance, a method similar to 5' RACE is available for
generating the missing 5' end of a desired full-length transcript. (Fromont-
Racine et
al., Nucleic Acids Res. 21(7):1683-1684 (1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a
population of RNA presumably containing full-length gene RNA transcripts. A
primer set containing a primer specific to the ligated RNA oligonucleotide and
a
primer specific to a known sequence of the gene of interest is used to PCR
amplify
the 5' portion of the desired full-length gene. This amplified product may
then be
sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source,
although poly-A+ RNA can be used. The RNA preparation can then be treated with
phosphatase if necessary to eliminate 5' phosphate groups on degraded or
damaged
RNA which may interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid pyrophosphatase in
order
to remove the cap structure present at the 5' ends of messenger RNAs. This
reaction
leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can
then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction is
used as a template for PCR amplification of the desired 5' end using a primer
specific
to the ligated RNA oligonucleotide and a primer specific to the known sequence
of
the gene of interest. The resultant product is then sequenced and analyzed to
confirm
that the 5' end sequence belongs to the desired gene.
Example 2: Isolation of Genomic Clones Corresponding to a Polynucleotide
A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR
using primers selected for the cDNA sequence corresponding to SEQ ID NO:X.,
according to the method described in Example 1. (See also, Sambrook.)
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Example 3: Tissue Distribution of Polypeptide
Tissue distribution of mRNA expression of polynucleotides of the present
invention is determined using protocols for Northern blot analysis, described
by,
among others, Sambrook et al. For example, a cDNA probe produced by the method
described in Example 1 is labeled with P32 using the rediprimeTM DNA labeling
system (Amersham Life Science), according to manufacturer's instructions.
After
labeling, the probe is purified using CHROMA SPIN-100TM column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The
purified labeled probe is then used to examine various human tissues for mRNA
expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H)
or human immune system tissues (IM) (Clontech) are examined with the labeled
probe using ExpressHybTM hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization and washing,
the
blots are mounted and exposed to film at -70 degree C overnight, and the films
developed according to standard procedures.
Example 4: Chromosomal MappinE of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5'
end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This
primer set is then used in a polymerase chain reaction under the following set
of
conditions : 30 seconds,95 degree C; 1 minute, 56 degree C; 1 minute, 70
degree C.
This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C.
Human, mouse, and hamster DNA is used as template in addition to a somatic
cell
hybrid panel containing individual chromosomes or chromosome fragments (Bios,
Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5 %
agarose
gels. Chromosome mapping is determined by the presence of an approximately 100
by PCR fragment in the particular somatic cell hybrid.
Example 5: Bacterial Expression of a Polypeptide
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A polynucleotide encoding a polypeptide of the present invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA
sequence, as outlined in Example 1, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHI and XbaI, at the S' end of the primers in order to clone the amplified
product
into the expression vector. For example, BamHI and XbaI correspond to the
restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc.,
Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Amps, a
bacterial origin of replication (ori), an IPTG-regulatable promoter/operator
(P/0), a
ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme
cloning
sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified
fragment is ligated into the pQE-9 vector maintaining the reading frame
initiated at
the bacterial RBS. The ligation mixture is then used to transform the E. coli
strain
M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4,
which
expresses the lacI repressor and also confers kanamycin resistance (Kan~.
Transformants are identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated
and
confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/I~ in liquid
culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml).
The O/N culture is used to inoculate a large culture at a ratio of 1:100 to
1:250. The
cells are grown to an optical density 600 (O.D.6oo) of between 0.4 and 0.6.
IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of 1
mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading
to
increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCl by stirnng for 3-4 hours at 4 degree C. The cell
debris
is removed by centrifugation, and the supernatant containing the polypeptide
is loaded
onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(available from
QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with
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high affinity and can be purified in a simple one-step procedure (for details
see: The
QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HC1, pH
8, the column is first washed with 10 volumes of 6 M guanidine-HCI, pH 8, then
washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide
is
eluted with 6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI.
Alternatively, the protein can be successfully refolded while immobilized on
the Ni-
NTA column. The recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCI, 20% glycerol, 20 mM Tris/HCl pH 7.4,
containing protease inhibitors. The renaturation should be performed over a
period of
1.5 hours or more. After renaturation the proteins are eluted by the addition
of 250
mM immidazole. Immidazole is removed by a final dialyzing step against PBS or
50
mM sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored
at
4 degree C or frozen at -80 degree C.
In addition to the above expression vector, the present invention further
includes an expression vector comprising phage operator and promoter elements
operatively linked to a polynucleotide of the present invention, called pHE4a.
(ATCC
Accession Number 209645, deposited on February 25, 1998.) This vector
contains:
1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli
origin of
replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences,
5) a
Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The
origin of replication (oriC) is derived from pUC 19 (LTI, Gaithersburg, MD).
The
promoter sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and
isolating
the larger fragment (the stuffer fragment should be about 310 base pairs). The
DNA
insert is generated according to the PCR protocol described in Example 1,
using PCR
primers having restriction sites for NdeI (5' primer) and XbaI, BamHI, XhoI,
or
Asp718 (3' primer). The PCR insert is gel purified and restricted with
compatible
enzymes. The insert and vector are ligated according to standard protocols.
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The engineered vector could easily be substituted in the above protocol to
express protein in a bacterial system.
Example 6: Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide,
expressed in E coli when it is present in the form of inclusion bodies. Unless
otherwise specified, all of the following steps are conducted at 4-10 degree
C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is cooled to 4-10 degree C and the cells harvested by continuous
centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected
yield
of protein per unit weight of cell paste and the amount of purified protein
required, an
appropriate amount of cell paste, by weight, is suspended in a buffer solution
containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a
homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate
is then mixed with NaCI solution to a final concentration of 0.5 M NaCI,
followed by
centrifugation at 7000 xg for 15 min. The resultant pellet is washed again
using O.SM
NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 xg centrifugation for 15 min.,
the
pellet is discarded and the polypeptide containing supernatant is incubated at
4 degree
C overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 xg) to remove insoluble particles,
the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract
with
20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA
by vigorous stirnng. The refolded diluted protein solution is kept at 4 degree
C
without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential
filtration unit equipped with 0.16 um membrane filter with appropriate surface
area
(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The
filtered sample is loaded onto a canon exchange resin (e.g., Poros HS-50,
Perseptive
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Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a
stepwise manner. The absorbance at 280 nm of the effluent is continuously
monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of
tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak
anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6Ø Both columns are washed with
40
mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using
a 10 column volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium
acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are
collected under constant AZBO monitoring of the effluent. Fractions containing
the
polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the
above refolding and purification steps. No major contaminant bands should be
observed from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified
protein is loaded. The purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml according
to LAL
assays.
Example 7: Cloning and Expression of a Polypeptide in a Baculovirus
Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a
polynucleotide into a baculovirus to express a polypeptide. This expression
vector
contains the strong polyhedrin promoter of the Autographs californica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as
BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40
("SV40")
is used for efficient polyadenylation. For easy selection of recombinant
virus, the
plasmid contains the beta-galactosidase gene from E. coli under control of a
weak
Drosophila promoter in the same orientation, followed by the polyadenylation
signal
of the polyhedrin gene. The inserted genes are flanked on both sides by viral
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sequences for cell-mediated homologous recombination with wild-type viral DNA
to
generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such
as pAc373, pVL941, and pAcIMI, as one skilled in the art would readily
appreciate,
as long as the construct provides appropriately located signals for
transcription,
translation, secretion and the like, including a signal peptide and an in-
frame AUG as
required. Such vectors are described, for instance, in Luckow et al., Virology
170:31-
39 (1989).
Specifically, the cDNA sequence contained in the deposited clone, including
the AUG initiation codon and the naturally associated leader sequence
identified in
Table 1, is amplified using the PCR protocol described in Example 1. If the
naturally
occurring signal sequence is used to produce the secreted protein, the pA2
vector does
not need a second signal peptide. Alternatively, the vector can be modified
(pA2 GP)
to include a baculovirus leader sequence, using the standard methods described
in
Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures," Texas Agricultural Experimental Station Bulletin No. 1555
(1987).
The amplified fragment is isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with appropriate restriction enzymes and again purified on a
1
agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue
(Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the
ligation
mixture and spread on culture plates. Bacteria containing the plasmid are
identified
by digesting DNA from individual colonies and analyzing the digestion product
by
gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA
sequencing.
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Five ug of a plasmid containing the polynucleotide is co-transfected with 1.0
ug of a commercially available linearized baculovirus DNA ("BaculoGoldT"'
baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
One ug
of BaculoGoldTM virus DNA and 5 ug of the plasmid are mixed in a sterile well
of a
microtiter plate containing 50 u1 of serum-free Grace's medium (Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 u1 Lipofectin plus 90 u1 Grace's
medium are
added, mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture is added drop-wise to Sf~ insect cells (ATCC CRL 1711)
seeded
in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The
plate is
then incubated for S hours at 27 degrees C. The transfection solution is then
removed
from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal
calf
serum is added. Cultivation is then continued at 27 degrees C for four days.
After four days the supernatant is collected and a plaque assay is performed,
as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life
Technologies Inc., Gaithersburg) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of
a "plaque assay" of this type can also be found in the user's guide for insect
cell
culture and baculovirology distributed by Life Technologies Inc.,
Gaithersburg, page
9-10.) After appropriate incubation, blue stained plaques are picked with the
tip of a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 u1 of Grace's medium and
the
suspension containing the recombinant baculovirus is used to infect Sf~ cells
seeded
in 35 mm dishes. Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf~ cells are grown in Grace's
medium supplemented with 10% heat-inactivated FBS. The cells are infected with
the recombinant baculovirus containing the polynucleotide at a multiplicity of
infection ("MOI") of about 2. If radiolabeled proteins are desired, 6 hours
later the
medium is removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Rockville, MD). After 42
hours, 5
uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are
added.
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The cells are further incubated for 16 hours and then are harvested by
centrifugation.
The proteins in the supernatant as well as the intracellular proteins are
analyzed by
SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of
purified protein may be used to determine the amino terminal sequence of the
produced protein.
Example 8: Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian
cell. A typical mammalian expression vector contains a promoter element, which
mediates the initiation of transcription of mRNA, a protein coding sequence,
and
signals required for the termination of transcription and polyadenylation of
the
transcript. Additional elements include enhancers, Kozak sequences and
intervening
sequences flanked by donor and acceptor sites for RNA splicing. Highly
efficient
transcription is achieved with the early and late promoters from SV40, the
long
terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
promoter of the cytomegalovirus (CMV). However, cellular elements can also be
used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention
include,
for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBCI2MI (ATCC 67109),
pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1,
Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
Alternatively, the polypeptide can be expressed in stable cell lines
containing
the polynucleotide integrated into a chromosome. The co-transfection with a
selectable marker such as dhfr, gpt, neomycin, hygromycin allows the
identification
and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful in
developing
cell lines that carry several hundred or even several thousand copies of the
gene of
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interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin, J.
L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and
Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection
marker
is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279
(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these
markers,
the mammalian cells are grown in selective medium and the cells with the
highest
resistance are selected. These cell lines contain the amplified genes)
integrated into a
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the
expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et
al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of
the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g.,
with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate
the
cloning of the gene of interest. The vectors also contain the 3' iritron, the
polyadenylation and termination signal of the rat preproinsulin gene, and the
mouse
DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate
restriction enzymes and then dephosphorylated using calf intestinal phosphates
by
procedures known in the art. The vector is then isolated from a 1 % agarose
gel.
A polynucleotide of the present invention is amplified according to the
protocol outlined in Example 1. If the naturally occurnng signal sequence is
used to
produce the secreted protein, the vector does not need a second signal
peptide.
Alternatively, if the naturally occurring signal sequence is not used, the
vector can be
modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with appropriate restriction enzymes and again purified on a
1
agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector
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are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment inserted
into plasmid
pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for
transfection. Five ~g of the expression plasmid pC6 a pC4 is cotransfected
with 0.5
ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid
pSV2-
neo contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including 6418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml 6418. After 2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in
alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1
mg/ml 6418. After about 10-14 days single clones are trypsinized and then
seeded in
6-well petri dishes or 10 ml flasks using different concentrations of
methotrexate (50
nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well plates
containing
even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained which grow at a
concentration of 100 - 200 uM. Expression of the desired gene product is
analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9: Protein Fusions
The polypeptides of the present invention are preferably fused to other
proteins. These fusion proteins can be used for a variety of applications. For
example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG
domains, and maltose binding protein facilitates purification. (See Example 5;
see
also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly,
fusion to
IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear
localization
signals fused to the polypeptides of the present invention can target the
protein to a
specific subcellular localization, while covalent heterodimer or homodimers
can
increase or decrease the activity of a fusion protein. Fusion proteins can
also create
chimeric molecules having more than one function. Finally, fusion proteins can
increase solubility and/or stability of the fused protein compared to the non-
fused
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protein. All of the types of fusion proteins described above can be made by
modifying the following protocol, which outlines the fusion of a polypeptide
to an
IgG molecule, or the protocol described in Example S.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified,
using primers that span the S' and 3' ends of the sequence described below.
These
primers also should have convenient restriction enzyme sites that will
facilitate
cloning into an expression vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc portion
can be ligated into the BamHI cloning site. Note that the 3' BamHI site should
be
destroyed. Next, the vector containing the human Fc portion is re-restricted
with
BamHI, linearizing the vector, and a polynucleotide of the present invention,
isolated
by the PCR protocol described in Example 1, is ligated into this BamHI site.
Note
that the polynucleotide is cloned without a stop codon, otherwise a fusion
protein will
not be produced.
If the naturally occurnng signal sequence is used to produce the secreted
protein, pC4 does not need a second signal peptide. Alternatively, if the
naturally
occurring signal sequence is not used, the vector can be modified to include a
heterologous signal sequence. (See, e.g., WO 96/34891.)
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGT
GGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG
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GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1)
Example 10: Production of an Antibody from a Polypeptide
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such methods,
cells
expressing a polypeptide of the present invention is administered to an animal
to
induce the production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the secreted protein is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonal antisera of greater specific
activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology. (Kohler et al., Nature
256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,
Eur. J.
Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures
involve immunizing an animal (preferably a mouse) with polypeptide or, more
preferably, with a secreted polypeptide-expressing cell. Such cells may be
cultured in
any suitable tissue culture medium; however, it is preferable to culture cells
in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated
at
about 56 degrees C), and supplemented with about 10 g/1 of nonessential amino
acids,
about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma
cells obtained through such a selection are then assayed to identify clones
which
secrete antibodies capable of binding the polypeptide.
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Alternatively, additional antibodies capable of binding to the polypeptide can
be produced in a two-step procedure using anti-idiotypic antibodies. Such a
method
makes use of the fact that antibodies are themselves antigens, and therefore,
it is
possible to obtain an antibody which binds to a second antibody. In accordance
with
this method, protein specific antibodies are used to immunize an animal,
preferably a
mouse. The splenocytes of such an animal are then used to produce hybridoma
cells,
and the hybridoma cells are screened to identify clones which produce an
antibody
whose ability to bind to the protein-specific antibody can be blocked by the
polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-
specific antibody and can be used to immunize an animal to induce formation of
further protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies of the present invention may be used according to the methods
disclosed
herein. Such fragments are typically produced by proteolytic cleavage, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2
fragments). Alternatively, secreted protein-binding fragments can be produced
through the application of recombinant DNA technology or through synthetic
chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using genetic constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric antibodies are
known in
the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et
al., EP 171496; Mornson et al., EP 173494; Neuberger et al., WO 8601533;
Robinson
et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,
Nature
314:268 (1985).)
Examine 11: Production Of Secreted Protein For High-Throughput Screening
As- says
The following protocol produces a supernatant containing a polypeptide to be
tested. This supernatant can then be used in the Screening Assays described
herein.
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First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution
(lmg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker)
for
a working solution of SOug/ml. Add 200 u1 of this solution to each well (24
well
plates) and incubate at RT for 20 minutes. Be sure to distribute the solution
over each
well (note: a 12-channel pipetter may be used with tips on every other
channel).
Aspirate off the Poly-D-Lysine solution and rinse with lml PBS (Phosphate
Buffered
Saline). The PBS should remain in the well until just prior to plating the
cells and
plates may be poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2 x 105 cells/well in .5m1
DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine
(12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx
Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 u1 Lipofectamine
(18324-012 Gibco/BRL) and Sml Optimem I (31985070 Gibco/BRL)/96-well plate.
With a small volume multi-channel pipetter, aliquot approximately tug of an
expression vector containing a polynucleotide insert, produced by the methods
described in Examples 8 or 9, into an appropriately labeled 96-well round
bottom
plate. With a mufti-channel pipetter, add SOuI of the Lipofectamine/Optimem I
mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45
minutes. After about 20 minutes, use a mufti-channel pipetter to add 150u1
Optimem
I to each well. As a control, one plate of vector DNA lacking an insert should
be
transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following
tasks. By tag-teaming, hands on time is cut in half, and the cells do not
spend too
much time on PBS. First, person A aspirates off the media from four 24-well
plates
of cells, and then person B rinses each well with .5-lml PBS. Person A then
aspirates
off PBS rinse, and person B, using alt-channel pipetter with tips on every
other
channel, adds the 200u1 of DNA/Lipofectamine/Optimem I complex to the odd
wells
first, then to the even wells, to each row on the 24-well plates. Incubate at
37 degrees
C for 6 hours.
While cells are incubating, prepare appropriate media, either 1%BSA in
DMEM with lx penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130
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mg/L CuS04-SHZO; 0.050 mg/L of Fe(N03)3-9H20; 0.417 mg/L of FeS04-7H20;
311.80 mg/L of Kcl; 28.64 mg/L of MgClz; 48.84 mg/L of MgS04; 6995.50 mg/L of
NaCI; 2400.0 mg/L of NaHC03; 62.50 mg/L of NaHZP04-H20; 71.02 mg/L of
Na2HP04; .4320 mg/L of ZnS04-7H20; .002 mg/L of Arachidonic Acid ; 1.022 mg/L
of Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of
Linoleic
Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100
mg/L of
Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of
D-
Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50
mg/ml
of L-Asparagine-H20; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-
HZO; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-
Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0
mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2HZ0; 99.65 mg/ml of L-
Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L
of
Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319
mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and
0.680 mg/L of Vitamin B,Z; 25 mM of HEPES Buffer; 2.39 mg/L of Na
Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20uM of
Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-
Cyclodextrin
complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed
with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal)
with 2mm glutamine and lx penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in
1L
DMEM for a 10% BSA stock solution). Filter the media and collect 50 u1 for
endotoxin assay in 15m1 polystyrene conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end
of the incubation period. Person A aspirates off the transfection media, while
person
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B adds 1.5m1 appropriate media to each well. Incubate at 37 degrees C for 45
or 72
hours depending on the media used: 1%BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one lml
deep well plate and the remaining supernatant into a 2m1 deep well. The
supernatants
from each well can then be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any of the
assays
described below using a supernatant, the activity originates from either the
polypeptide directly (e.g., as a secreted protein) or by the polypeptide
inducing
expression of other proteins, which are then secreted into the supernatant.
Thus, the
invention further provides a method of identifying the protein in the
supernatant
characterized by an activity in a particular assay.
Example 12: Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and
proliferation of cells is called the Jaks-STATs pathway. Activated proteins in
the
Jaks-STATs pathway bind to gamma activation site "GAS" elements or interferon-
sensitive responsive element ("IS1RE"), located in the promoter of many genes.
The
binding of a protein to these elements alter the expression of the associated
gene.
GAS and ISIRE elements are recognized by a class of transcription factors
called Signal Transducers and Activators of Transcription, or "STATs." There
are six
members of the STATs family. Statl and Stat3 are present in many cell types,
as is
Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and
is not in
many cell types though it has been found in T helper class I, cells after
treatment with
IL-12. StatS was originally called mammary growth factor, but has been found
at
higher concentrations in other cells including myeloid cells. It can be
activated in
tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus
upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase
("Jaks")
family. Jaks represent a distinct family of soluble tyrosine kinases and
include Tyk2,
Jakl, Jak2, and Jak3. These kinases display significant sequence similarity
and are
generally catalytically inactive in resting cells.
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The Jaks are activated by a wide range of receptors summarized in the Table
below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-
51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided
into two
groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-
12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)
Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a
conserved
cysteine motif (a set of four conserved cysteines and one tryptophan) and a
WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID N0:2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
activate STATs, which then translocate and bind to GAS elements. This entire
process is encompassed in the Jaks-STATs signal transduction pathway.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the GAS or the ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth factors and
cytokines
are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by
using
GAS elements linked to reporter molecules, activators of the Jaks-STATs
pathway
can be identified.
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