CA 02364209 2001-09-24
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49 Human Secreted Proteins
Field of the Invention
This invention relates to newly identified polynucleotides and the
polypeptides encoded by these polynucleotides, uses of such polynucleotides
and
polypeptides, 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
IS redculum (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
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growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of
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
S 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 poiynucleotides 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, andlor conditions.
The
IS 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
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preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
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 polynucieotides 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, 1S
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., S' 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 S' 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.
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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
S 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,
Mantissas, 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 ~g/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 NaHZP04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, I00 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with IXSSPE,
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 andlor substitution of alternate blocking reagents used to suppress
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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 hybridisation conditions described
above,
due to problems with compatibility.
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 polynuclevtide 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 mare 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
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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
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, amffdation, 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
phosphotidylinositot,
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
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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.)
Many proteins (and translated DNA sequences) contain regions where the
amino acid composition is highly biased toward a small subset of the available
residues. For example, membrane spanning domains and signal peptides (which
are
also membrane spanning) typically contain long stretches where Leucine (L),
Valine
(V), Alanine (A), and Isoleucine (I) predominate. Poly-Adenosine tracts
(polyA) at
the end of cDNAs appear in forward translations as poly-Lysine (poly-K) and
poly-
Phenylalanine (poly-F) when the reverse complement is translated. These
regions are
often referred to as "low complexity" regions.
Such regions can cause database similarity search programs such as BLAST to
find high-scoring sequence matches that do not imply true homology. The
problem is
exacerbated by the fact that most weight matrices (used to score the
alignments
generated by BLAST) give a match between any of a group of hydrophobic amino
acids (L,V and I) that are commonly found in certain low complexity regions
almost
as high a score as for exact matches.
In order to compensate for this, BLASTX.2 (version 2.Oa5MP-WashU)
employs two filters ("seg" and "xnu") which "mask" the low complexity regions
in a
particular sequence. These filters parse the sequence for such regions, and
create a
new sequence in which the amino acids in the low complexity region have been
replaced with the character "X". This is then used as the input sequence
(sometimes
referred to herein as "Query" and/or "Q") to the BLASTX program. While this
regime helps to ensure that high-scoring matches represent true homology,
there is a
negative consequence in that the BLASTX program uses the query sequence that
has
been masked by the filters to draw alignments.
Thus, a stretch of "X"s in an alignment shown in the following application
does not necessarily indicate that either the underlying DNA sequence or the
translated protein sequence is unknown or uncertain. Nor is the presence of
such
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8
stretches meant to indicate that the sequence is identical or not identical to
the
sequence disclosed in the alignment of the present invention. Such stretches
may
simply indicate that the BLAffX program masked amino acids in that region due
to
the detection of a tow complexity region, as defined above. In all cases, the
reference
sequences) (sometimes referred to herein as "Subject", "Sbjct", and/or "S")
indicated
in the specification, sequence table (Table 1 ), andlor the deposited clone is
(are) the
definitive embodiments) of the present invention, and should not be construed
as
limiting the present invention to the partial sequence shown in an alignment,
unless
specifically noted otherwise herein.
Po~ynucleotides and Po,~rneutides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Ovarian Tumor l0-3-95; Spleen, Chronic lymphocytic
leukemia.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 60 as residues: Ser-21 to Phe-31.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:11 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1712 of SEQ ID NO:
I 1, b
is an integer of 15 to 1726, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO: I l, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
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y
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gnlIPIDIe134S132 (all information available through the recited accession
number is
S incorporated herein by reference) which is described therein as "cDNA EST
yk42Sa6.3 comes from this gene; cDNA EST yk406e6.3 comes from this gene;
cDNA EST yk42Sa6.S comes from this gene: eDNA EST yk480c6.S comes from this
gene; cDNA EST yk406e6.5 comes from this gene (Caenorhabditis elegans]". A
partial alignment demonstrating the observed homology is shown immediately
below.
>gnllPIDle1345132 cDNA EST yk425a6.3 comes from this gene; cDNA EST
yk406e6.3
cDNA
comes from this gene; cDNA EST yk425a6.5 comes from this gene;
IS EST yk480c6.5 comes from this gene; cDNA EST yk406e6.5 comes
from
F02A9.4B
this gene [Caenorhabditis elegans] >spIE13451321E1345132
PROTEIN.
Length = 414
Plus Strand HSPs:
Score = 192 (67.6 bits), Expect = 3.7e-16, Sum P(2) = 3.7e-16
2S Identities = 53/236 (228), Positives = 109/236 (468), Frame = +1
Q: 331 QNLGIEKEAVLLNLKDNEELAEQGTSFSKTCLTQFLEDAYPDLPTEGIKSLVDFLTSEEV 510
+N+ + + + N EL ++G L ++L P E I+++ L +E
S: 95 ENVESAQPGAEVGTEHNAELVKRGEQNLSIWLKRYLRFHLAKAPEELIEAIDSHLLDDEC 154
Q: 511 VCHVARNLAVEQLTLSAEFPVPPPVLQKTFFAVIGALLQSSGPERTALFIRDFLIPQMTG 690
+ +A +L ++ L + EFP+ F A+ G E+ + DF++PQ+
S: 155 LAGIASHLGIDHLVRTKEFPISQESSADAFRALAGVF----SDEKVKNLVIDFIVPQLVD 210
3S Q: 691 KELF--EIWRIINPMGLLVEELKKRNISPPESRLTRQSGSTTALPVFFVGLYCDK-RLIA 861
+ +I+ + +p+ ++ + LK ++ E R+ R +G +A P++ V +y DK K +
S: 211 IDFAGNDIYPLADPLAWTDLLKSNGVTEIEPRVLRSAGENSAEPIYWAIYADKLKNVG 270
Q: 862 EGPGETXXXXXXXXXXXXXXKLYGFTENRRPWDYSKPKENLRVEKTSLPA-NLSK*WQPG 1038
4Q + GE+ +L+ T + + + N+ +E+ S P +LS+ PG
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S: 271 QSAGESLAIAVDMAAREALLRLWDITSEKVLF-FGDRAANVPLERYSQPNFSLSQKCSPG 329
The segment of gnIIPIDle1345132 that is shown as "S" above is set out in the
sequence listing as SEQ ID NO. 109. Based an the structural similarity, these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 110 which
corresponds to the "Q" sequence in the alignment shown above (gaps introduced
in a
sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in H. Ovarian
Tumor, II, OV5232.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 61 as residues: Pro-50 to Trp-62, Lys-95 to Ser-114.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:12 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1173 of SEQ ID
N0:12, b
is an integer of 15 to 11$7, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:12, and where b is greater than or
equal to a
+ l4.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Stratagene liver (#937224); Human Synovial Sarcoma;
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Activated T-cell( 12h)/Thiouridine-re-excision and to a lesser extent in H.
Epididiymus, caput & corpus; Liver, Hepatoma: Saares ovary tumor NbHOT; Human
Adult Heart; LNCAP untreated; Human OB HOS control fraction l; Human Fetal
Spleen; CHME Cell Line,untreated; T-Cell PHA 16 hrs; Human Fetal Dura Mater;
Human Rhabdomyosarcoma; NTERA2, control: Pancreas Islet Cell Tumor; Human
Gall Bladder; human tonsils; Endothelial-induced; Human Microvascular
Endothelial
Cells, fract. A; Human 8 Week Whole Embryo; Human Cerebellum; Soares placenta
Nb2HP and Soares fetal liver spleen 1NFLS.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 62 as residues: Lys-30 to Pro-36.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:13 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between i to I 135 of SEQ ID
N0:13, b
is an integer of IS to I 149, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID NO: l3, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gi11710216 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "Homo sapiens
protein". A partial alignment demonstrating the observed homology is shown
immediately below.
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12
>gi~1710216 unknown [Homo Sapiens] >sp~Q99770~Q99770 HYPOTHETICAL 15.4 KD
PROTEIN.
Length = 139
S Hinus Strand HSPs:
Score = 159 (56.0 bits), Expect = 2.8e-12, Sum P(2] = 2.8e-12
Identities = 38/79 (48%), Positives = 49/79 (62%), Frame = -3
lO Q: 1346 FFFFLRQGLALLSRHECSGMVMAYCILDLLGSSNPHASASQVAGTTGVGHYARLLFLFFV 1167
FFFF Q ++ +R ECSG + A+C L L GSSN ASAS+VAGT G A+L+F+F
S: SO FFFFETQSHSV-TRLECSGTISAHCNLCLPGSSNSPASASRVAGTAGTCRRAQLIFVFLA 108
Q: 1166 ETGSHYVRPVQYSLVLNLH 1110
IS E G H+V L L +H
S: 109 EMGFHHVGRDGLDLNLVIH 127
The segment of gi117102I6 that is shown as "S" above is set out in the
sequence listing as SEQ ID NO. 111 .
20 Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 112 which
corresponds to the "Q" sequence in the alignment shown above (gaps introduced
in a
sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
2S tissues/eDNA libraries: Primary Dendritic cells,frac 2; Primary Dendritie
Cells, lib 1
and to a lesser extent in L428; Rejected Kidney, lib 4; Human Bone Marrow,
treated;
Hodgkin's Lymphoma II; Keratinocyte; Human Lung; Myoloid Progenitor Cell Line;
Mole Cell Line GM-CSF treated ( 1 ng/ml); Human Thymus Stromal Cells; NTERA2,
control; Human Liver, normal; H. Leukocytes, normalized cot S00 A; Nasal
polyps;
30 Hodgkin's Lymphoma 1; Human T'onsiis, Lib 2; pBMC stimulated w/ poly I/C;
Ovarian Tumor l0-3-9S; I-I. Ovarian Tumor, II, OVS232; Human Heart; Liver,
Hepatoma; Bone Marrow Stromal Cell, untreated; Human Placenta; Dendritic
cells,
pooled and T Cell helper I.
Many polynucleotide sequences, such as EST sequences, are publicly
3S available and accessible through sequence databases. Some of these
sequences are
related to SEQ ID N0:14 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
CA 02364209 2001-09-24
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!3
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1334 of SEQ ID
N0:14, b
is an integer of 15 to 1348, where both a and b correspond to the positions of
nucleotide residues shown in SEQ iD N0:14, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY G ENE NO: 5
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: H, Frontal Cortex. Epileptic; Human Whole Brain #2 -
Oligo
dT > I.SKb.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:15 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1934 of SEQ ID
NO:15, b
is an integer of 15 to 1948, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO: I5, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
It has been discovered that this gene is expressed primarily in the following
tissuesIcDNA libraries: CD34 positive cells (Cord Blood) and to a lesser
extent in
Frontal lobe,dementia,re-excision; Human adult small intestine,re-excision;
Human
Stomach,re-excision; Soares_fetal_heart_NbHH 19W; Human Manic Depression
Tissue; KMH2; Human Adult Pulmonary.re-excision; HUMAN B CELL
LYMPHOMA and T cell helper II.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
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1-1
related to SEQ ID NO: l6 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between i to 1618 of SEQ iD
NO:16, b
is an integer of 15 to 1632, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:16. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
It has been discovered that this gene is expressed primarily in LNCAP prostate
cell line.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Sorne of these sequences
are
related to SEQ 1D N0:17 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1132 of SEQ ID
N0:17, b
is an integer of 15 to 1146, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:17, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
bbsl 121221 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "orf 1 5' of
EpoR
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li
(mice, Peptide, 85 aa( BMus sp. ~". A partial alignment demonstrating the
observed
homology is shown immediately below.
>bbs1121221 orfl 5' of EpoR (mice, Peptide, 85 aa] [Mus sp.)
S >sp~Q64238~Q69238
ORF1 5' OF EPOR.
Length = 85
I0
Plus Strand HSPs:
Score = 114 (40.1 bits),. Expect = 2.2e-U5, P = 2.2e-05
Identities = 24/68 (35%), Positives = 39/68 (57%), Frame = +2
Q: 92 VC--ICTHIYLCVYAYIYTCVYMHTYILVCVCTYAYIYTCMCMYICIHTYIHTRTHMHIS 265
IS +C IC + C+yAy V +TY+ +C+(; y Y+ C+ MYtCIH K M+t
S: 13 ICLPICPWVCQCMYAY-~---VNQYTYLYLCICVYPYM--CLFMYVCIHASTHVC--MYVR 64
Q: 266 SLNLARAGFA 295
++ L +A
2~ S: 65 TMYLCMYMYA 74
The segment of bbsl121221 that is shown as "S" above is set out in the
sequence listing as SEQ ID NO. 113. Based on the structural similarity, these
homologous polypeptides are expected to share at least some biological
activities.
25 Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 1 I4 which
30 corresponds to the "Q" sequence in the alignment shown above (gaps
introduced in a
sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Synovial ~broblasts (II1/TNF~, subt; PC3 Prostate cell
line.
Many polynucleotide sequences, such as EST sequences, are publicly
3S available and accessible through sequence databases. Some of these
sequences are
related to SEQ ID N0:18 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
40 are one or more polynucleotides comprising a nucleotide sequence described
by the
CA 02364209 2001-09-24
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16
general formula of a-b, where a is any integer between 1 to 1197 of SEQ ID
N0:18, b
is an integer of 15 to 1211, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:18. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 9
This invention relates to newly identified polynucleotides, polypeptides
encoded by such polynucleotides, the use of such polynucleotides and
polypeptides,
as well as the production of such polynucleotides and polypeptides. The
polypeptide
IO of the present invention has been putatively identified as a human
transforming
growth factor alpha homolag derived from prostate cell lines. More
particularly, the
polypeptide of the present invention has been putatively identified as
transforming
growth factor alpha prostate-derived protein, sometimes hereafter referred to
as
'TGFa-P". The invention also relates to inhibiting the action of such
polypeptides.
Cellular growth and differentiation appear to be initiated, promoted,
maintained and regulated by a multiplicity of stimulatory, inhibitory and
synergistic
factors and hormones. The alteration andlor breakdown of the cellular
homeostasis
mechanism seems to be a fundamental cause of growth related diseases,
including
neoplasia. Growth modular factors are implicated in a wide variety of
pathological
and physiological processes including signal transduction, cell communication,
growth and development, embryogenesis. immune response, hematopoiesis cell
survival and differentiation, inflammation. tissue repair and remodeling,
atherosclerosis and cancer.
Epidermal growth factor (EGF), transforming growth factor alpha (TGFa),
betacellulin, amphiregulin, and vaccinia growth factor among other factors are
growth
and differentiation modulatory proteins produced by a variety of cell types
either
under normal physiological conditions or in response to exogenous stimuli and
are
members of the EGF family. These peptide growth factors influence wound cells
through autocrine and paracrine mechanisms. They also play important roles in
normal wound healing in tissues such as skin, cornea and gastrointestinal
tract and all
share substantial amino acid sequence homology including the conserved
placement
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17
of three intro-chain disulfide bonds. In addition, all the factors of this
family bind to a
170,000 molecular weight transmembrane glycoprotein receptor and activate the
tyrosine kinase activity in the receptor's cytoplasmic domain (Buhrow, S.A. et
al.,
J.Bio.Chem., 258:7824-7826 ( 1983)).
The receptors are expressed by many types of cells including skin
keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells
of the GI
tract. These peptide growth Factors are synthesized by several cells involved
in wound
healing including platelets, keratinocytes, and activated macrophages. These
growth
factors have also been implicated in both the stimulation of growth and
differentiation
of certain cells, for example, neoplasia, and the inhibition of other types of
cells.
Betacellulin is a 32-kilodalton glycoprotein that appears to be processed from
a larger
transmembrane precursor by proteolytic cleavage. The carboxyl-terminal domain
of
betacellulin has 50% sequence similarity with that of rat transforming growth
factor a.
Betacellulin is a potent mitogen for retinal pigment epithelial cells and
vascular smooth muscle cells. Amphireguiin is a bifunctional cell growth
regulatory
factor which exhibits potent inhibitory activity on DNA synthesis in
neoplastic cells,
yet promotes the growth of certain normal cells. A wide variety of uses for
amphiregulin have been assigned including the treatment of wounds and cancers.
For
example, amphiregulin has potent anti-proliferative effects in vitro on
several human
cancer cell lines of epithelial origin. AmphireQulin also induces the
proliferation of
human foreskin fibroblasts as shown in United States Patent Application No.
5,115,096.
TGFa has pleiotropic biological effects. The production of certain members of
TGFa is synthesized by a number of oncogenically transformed fibroblasts
(Ciardiello
et al., J.Cell.Biochem., 42:45-57 ( 1990)), as well as by a variety of tumors,
including
renal, breast and squamous carcinomas, melanomas and glioblastomas (Derynck,
R.
et al., Cancer Res., 47:707-712 ( 1987)). There is direct evidence that TGFa
expression can be a contributing factor in the conversion of a normal cell to
its
tumorigenic counterpart by analyzing trans~enic mice in which tumor cells
express
high levels of TGFa. TGFa transgenic animals display a variety of neoplastic
lesions,
depending on the strain of mouse and the choice of promotor regulating TGFa
CA 02364209 2001-09-24
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18
expression (Sandgren, et al., Cell, 61:1121- I 135 ( 1990)). TGFa also plays a
role in
normal embryonic development and adult physiology (Derynck, R. Adv.Cancer
Res.,
58:27-5 ( 1992)).
TGFa has been expressed in many tissues including skin, brain,
gastrointestinal mucosa and activating macrophages. Accordingly, TGFa is an
important factor in controlling growth of epithelial cells and has a role in
wound
healing. TGFa has also been found to be angiogenic (Schreiber, et al.,
Science,
232:1250-1253 (1986)). The polypeptide of the present invention has been
putatively
identified as transforming growth factor TGFa-P. This identification has been
made as
l0 a result of amino acid sequence homology to pig TGFa, human epiregulin, and
human
heparin-binding EGF, in combination with its isolation from a prostate cDNA
library
Figures lA-B show the nucleotide (SEQ ID N0:19) and deduced amino acid
sequence {SEQ 1D N0:68) of TGFa-P. Predicted amino acids from about I to about
20 constitute the predicted signal peptide (amino acid residues from about 1
to about
20 in SEQ ID N0:68) and are represented by the underlined amino acid regions;
amino acids from about 21 to about 112 constitute the predicted mature protein
(amino acid residues from about 12 to about 112 in SEQ ID N0:68); amino acid
residues 53 to about 102 constitute the active/EGF-like domain of the protein
(amino
acid residues 53 to about lU2 of SEQ ID N0:68) and are represented by the
double
underlined amino acids.
Figure 2 shows the regions of similarity between the amino acid sequences of
the TGF-aP protein (SEQ ID N0:68), the TGF-alpha protein from pig (SEQ ID
NO: l t7), the human epiregulin protein (SEQ ID N0:118), and the human heparin-
binding EGF protein (SEQ ID N0:119).
Figure 3 shows an analysis of the TR13 amino acid sequence. Alpha, beta,
turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions;
flexible regions; antigenic index and surface probability are shown.
A polynucleotide encoding a polypeptide of the present invention may be
obtained from human prostate and keratinocyte tissue, in addition to human
prostate
cell lines. The polynucleotide of this invention was discovered in a human
prostate
cDNA library. Its translation product has homology to the characteristic EGF
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19
domains. As shown in Figures t A-B, TGF-aP has a single EGF domain (the EGF
domain comprises amino acids 53-!02 of SEQ ID N0:68; which corresponse to
amino acids 53-102 of Figures lA-B) with strong conservation between other
members of the EGF and TCJF-alpha family. See, for example, Lecka-Czernik, et
al.,
Molecular and Cellular Biology, 15( I ):120 ( 1995), and in particular the
consensus
EGF domain shown in Figure SC on page 125.
The polynucleotide contains an open reading frame encoding a complete TGF-
aP polypeptide of 112 amino acids. TGF-aP exhibits a high degree of homology
at the
amino acid level to the pig TGF-alpha, human epiregulin, and human heparin-
binding
EGF protein (as shown in Figure 2). The present invention provides isolated
nucleic
acid molecules comprising a polynucleotide encoding the TGF-aP polypeptide
having the amino acid sequence shown in Figures lA-B (SEQ ID N0:19), which was
determined by sequencing a cloned cDNA_
The nucleotide sequence shown in Figures lA-B (SEQ ID N0:19) was
obtained by sequencing a cl:)NA clone, which was deposited on April 08, 1999
at the
American Type Culture Collection, and given Accession Number 203918. The
deposited clone is inserted in the pBluescript plasmid (Life Technologies,
Rockville,
MD) using the EcoRI/Xhol restriction endonuclease cleavage sites.
The present invention is further directed to fragments of the isolated nucleic
acid molecules described herein. By a fragment of an isolated DNA molecule
having
the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown
in
SEQ ID N0:19 is intended DNA fragments at least about ISnt, and more
preferably
at least about 20 nt, still more preferably at least about 30 nt, and even
more
preferably, at least about 40 nt in length which are useful as diagnostic
probes and
primers as discussed herein.. Of course, larger fragments 50-1500 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 the deposited cDNA or as shown in SEQ
ID
N0:19. 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 the
deposited cDNA or the nucleotide sequence as shown in SEQ ID N0:19. In this
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WO 00/63230 PCT/US00/07677
context "about" includes the particularly recited size, larger or smaller by
several (S,
4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Representative examples of TGF-aP polynucteotide fragments of the invention
include, for example, fragments that comprise, or alternatively, consist of, a
sequence
S from about nucleotide 1 to about S0, from about S I to about 100, from about
101 to
about 150, from about 1 S 1 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, from about 501 to about 550,
from
about SS 1 to about 600, from about 601 to about 650, from about 6S 1 to about
700,
10 from about 701 to about 750, from about 7S 1 to about 800, from about 801
to about
850, from about 851 to about 900, from about 901 to about 950, from about 9S 1
to
about 1000, from about 10(11 to about IOSO, from about 1051 to about 1100,
from
about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to
about
1250, from about 1251 to about 1300, from about 1301 to about 1350, from about
1S 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about
1500,
from about 1501 to about 1550, from about 1551 to about 1600, from about 1601
to
about 1650, from about 165 t to about 1699, from about 83 to about 358, from
about
179 to about 328, and from about 23 to about 82 of SEQ ID N0:19, or the
complementary strand thereto, or the cDNA contained in the deposited clone. In
this
20 context "about" includes the particularly recited ranges, larger or smaller
by several
(S, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferred nucleic acid fragments of the present invention include nucleic acid
molecules encoding a member selected from the group: a polypeptide comprising
or
alternatively, consisting of, the active/EGF-like domain (amino acid residues
from
2S about S3 to about 102 in Figures lA-B (amino acids from about 53 to about
102 in
SEQ ID N0:68), and a polypeptide that is 9S % identical to the polypeptide
sequence
of the active/EGF-like domain of TGF-aP.
Additional preferred fragments of the present invention also include a member
selected from the group : a polynucleotide that hybridizes to the complement
of the
polynucleotide encoding the active/EGF-like domain, a polypeptide encoded
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21
by said complement, and a polynucleotide that encodes a polypeptide that is 95
%
identical to the polypeptide sequence of the activelEGF-like domain of TGF-aP.
Since the location of these domains have been predicted by computer
analysis, one of ordinary skill would appreciate that the amino acid residues
constituting these domains may vary slightly (e.g., by about l to 15 amino
acid
residues) depending on the criteria used to define each domain. In additional
embodiments, the polynucleotides of the invention encode functional attributes
of
TGF-aP. Preferred embodiments of the invention in this regard include
fragments that
comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-
sheet
and beta-sheet forming regions ("beta-regions"), turn and tum-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 of TGF-aP.
Preferred polypeptides of the present invention comprise, or alternatively
IS consist of, one, two, or all three of the immunogenic epitopes shown in SEQ
ID
N0:68 as residues: Asn-41 to Asp-46, Glu-62 to Tyr-67, Val-104 to Leu-112.
Polynucleotides encoding these polypeptides are also encompassed by the
invention,
as are antibodies that bind one or more of these peptides.
Preferred nonexclusive embodiments also include antibodies that bind to any
of immunogenic epitopes of the invention. as well as, antibodies that bind the
active/EGF-like domain of TGF-aP.
Additionally, nonexclusive embodiments of the invention include polypeptide
fragments that comprise, or alternatively, consist of, a sequence selected
from the
group: M-1 to Y-102, E-21 to I-52, A-53 to Y-102, A-52 to L-112, N-41 to Y-67,
and
E-62 to L-112 of SEQ ID N0:68. Polynucleotides that encode these fragments are
also encompassed by the invention.
The data representing the structural or functional attributes of TGF aP set
forth in Figure 3 and/or Table 1i1, 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 VI11, IX, XI11, and XIV of Table III
can
be used to determine regions of TGF-aP which exhibit a high degree of
potential for
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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. As set out in Figure 3 and in fable III, such preferred
regians
include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-
regions,
Chou-Fasman alpha-regions, beta-regions. and turn-regions, Kyte-Doolittle
hydrophilic regions and Hopp-Woads hydrophobic regions, Eisenberg alpha- and
beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf
regions of
high antigenic index and Ernini surface-forming regions. Even if deletion of
ane or
more amino acids from the N-terminus of a protein results in modification of
loss of
one or more biological functions of the protein, other functional activities
(e.g.,
biological activities, ability to multimerize, etc.) may still be retained.
For example,
the ability of shortened TGF-aP muteins to induce and/or bind to antibodies
which
recognize the complete or mature forms of the polypeptides generally will be
retained
when less than the majority of the residues of the complete or mature
polypeptide are
removed from the N-terminus.
The data presented in Figure 3 are also represented in tabular form in Table
III. 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 3, and Table III: "Res": amino acid residue
of SEQ
lD NO: 68 and Figures 1 A and 1 B; "Position": position of the corresponding
residue
within SEQ ID NO: 68 and Figures 1 A and I B; I: Alpha, Regions - Gamier-
Robson;
II: Alpha, Regions - Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV:
Beta,
Regions - Chou-Fasman; V: Turn, Regions - Garnier-Robson; VI: Turn, Regions
Chou-Fasman; VII: Coil, Regions - Garnier-:Robson; VIII: Hydrophilicity Plot
Kyte-Doolittle; IX: Hydrophobicity Plot - Happ-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.
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23
Certain preferred regions in these regards are set out in Figure 3, but may,
as
shown in Table III, be represented or identified by using tabular
representations of the
data presented in Figure 3. 'the DNA'~STAR computer algorithm used to generate
Figure 3 (set on the original default parameters) was used to present the data
in Figure
3 in a tabular format (See Table I(1). The tabular format of the data in
Figure 3 may
be used to easily determine specific boundaries of a preferred region.
The above-mentioned preferred regions set out in Figure 3 and in Table III
include, but are not limited to, regions of the aforementioned types
identified by
analysis of the amino acid sequence set out in Figure 1. Whether a particular
polypeptide lacking N-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 an TGF-aP
mutein with a
large number of deleted N-terminal amino acid residues may retain some
biological or
immunogenic activities. In fact, peptides composed of as few as six TGF-aP
amino
acid residues may often evoke an immune response. Accordingly, the present
invention further provides polypeptides having one or more residues deleted
from the
amino terminus of the TGF-aP amino acid sequence shown in Figures lA-B, up to
the
glutamine residue at positian number 108 and polynucleotides encoding such
polypeptides. In particular, the present invention provides polypeptides
comprising
the amino acid sequence of residues nl-112 of Figures lA-B, where nl is an
integer
from 2 to 108 corresponding to the position of the amino acid residue in
Figures lA-B
(which is identical to the sequence shown as SEQ ID N0:68).
In another embodiment, N-terminal deletions of the TGF-aP polypeptide can
be described by the general formula n2-I 12, where n2 is a number from 2 to
108,
corresponding to the position of amino acid identified in Figures lA-B. N-
terminal
deletions of the TGF-aP polypeptide of the invention shown as SEQ ID N0:68
include polypeptides comprising, or alternatively consisting of, an amino acid
sequence selected from the group: A-2 to L-1 l2; L-3 to L-112; G-4 to L-112; V-
5 to
L-112; P-6 to L-112; I-7 to L-112; S-8 to L-I 12; V-9 to L-l 12; Y-10 to L-
112; L-11
to L-1 l2; L-12 to L-112; F'-13 to L-112; N-14 to L-I 12; A-15 to L-112; M-16
to L-
I 12; T-17 to L- I 12; A-18 to L-112; L- I 9 to L-112; T-20 to L-112; E-21 to
L-1 ! 2; E-
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y
22 to L-112; A-23 to L-112; A-24 to L- t I 2; V-25 to L- I 12; T-26 to L-112;
V-27 to
L- I I 2; T-28 to L-1 I 2; P-29 to L-112; P-30 to L- I 12;1-31 to L-112; T-32
to L- l 12;
A-33 to L-1 I 2; Q-34 to L- l 12; Q-35 to L- l 12; G-36 to L- I 12; N-37 to L-
I 12; W-38
to L-112; T-39 to L- I 12; V -.40 to L-112; N-41 to L- I I 2; K-4.2 to L-112;
T-43 to L-
112; E-44 to L-112; A-45 to L- I I 2 ; D-46 to L-1 12; N-47 to L-112; I-48 to
L- l 12; E-
49 to L-112; G-50 to L-112; P-5 I to L-112; 1-52 to L-112; A-53 to L-112; L-54
to L-
112; K-55 to L- I 12; F-56 to L-112; S-57 to L-112; H-58 to L- I I 2; L-59 to
L-112; C-
60 to L-112; L-61 to L-112; E-62 to L-1 I 2; D-b3 to L-112; H-64 to L-112; N-
65 to L-
112; S-66 to L-112; Y-67 to L-112; C-68 to L-1 I 2; I-69 to L- I 12; N-70 to L-
1 I 2; G-
I 0 71 to L- I 12; A-72 to L-11'2 ; C-73 to L- I 12; A-74 to L-112; F-75 to L-
112; H-76 to L-
I 12; H-77 to L-112; E-78 to L-112; L-79 to L-I I2; E-80 to L-112; K-81 to L,-
112; A-
82 to L-112; I-83 to L-112; C-84 to L-112; R-85 to L-112; C-86 to L-112; F-87
to L-
112; T-88 to L-112; G-89 to L-112; Y-90 to L-112; T-91 to L-112; G-92 to L-
112; E-
93 to L-112; R-94 to L-112; C-95 to L- i 12; L-96 to L-112; K-97 to L-112; L-
98 to L-
112; K-99 to L-112; S-100 to L- I 12; P-101 to L- I 12; Y-102 to L-112; N-103
to L-
112; V-104 to L-112; C-105 to L-I 12; S-106 to L-112; and G-107 to L-112 of
SEQ
ID N0:68. 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 or loss of one or more
biological
functions of the protein, other functional activities (e.g., biological
activities (e.g.,
ability to illicit mitogenic activity, induce differentiation of normal or
malignant cells,
bind to EGF receptors, etc.), ability to multimerize, ability to bind EGF
receptors may
still be retained. For example the ability of the shortened TGF-aP mutein to
induce
andlor 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 an
TGF-aP mutein with a large number of deleted C-terminal amino acid residues
may
CA 02364209 2001-09-24
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2~
retain some biological or immunogenic activities. In fact, peptides composed
of as
few as six TGF-aP 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
TGF-aP polypeptide shown in Figures IA-B, up to the proline residue at
position
number 6, and polynucleotides encoding such polypeptides. In particular, the
present
invention provides polypeptides comprising the amino acid sequence of residues
1-
ml of Figures lA-B, where ml is an integer from 7 to 112 corresponding to the
position of the amino acid residue in Figures lA-B. Moreover, the invention
provides
polynucleotides encoding polypeptides comprising, or alternatively consisting
of, the
amino acid sequence of C-terminal deletions of a TGF-aP polypeptide (SEQ ID
N0:68) selected from the group: M-1 to P-111; M- I to R-110; M-1 to R-109; M-1
to
E-108; M-1 to G-107; M-1 to S-106; M-1 to C-105; M-1 to V-104; M-1 to N-103; M-
I to Y-102; M-1 to P-101; M-1 to S-100: M-1 to K-99; M-l to L-98; M-I to K-97;
M-
I to L-96; M-1 to C-95 ; M- I to R-94; M- I to E-93 ; M- I to G-92; M-1 to T-
91; M- I
to Y-90; M-1 to G-89; M-1 to T-88; M- I to F-87 ; M- I to C-86; M-1 to R-85 ;
M-1 to
C-84; M-I to I-83; M-1 to A-82; M-1 to K-81; M-I to E-80; M-1 to L-79; M-1 to
E-
78; M-1 to H-77; M-I to H-76; M-1 to F-75; M-1 to A-74; M-1 to C-73; M-1 to A-
72;
M-1 to G-71; M-I to N-70; M-I to I-69; M-1 to C-68; M-I to Y-67; M-I to S~-66;
M-
1 to N-65; M-1 to H-64; M-1 to D-63 ; M-1 to E-62; M-1 to L-61; M- I to C-60;
M-1
to L-59; M-1 to H-58; M-1 to S-57; M- I to F-56; M- I to K-55; M-1 to L-54; M-
I to
A-53; M-I to I-52; M-1 to P-51; M-1 to G-50; M-1 to E-49; M-I to I-48; M-I to
N-
47; M-1 to D-46; M-1 to A-45 ; M- I to E-44; M-1 to T-43 ; M-1 to K-4.2; M-1
to N-41;
M-1 to V-40; M-I to T-39; M-I to W-38; M-1 to N-37; M-1 to G-36; M-I to Q-35;
M-1 to Q-34; M-1 to A-33; M-1 to T-32; M-1 to I-31; M-1 to P-30; M-1 to P-29;
M-1
to T-28; M-1 to V-27; M-1 to T-26; M-1 to V-25; M-I to A-24; M-1 to A-23; M-1
to
E-22; M-1 to E-21; M-1 to °f-~20; M-1 to L--19; M-1 to A-18; M-1 to T-
17; M-1 to M-
16; M-l to A-15; M-I to N-14; M-1 to F-13; M-1 to L-12; M-1 to L-1 l; M-1 to Y-
10;
M-1 to V-9; M-1 to S-8; M-1 to I-7; and M-1 to P-6; of SEQ ID N0:68.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
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Moreover, the invention provides polynucleotides encoding polypeptides
comprising, or alternatively consisting of, the amino acid sequence of N-
terminal
deletions of the TGF-aP EGF-like domain polypeptides of the invention shown as
SEQ ID N0:120 (amino acid residues 53 to l02 in Figure 1) include polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the
group: L-2 to Y-50; K-3 to Y-50; F-4 to Y-50; S-5 to Y-50; H-6 to Y-50; L-7 to
Y-50;
C-8 to Y-50; L-9 to Y-50; E-10 to Y-50; D-11 to Y-50; H-12 to Y-50; N-13 to Y-
50;
S-14 to Y-50; Y-IS to Y-50; C-16 to Y-50; I-I7 to Y-50; N-18 to Y-50; G-19 to
Y-
50; A-20 to Y-50; C-21 to Y-50; A-22 to Y-50; F-23 to Y-50; H-24 to Y-50; H-25
to
Y-50; E-26 to Y-50; L-27 to Y-50; E-28 to Y-50; K-29 to Y-50; A-30 to Y-50; I-
31 to
Y-50; C-32 to Y-50; R-33 to Y-50; C-34 to Y-50; F-35 to Y-50; T-36 to Y-50; G-
37
to Y-50; Y-38 to Y-50; T-39 to Y-50; G-40 to Y-50; E-4.1 to Y-50; R-42 to Y-
50; C-
43 to Y-50; L-44 to Y-50; and K-45 to Y-50 of SEQ ID N0:120. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
Moreover, the invention provides polynucleotides encoding polypeptides
comprising, or alternatively consisting of, the amino acid sequence of C-
terminal
deletions of the TGF-aP EGF-like domain polypeptides of the invention shown as
SEQ ID NO: (amino acid residues 53 to 102 in Figure I) include polypeptides
comprising, or alternatively consisting of, an amino acid sequence selected
from the
group: A-1 to P-49; A-1 to S-48; A-1 to K-47 ; A- I to L-46; A-1 to K-45; A-1
to L-44;
A-1 to C-43; A-1 to R-42; A-1 to E-41; A-1 to G-40; A-1 to T-39; A-1 to Y-38;
A-1
to G-37; A-1 to T-36; A-1 to F.-35; A-I to C-34; A-1 to R-33; A-1 to C-32; A-1
to I-
31; A-1 to A-30; A-1 to K-29; A-1 to E-28; A-1 to L-27; A- I to E-26; A-1 to H-
25;
A-1 to H-24; A-1 to F-23 ; A-1 to A-22; A- ! to C-21; A-1 to A-20; A-1 to G-
19; A-1
to N-18; A-1 to I-17; A-1 to C-16; A-1 to Y-15; A-1 to S-14; A-1 to N-13; A-1
to H-
12; A-1 to D-1 I ; A-1 to E-10; A- I to L-9; A-1 to C-8 ; A- I to L-7; and A-
I to H-6 of
SEQ ID N0:120. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
In addition, the invention provides nucleic acid molecules having nucleotide
sequences related to extensive portions of SEQ ID N0:19 which have been
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27
determined from the following related cDNA clones: HPJBS 16R {SEQ ID N1~:120)
and HKAJV85R (SEQ ID N0:121).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: PC3 Prostate cell line: prostate, and Keratinocyte_
S Based on the sequence similarity to TGF-a, the human Epiregulin, and Human
Heparin-binding EGF proteins, the translation product of this clone is
expected to
share at least some biological activities with EGF domain-containing proteins,
and
particularly those referenced above. Such activities are known in the art,
some of
which are described elsewhere herein. Specifically, polynucleotides and
polypeptides
of the invention are useful for modulatin= mitogenic activity for a variety of
cell
types, which include, but are not limited to, prostate, colon, and mesenchymal
cells.
Polynucleotides and polypeptides of the invention are also useful for
modulating the differentiation of normal and malignant cells, binding to and
activating EGF receptor proteins, in addition to other novel receptors, and
modulating
the proliferation and/or dedifferentiation of cancer and neoplastic cells. For
the
purposes of the present invention, modulate may be defined as increasing,
decreasing,
or maintaining stasis of some cellular process, biochemical pathway, or
biochemical
reaction. Polynucleotides and poiypeptides of the invention may play an
important
role in the pathogenesis of human cancers and cellular transformation,
particularly
those of the prostate and integument, including melanomas.
Polynucleotides and polypeptides of the invention may also be involved in the
pathogenesis of developmental abnormatlities based upon its potentail effects
on
proliferation and differentiation of cells and tissue cell types. Due to the
potential
proliferating and differentiating activity of said polynucleotides and
polypeptides, the
invention may be useful as a therapeutic anent in inducing tissue
regeneration,
particularly of cardiomyocytes, the integument, and epithelial cells (e.g.,
kidney
proximal tubule cells).
The tissue distribution within prostate , prostate cell lines, in combination
with
the homology to TGF-alpha family members, indicates polynucleotides and
polypeptides of this clone, and fragments and variants thereof, are useful for
the
treatment, diagnosis, and/<ar prevention of various proliferative diseases
and/or
CA 02364209 2001-09-24
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disorders, particularly prostate cancer. Representative uses are described in
the
"Biological Activity", "Hyperproliferative Disorders", "Infectious Disease",
and
"Regeneration" sections below, in Example 1 I, 19, and 20, and elsewhere
herein.
Alternatively, based upon the expression of this protein within keratinocytes,
indicates that the protein is useful in detecting, treating, and/or preventing
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, autoirnmune disorders (i.e. lupus erythematosus, vitiligo,
dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus), keloids,
striae,
erythema, petechiae, purpura, and xanthelasma. In addition, 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).
Moreover, the protein product of this clone may also be useful for the
treatment or diagnosis of various connective tissue disorders (i.e.,
arthritis, trauma,
tendonitis, chrondomalacia and inflammation, etc.), autoimmune disorders
(i.e.,
rheumatoid arthritis, lupus, scleroderma, dermatomyositis, etc.), dwa~sm,
spinal
deformation, joint abnormalities, amd chondrodysplasias (i.e.
spondyloepiphyseal
dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II,
metaphyseal
chondrodysplasia type Schmid). Furthermore, the protein may also be used to
determine biological activity, to raise antibodies, as tissue markers, to
isolate cognate
ligands or receptors, to identify agents that modulate their interactions, in
addition to
its use as a nutritional supplement. 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.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:19 and may have been publicly available prior to
conception of
CA 02364209 2001-09-24
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?9
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1689 of SEQ ID NO:
l9, b
is an integer of 15 to 1703, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:19, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
It has been discovered that this gene is expressed primarily in PC3 Prostate
cell line.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 69 as residues: Gln-26 to Ala-33.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:20 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1487 of SEQ ID
N0:20, b
is an integer of 15 to 1501, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:20, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gi12736153 (all information available through the recited accession number is
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5
incorporated herein by reference} which is described therein as "(AF021936)
myotonic dystrophy kinase-related Cdc42-binding kinase MRCK-beta (Rattus
norvegicus~". A partial alignment demonstrating the observed homology is shown
immediately below.
>gi~2736153 (AF021936) myotonic dystrophy kinase-related Cdc42-binding
kinase
MRCK-beta [Rattus norvegicus] >sp~054875~054875 MYOTONIC
DYSTROPHY
IO KINASE-RELATED CDC42-BINDING KINASE MRCK-BETA.
Length = 1702
Plus Strand HSPs:
15 Score = 376 (132.4 bits), Expect = 3.6e-39, Sum P(2) = 3.6e-39
Identities = 77/89 (86%), Positives = 82/89 (92%), Frame = +3
Q: 2685 SCFSDRGSLKSIMQSNTLTKDEDVQRDLEHSLQMEAYERRIRRLEQEKLELSRKLQESTQ 2864
SCFSDRGSLKS++QSNTLTKDEDVQRDLE+SLQ+EAYERRIRRLEQEKLELSRKLQESTQ
2O S: 407 SCFSDRGSLKSMIQSNTLTKDEDVQRDLENSLQIEAYERRIRRLEQEKLELSRKLQESTQ 466
Q: 2865 TVQSLHGSSRALSNSNRDKEIXXXXXXLE 2951
TVQSLHGS+RAL NSNRDREI LE
S: 467 TVQSLHGSTRALGNSNRDKEIKRLNEELE 495
Score = 143 (50.3 bits), Expect = 3.6e-39, Sum P(2) = 3.6e-39
Identities = 26/26 (100%), Positives = 26/26 (100%), Frame = +2
Q: 533 EILPPGSHTGFSGLHLPFIGFTFTTE 610
3O EILPPGSHTGFSGLHLPFIGFTFTTE
S: 381 EILPPGSHTGFSGLHLPFIGFTFTTE 406
Score = 62 (21.8 bits), Expect = l.Oe-05, Sum P(2) = l.Oe-05
Identities = 21/62 (33%), Positives = 38/62 (61%), Frame = +3
Q: 2709 LKSZM-QSNTLTKD-ED---VQRDLEHSLQ-MEAYERRIRRLEQEKLELSRKLQESTQTV 2870
+KS M SN L + ED ++++ E S Q ++ E++ R QEK EL ++L E+++ +
S: 497 MKSKMADSNRLERQLEDTVTLRQEHEDSTQRLKGLEKQYRLARQEKEELHKQLVEASERL 556
Q: 2871 QS 2876
+S
S: 557 KS 558
The segments of gi12736153 that are shown as "S" above are set out in the
sequence listing as SEQ ID NO. 123.SEQ ID NO. 125 and SEQ ID NO. 127. Based
on the structural similarity, these homologous polypeptides are expected to
share at
least some biological activities. Such activities are known in the art, some
of which
are described elsewhere herein. Assays for determining such activities are
also known
in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 124,SEQ ID
NO.
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31
126 and/or SEQ ID NO. l28 which correspond to the "Q" sequences in the
alignment
shown above (gaps introduced in a sequence by the computer are, of course,
removed).
It has been discovered that this gene is expressed primarily in PC3 Prostate
cell line.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 70 as residues: His-35 to His-40.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:21 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
IS general formula of a-b, where a is any integer between I to 2957 of SEQ ID
N0:21, b
is an integer of 15 to 2971, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:21, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Spleen metastic melanoma; Human Thymus Stromal Cells;
Human Testes Tumor; Anergic T-cell; Human Endometrial Tumor.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:22 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1376 of SEQ ID
N0:22, b
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32
is an integer of 15 to 1390, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:22, and where b is greater than or
equal to a
+ l4.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
It has been discovered that this gene is expressed primarily in Human
Synovium.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:23 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 730 of SEQ ID
NO:23, b
is an integer of IS to 744, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:23, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
It has been discovered that this gene is expressed primarily in Human
Synovium.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 73 as residues: Lys-2 to Trp-8.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:24 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
CA 02364209 2001-09-24
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33
general formula of a-b, where a is any integer between l to 358 of SEQ 1D
NO:24, b
is an integer of I S to 372, where both a and b correspond to the positions of
nucleotide residues shown in SEQ 1D N0:24, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15
It has been discovered that this gene is expressed primarily in Human
Synovium.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:25 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 386 of SEQ ID
N0:25, b
is an integer of 15 to 400, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:25, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 1G
It has been discovered that this gene is expressed primarily in Human
Synovium.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:26 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any inte_er between 1 to 418 of SEQ ID
N0:26, b
CA 02364209 2001-09-24
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3.t
is an integer of 15 to 432, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:26, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 17
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Synovial hypoxia-RSF subtracted and to a lesser extent
in
Soares fetal liver spleen 1 NFLS; Soares_senescent fibroblasts_NbHSF; Soares
placenta Nb2HP; Soares fetal lung NbHLI9W; NTERA2, control; Soares
melanocyte 2NbHM; T cell helper II; Human Fetal Brain;
Soares fetal_liver_spleen_ I NFLS__S I ; Activated T-cell( 12h)rChiouridine-re-
excision; Stratagene hNT neuron (#937233); Human 8 Week Whole Embryo; Soares
infant brain 1NIB; Stratagene HeLa cell s3 937216; Human Frontal Cortex,
Schizophrenia; Jurkat T-cell G 1 phase; Human Prostate; Macrophage-oxLDL; H
IS Macrophage (GM-CSFtreated), re-excision; Activated T-Cell
(l2hs)/Thiouridine
labelledEco; Endothelial cells-control; Bone Marrow Cell Line (RS4,11); Soares
ovary tumor NbHOT; Soares,_multiple_sclerosis_2NbHMSP; LNCAP + o.3nM
81881; Weizmann Olfactory Epithelium; Human Aortic Endothelium; Soares retina
N2b4HR; Human Skin Tumor; Soares_fetal_heart_NbHHI9W; Stratagene fibroblast
(#937212); Smooth Muscle- HASTE normalized; Messangial cell, frac 2; Soares
adult brain N2b4HB55Y; S'TROMAL -OSTEOCLASTOMA; Hepatocellular Tumor;
Stratagene pancreas (#937208); Human Stomach,re-excision; Myoloid Progenitor
Cell Line; L428; Stratagene fetal spleen (#937205); Human Fetal Dura Mater;
Human
Adipose; PERM TF274; Human T-Cell Lymphoma; Colon Tumor II; Human
Synovial Sarcoma; Human Fetal Heart; Soares_pregnant uterus_NbHPU; HUMAN
B CELL LYMPHOMA; Hodgkin's Lymphoma II; Keratinocyte; Nine Week Old
Early Stage Human; Soares_fetal_heart_NbHHI9W; Primary Dendritic Cells, lib l;
Human adult lymph node, subtracted; Human Lung Cancer, subtracted; Human
Placenta, subtracted; Larynx Normal; Human Normal Cartilage,Fraction 1; Human
B
Cell 8866; Human Prostate, subtracted: Ku 812F Basophils Line; Human Prostate
Cancer, Stage B2 fraction; Human Fetal Luno; Whole 6 Week Old Embryo; Human
CA 02364209 2001-09-24
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3~
(Caco-2) cell line, adenocarcinoma, colon, remake; Resting T-Cell; Morton
Fetal
Cochlea; LI Cell line; Human OB HOS control fraction I; Human Colon; Human
Colon Carcinoma (HCC) cell line; Human Adult Retina; Human Lung; Early Stage
Human Lung, subtracted; Human Quadriceps;
Soares_placenta 8to9weeks 2NbHP8to9W: Soares fetal lung_NbHLI9W; Healing
groin wound - zero hr post-incision (control); Soares_pineal_gland_N3HPG; HEL
cell line; Pancreas Tumor PCA4 Tu; Human Synovium; human corpus colosum;
Alzheimers, spongy change; NTERA2 + retinoic acid, l4 days; Human Fetal
Epithelium (Skin); Stratagene endothelial cell 937223; Stratagene ovarian
cancer
(#937219); Soares fetal lung_NbHLI9W: H. Kidney Cortex, subtracted;
Glioblastoma; Salivary Gland, Lib 2; HL-60. PMA 4H, re-excision; Synovial
hypoxia; Jurkat T-Cell, S phase; H. Lymph node breast Cancer; Human Chronic
Synovitis; Fetal Liver, subtraction II; T-Cell PHA 16 hrs; Breast Cancer Cell
line,
angiogenic; Human Thymus; Human Brain, Striatum; Human Fetal Kidney; human
ovarian cancer; HUMAN JURKA'r MEMBRANE BOUND POLYSOMES; Human
Activated T-Cells; Human Primary Breast Cancer Reexcision; T-Cell PHA 24 hrs;
Human Heart; Stromal cell TF274; Human Hippocampus; Spinal cord; Human
Chondrosarcoma; Bone Marrow Stromal Cell, untreated; Hemangiopericytoma;
Human Whole Six Week Old Embryo; Stratagene lung (#937210); Hepatocellular
Tumor, re-excision; Macrophage-oxLDL, re-excision; Resting T-Cell Library,II;
12
Week Old Early Stage Human; Colon Normal II; breast lymph node CDNA library;
Adipocytes; Human Testes Tumor; Dendritic cells, pooled;
Soares multiple sclerosis 2NbHMSP; Primary Dendritic celis,frac 2; human
tonsils;
Human Adult Pulmonary,re-excision; Human Osteoclastoma; Human Amygdala;
Soares fetal heart NbHHI9W; Human Microvascular Endothelial Cells, fract. A;
Smooth muscle,control; Human Bone Marrow, treated; Spleen, Chronic lymphocytic
leukemia; Osteoblasts; Human Cerebellum: Pancreatic Islet and
Soares_multiple_sclerosis_2NbHMSP.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:27 and may have been publicly available prior to
conception of
CA 02364209 2001-09-24
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36
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 780 of SEQ 1D
N0:27, b
is an integer of 15 to 794, where bath a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:27, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares_parathyroid_tumor NbHPA; human pleural cancer
and to a lesser extent in NC.".1_CGAP_Br2; NCI CGAP GC4 and Human Amygdala.
Many polynucleotide sequences, such as EST sequences, are publicly
IS available and accessible through sequence databases. Some of these
sequences are
related to SEQ ID N0:28 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any inteser between 1 to 1814 of SEQ ID
N0:28, b
is an integer of IS to 1828, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:28, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 19
It has been discovered that this Qene is expressed primarily in the following
tissues/cDNA libraries: Prostate; Human Manic Depression Tissue; 12 Week Old
Early Stage Human; T cell helper II: Soares fetal liver spleen I NFL,S.
Many poiynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
CA 02364209 2001-09-24
WO 00/63230 PC'T/US00/07677
related to SEQ ID N0:29 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 892 of SEQ ID
NO:29, b
is an integer of 15 to 906, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:29, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 20
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soaves fetal liver spleen 1 NFLS and to a lesser
extent in
Soaves testis_NHT; Stromal cell TF274; Stratagene lung (#937210);
Soaves fetal heart NbHHI9W; Soaves NhHMPu S1; Soaves retina N2b5HR;
Healing groin wound, 6.5 hours post incision; Soares_NhHMPu S1;
Soaves multiple sclerosis 2NbHMSP; Colon Normal III; NCI CGAP_Lei2; Human
Osteoclastoma; Nine Week Old Early Stage 1-iuman; NCI CGAP_GCB 1;
Soares_multiple sclerosis 2NbHMSP; Human Cornea; Human Fetal Brain,
normalized AC5002; Frontal Lobe, Dementia; prostate-edited; stomach cancer
(human); Soaves testis NH°f; Human Liver; Salivary Gland, Lib 2; Human
Adipose
Tissue, re-excision; Human Manic Depression Tissue; Human Prostate;
Soaves senescent_fibroblasts_NbHSF; normalized infant brain cDNA;
Hepatacellular
Tumor, re-excision; 12 Week Old Early Stage Human; Smooth muscle, serum
treated;
Soaves melanocyte 2NbHM; 12 Week Early Stage Human II, Reexcision; Human
Placenta; Human Adult Pulrnonary,re-excision; Endothelial-induced;
NCl CGAP_LuS; NCI CGAP_GCB 1; NCI_CGAP_Prl2; Human Amygdala;
Soares_pregnant_uterus_NbHPU; Monocyte activated; Osteoblasts; Human 8 Week
Whole Embryo; Soares_pregnant uterus_NbHPU;
Soaves fetal liver spleen_1 NFLS_S 1; Primary Dendritic Cells, lib 1 and
Soaves
infant brain INIB.
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38
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:30 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
$ excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 3470 of SEQ ID
N0:30, b
is an integer of 15 to 3484, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:30, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 21
The computer algorithm BLASTX has been used to determine that the
1$ translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gi13108093 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "(AF0612$8)
LIM
protein [Homo Sapiens)". A partial alignment demonstrating the observed
homology
is shown immediately below.
>gi~3108093 (AF061258) LIM protein [Homo Sapiens] >sp10607051060705 LIM
PROTEIN.
Length = 596
2$
Plus Strand HSPs:
Score = 342 (120.4 bits), Expect = 8.4e--30, P = 8.4e-30
Identities = 65/65 (100%), Positives = 65/65 (100%), Frame = +2
Q: 254 HIVERYTEFYHVPTHSDASKKRLIEDTEDWRPRTGTTQSRSFRILAQITGTEHLKESEAD 433
HIVERYTEFYHVPTHSDASKRRLIEDTEDWRPRTGTTQSRSFRILAQITGTEHLRESEAD
S: 242 HIVERYTEFYHVPTHSDASKKRLIEDTEDWRPRTGTTQSRSFRILAQITGTEHLKESEAD 301
3$ Q: 434 NTKKA 448
NTRKA
S: 302 NTKXA 306
The segment of gi13108093 that is shown as "S" above is set out in the
sequence listing as SEQ ID NO. I29. Based on the structural similarity, these
CA 02364209 2001-09-24
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39
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 130 which
corresponds to the "Q" sequence in the alignment shown above (gaps introduced
in a
sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues/eDNA libraries: Stratagene muscle 937209 and to a lesser extent in
Soares NhHMPu S 1; Soares fetal heart_NbHH 19W; Human Cardiomyopathy,
subtracted; 12 Week Old Early Stage Human. II; Soares total fetus Nb2HF8 9w;
Human Heart; 12 Week Old Early Stage Human; Soares fetal lung_NbHLI9W and
Soares fetal liver spleen 1NFLS.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:31 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleatides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1671 of SEQ ID
N0:31, b
is an integer of 15 to 1685, where both a and b correspond to the positions of
nucleotide residues shown in SEQ lD N0:31. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 22
1t has been discovered that this gene is expressed primarily in Human
Synovium.
Many polynucieotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
related to SEQ ID N0:32 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleatides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 41 I of SEQ ID
N0:32, b
is an integer of IS to 425, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:32, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 23
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Human Prostate Cancer, Stage C fraction.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 82 as residues: Met-17 to Glu-24.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:33 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accardingly, preferably excluded from the present
invention
are one or more polynucleatides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 472 of SEQ ID
N0:33, b
is an integer of I S to 486, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:33, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY ('sENE NO: 24
It has been discovered that this gene is expressed primarily in Prostate.
Many poiynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
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-i 1
related to SEQ 1D N0:34 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be curnbersorne. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1222 of SEQ ID
N0:34, b
is an integer of 15 to 1236, where both a and b correspond to the positions of
nucleotide residues shown in SEQ 1D N0:34. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 25
It has been discovered that this gene i5 expressed primarily in Human 8 Week
Whole Embryo.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:35 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1845 of SEQ ID
N0:35, b
is an integer of 15 to 1859, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:35, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
It has been discovered that this gene is expressed primarily in Supt Cells,
cyclohexamide treated.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:36 and may have been publicly available prior to
conception of
CA 02364209 2001-09-24
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-t2
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 791 of SEQ ID
NO:36, b
is an integer of IS to 805, where both a and b correspond to the positions of
nucleotide residues shown in SEQ 1D N0:36, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 27
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares_pregnant_uterus__NbHPU and to a lesser extent
in
Soares fetal lung_NbHLI9W; Soares fetal~heart NbHHI9W; H. Meningima, M1;
Human Fetal Lung III; Human Synovium; Human Stomach,re-excision; Early Stage
Human Brain; human tonsils and Soares placenta Nb2HP.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 86 as residues: Ser-13 to Gly-18~
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:37 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of" the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1760 of SEQ ID
N0:37, b
is an integer of 15 to 1774, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:37, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTE><N ENCODED BY C~ENE NO: 28
It has been discovered that this gene is expressed primarily in human tonsils.
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.t3
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:38 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1605 of SEQ ID
N0:38, b
is an integer of 15 to 1619, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:38, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares fetal liver spleen INhL,S and to a Lesser
extent in B
Cell Lymphoma; Ovarian Tumor 10-3-95 and Merkel Cells.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:39 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynuclec>tides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to 1620 of SEQ ID
N0:39, b
is an integer of 15 to 1634, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:39, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 30
It has been discovered that this gene is expressed primarily in H. Ovarian
Tumor, Il, OV5232.
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Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:4U and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 607 of SEQ ID
N0:40, b
is an integer of 15 to 621, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:40, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 31
It has been discovered that this gene is expressed primarily in H. Ovarian
Tumor, Il, OV 5232.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:41 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynueleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 2130 of SEQ ID
N0:41, b
is an integer of 15 to 2144" where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:41, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 32
It has been discovered that this gene is expressed primarily in Ovarian Tumor
10-3-95.
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-t~
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 91 as residues: Glu-35 to Glu-50.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:42 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 720 of SEQ ID
NO:42, b
is an integer of 15 to 734, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:42, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 33
It has been discovered that this gene is expressed primarily in H. Ovarian
Tumor, II, OV5232.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 92 as residues: Phe-32 to Asn-37.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:43 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to ( 183 of SEQ ID
N0:43, b
is an integer of I5 to 1 I97, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:43, and where b is greater than or
equal to a
+ 14.
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-16
FEATURES OF PROTEIN E NCODED BY GENE NO: 34
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Messangial cell, frac 1; IiUMAN STOMACH: B Cell
lymphoma; Hepatocellular 'Tumor; H Female Bladder, Adult; Healing groin wound,
6.5 hours post incision; Brain Frontal Cortex, re-excision; Human Adult Small
Intestine; Human Osteoblasts II; Human Uterine Cancer; Human Hippocampus;
Rejected Kidney, lib 4; 12 Week Old Early Stage Human; H Macrophage (GM-CSF
treated), re-excision; HTCDL1; Human pancreatic islet; NCI CGAP_GCB1;
NCI CGAP_Prl2; NCI C<~AP_Pr22; NCI CGAP Brn23; Colon Tumor 11; Soares
melanocyte ZNbHM; Hodgkin's Lymphoma II; Keratinocyte.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:44 and may have been publicly available prior to
conception of
the present invention. Preferably, such related palynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to 2096 of SEQ ID
N0:44, b
is an integer of 15 to 2110, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:44, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 35
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Activated T-Cells, 8 hrs., ligation 2; Human Uterine
Cancer;
Human Fetal Lung III and to a lesser extent in Supt cells, cyclohexamide
treated,
differentially expressed; Kidney medulla; Larynx carcinoma II; Activated T-
Cells, 8
hrs, subtracted: NCI CGAP.._ AA t ; NCI CGAP_-Co 1; NCI CGAP_Pr3; Saos2 Cells,
Vitamin D3 Treated; Human Aortic Endothelium; Supt Cells, cyclohexamide
treated;
HSA 172 Cells: Human Adult Heart,re-excision; Human Prostate; H. Kidney
Medulla, re-excision; T-Cell PHA I6 hrs: Human Chondrosarcoma; Human 'Thymus
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Stromal Cells; Human Whole Six Week Old Embryo; Stratagene liver (#937224);
breast lymph node CDNA library; Soares breast 3NbHBst; Soares melanocyte
2NbHM; Human Amygdala; Human Microvascular Endothelial Cells, fract. A;
Stratagene muscle 937209.; Stratagene endothelial cell 937223; Smooth
muscle,controi; Activated T-cell( l2h)IThiouridine-re-excision and T cell
helper II.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:45 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 942 of SEQ ID
N0:45, b
is an integer of IS to 956, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:45. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 36
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Healing bruin wound. 7.5 hours post incision; Rejected
Kidney, lib 4.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 95 as residues: Pro-2 to Gln-7.
Many polynucleoti<le sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:4fi and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucieotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more pvlynuclec~tides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 580 of SEQ ID
N0:46, b
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WO 00/63230 PCT/US00/07677
is an integer of 15 to 594, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:46. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 37
It has been discovered that this gene is expressed primarily in Rejected
Kidney, lib 4.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:47 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of' tire present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 919 of SEQ ID
N0:47, b
is an integer of 15 to 933, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:47, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 38
It has been discovered that this gene is expressed primarily in Rejected
Kidney, lib 4.
Many polynucleotide sequences. such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:48 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to I l28 of SEQ ID
N0:48, b
is an integer of I S to t 142, where both a and b correspond to the positions
of
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~9
nucleotide residues shown in SEQ ID N0:48. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 39
S The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gi13746338 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "(A1~023259)
RNA-
binding protein Homo sapiens~". A partial alignment demonstrating the observed
homology is shown immediately below.
>gi~3746338 (AF023259) RNA-binding protein [Homo Sapiens] >sp~075876~a75876
RNA-BINDING PROTEIN.
1S Length = 411
Plus Strand HSPs:
Score = 324 (114.1 bits), Expect = 1.6e-28, P = 1.6e-28
2~ Identities = 65/72 (90%), Positives = 67/72 (93%), Frame = +3
Q: 105 VRANGVQAQMAKQQEQDPTNLYISNLPISMDEQELENMLKPFGHVISTRILRDANGVSRG 284
++ANGVQAQMAKQQEQDPTNLYISNLPISMDEQELENMLKPFGHVISTRILRDANGVSRG
S: I13 LKANGVQAQMAKQQEQDPTNLYISNLPISMDEQELENMLKPFGHVISTRILRDANGVSRG 172
2S
Q: 285 VGFAR~NSFFVC 3201
VGFAR S C
5: 173 VGFARMESTEKC 184
30 Score = 93 (32.7 bits), Expect = 0.0040, P = 0.0040
Identities = 18/51 (35%), Positives = 30/51 (58%), Frame = +3
Q: 144 QEQDPTNLYISNLPISMDEQELENMLKPFGHVISTRILRDAN-GVSRGVGF 293
++ TNLYI LP +Q+L + +p+G ++ST+ + D N +G GF
3S S: 47 EQLSKTNLYIRGLPPGTTDQDLIKLCQPYGKIVSTKAILDKNTNQCKGYGF 97
The segments of gi13746338 that are shown as "S" above are set out in the
sequence listing as SEQ ID NO. i37 and SEQ ID NO. 139. Based on the structural
similarity, these homologous polypeptides are expected to share at least some
40 biological activities. Such activities are known in the art, some of which
are described
elsewhere herein. Assays for determining such activities are also known in the
art,
some of which have been described elsewhere herein.
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
~0
Preferred polypeptides of the invention comprise a polypeptide having the
amino acid sequence set out in the sequence listing as SEQ ID NO. 138 and/or
SEQ
ID NO. 140 which correspond to the "Q" sequences in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in Rejected
Kidney, lib 4.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 98 as residues: Lys-44 to Gly-50.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:49 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 406 of SEQ ID
N0:49, b
is an integer of 15 to 420, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:49, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 40
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Human Placenta; B Cell lymphoma; PERM TF274.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:50 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. Ta list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 578 of SEQ ID
NO:50, b
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
pl
is an integer of 15 to 592, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:SO, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 41
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Svares fetal heart NbHH 19W and to a lesser extent in
Snares placenta Nb2HP; Snares fetal liver spleen 1NFLS; Human Endometrial
Tumor; Snares NhHMPu_S1; Stratagene Iuna (#937210); Human Bone Marrow,
treated; Snares fetal_I i ver spleen_ 1 NFLS_S t ;
Snares senescent fibroblasts_,NbHSF; Activated T-cells;
Snares total_fetus_Nb2HF8_ 9w; NTERA2 teratocarcinoma cell line+retinoic acid
(14 days); Human Pineal Gland; HEL cell line;
Snares multiple sclerosis 2NbHMSP; Soares_placenta_8to9weeks 2NbHP8to9W;
Human Adrenal Gland Tumor; 12 Week Old Early Stage Human; Human Testes;
Stratagene NT2 neuronal precursor 937230; Keratinocyte and
Soares_pregnant_uterus NbHPU.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:51 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To List every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1713 of SEQ ID
N0:51, b
is an integer of 15 to 1727, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:51, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 42
CA 02364209 2001-09-24
WO 00/63230 PCT/L1S00/07677
s2
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Prostate BPH,Lib 2, subtracted; Human Fetal Brain,
random
primed; Human Placenta; Amniotic Cells - TNF induced; CD34 depleted Buffy Coat
(Cord Blood); Hodgkin's Lymphoma II.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 101 as residues: Pro-26 to Asn-34, Asp-45 to Arg-64, Pro-93 to
Pro-
108.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:52 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1093 of SEQ ID
N0:52, b
is an integer of 15 to 1107, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:52, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 43
It has been discovered that this gene is expressed primarily in L8 cell line.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 102 as residues: Ser-21 to Phe-32.
Many polynueleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ 1D N0:53 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to 258 of SEQ ID
NO:53, b
CA 02364209 2001-09-24
WO OOI63230 PCT/US00/07677
i3
is an integer of 15 to 272, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:53,. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 44
It has been discovered that this gene is expressed primarily in A1-CELL
LINE.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 103 as residues: Thr-22 to Lys-31.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:54 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to 161 of SEQ ID
NO:54, b
is an integer of 15 to 175, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:54, and where b is greater than or
equal to a
+ l4.
FEATURES OF PROTEIN ENCODED BY GENE NO: 45
It has been discovered that this gene is expressed primarily in Testes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:55 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucieotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between I to 1000 of SEQ ID
NO:55, b
CA 02364209 2001-09-24
WO 00/63230 PCTlUS00/07677
is an integer of 15 to 1014, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:~S, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 46
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Testes; HUMAN B CELL LYMPHOMA.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:56 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between i to 1628 of SEQ ID
N0:56, b
is an integer of 15 to 1642, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:56, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 47
It has been discovered that this Gene is expressed primarily in the following
tissues/cDNA libraries: Soares infant brain 1 NIB and to a lesser extent in
Human
Prostate Cancer, Stage B2 fraction; Human Adult Pulmonary; Human Pancreatic
Carcinoma; wilm's tumor; Pancreas Islet Cell Tumor;
Soares_pregnant_uterus NbHPU and Soares_parathyroid tumor NbHPA.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:57 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
CA 02364209 2001-09-24
WO 00163230 PCT/L1S00/07677
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 294 of SEQ ID
N0:57, b
is an integer of 15 to 308, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:57. and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 48
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Human adult testis, large inserts and to a lesser
extent in
Human Adult Testes, Large Inserts, Reexcision; Human Fetal Heart; Spleen,
Chronic
lymphocytic leukemia and Human Testes.
Preferred immunogenic epitopes include those comprising a sequence shown
in SEQ ID NO. 107 as residues: Ala-29 to Thr-36.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:58 and may have been publicly available prior to
conception of
the present invention. Preferably, such related palynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleatides camprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1573 of SEQ ID
N0:58, b
is an integer of 15 to 1587, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:58, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY CrENE NO: 49
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in Soares
ovary
tumor NbHOT; pBMC stimulated w/ poly IIC; NCI CGAP_GCB1;
Soares total fetus_Nb2HF8__9w; Morton Fetal Cochlea; NCI CGAP_LuS;
Soares fetal_heart_NbHH 19W; Hodgkin's Lymphoma I; Myoloid Progenitor Cell
CA 02364209 2001-09-24
WO 00/63230 PCT/1US00/07677
~6
Line; Human Prostate; Stratagene colon (#937204); Stratagene pancreas
(#937208);
Human Bone Marrow, re-excision; Human Osteoblasts II; Stratagene pancreas
(#937208); Smooth muscle, serum induced.re-exc; HUMAN B CELL LYMPHOMA;
NCI CGAP_Pr21 and Keratinocyte.
Many polynucieotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:59 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 2127 of SEQ ID
N0:59, b
is an integer of 15 to 2141, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:~9, and where b is greater than or
equal to a
+ 14.
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
57
a ~ M '~ ~
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CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
58
Q
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vo r~ oo ~ o ...
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
59
O ~ M ~ d' ~ N M
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CA 02364209 2001-09-24
WO 00/63230 PCT/I7S00/07677
a ~ M ~ _- -
0
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v~ w a_~tz.,
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x x x x x x
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CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
61
Ct
0
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CA 02364209 2001-09-24
WO 00/63230 PC1'/US00/07677
62
_. -
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CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
63
a O O M ~ '~' v1 00 N
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CA 02364209 2001-09-24
WO 00/63230 PCT/i1S00/07b77
64
u_ _
Q O C Q M M M M M
c~.,~ C
'' ~ N N ~ M M N
a
Q ~O ~ ~ N N ~ M M N
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CA 02364209 2001-09-24
WO 00/63230 PCT/LJS00l07677
a o ~ ~ o
0
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a
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Q ~ o o ~. d a
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CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
66
Table I 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 t 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 identifr.ed as SEQ ID NO:X.
The cDNA Clone 1D 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." T'he 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 ID 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
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any of the polypeptide sequences disclosed in the sequence listing) are
sufficiently
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 lD 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 identifiied in Table I.
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 I. 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.
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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
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
l0 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.
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 67:3/-40
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(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 1D NO:Y and/or a
polypeptide encoded by the cDNA contained in A'TCC deposit Z. Polynucleotides
encoding a polypeptide coml>rising, 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.
ignal Sequences
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
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)
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protein. The method of vor~ 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%. (vcyn 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
shown in SEQ ID NO:Y which have an N-terminus beginning within 5 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. 1~or example, the
naturally occurring signal sequence may be further upstream from the predicted
signal
sequence. However, it is likely that the predicted signal sequence wilt 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 ID
NO:X and/or the polynucteotide sequence contained in the cDNA of a deposited
clone, in a mammalian cell (e.g., COS cells. as desribed below). These
polypeptides,
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and the polynucleotides encoding such polypeptides, are contemplated by the
present
invention.
Polpnucleotide and Polypeptide Variants
S The present invention is directed to variants of the polynucleotide sequence
disclosed in SEQ ID 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 ID NO:~'' 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
IS 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
complementary strand thereto, a nucleotide sequence encoding the polypeptide
of
SEQ ID 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).
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%,
95%, 96%, 97%, 98%, 99°~o identical to, for example, the polypeptide
sequence
shown in SEQ ID 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).
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By a nucleic acid having a nucleotide sequence at least, for example, 95%
"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
9S%
identical to a reference nucleatide 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
as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 80%, 8S%, 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
overall match between a query sequence (a sequence of the present invention)
and a
subject sequence, also referred to as a glabal 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 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=I,
Joining Penalty=30, Randamization 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 S' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the f~ASTDB program does not account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
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sequences truncated at the ~' 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 matchedlaligned 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,
l0 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
IS 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
20 subject sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the S' or 3' of
the subject
sequence which are not matched/aiigned 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
25 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
30 except that the subject polypeptide sequence may include up to five amino
acid
alterations per each l00 amino acids of the query amino acid sequence. In
other
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7~
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 rnay 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, far 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
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/ali~ned is
determined by
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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
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, I-5, or 1-2 amino acids are substituted,
deleted, or
added in any combination are also preferred. Polynucleotide variants can be
produced
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76
for a variety of reasons, e.g., to optimize colon expression for a particular
host
(change colons 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 Vfiley & 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. F'or example,
Gayte 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
3,500 individual IL-la mutants that avera=ed 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 "jmjost of the
molecule
could be altered with little effect on either binding or biological activity
j." (See,
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.
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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
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 (~-terminal residues of a protein retains such
immunogenic
activities can readily be determined by routirre 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
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 ai.,
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
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
substitution could be modified while still maintaining biological activity of
the
protein.
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-IU85 (1989).) The resulting mutant
molecules can then be tested for biological activity.
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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 mare of the non-conserved amino acid
residues,
where the substituted amino acid residues may yr 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. Such variant polypeptides
are
deemed to be within the scope of those skilled in the art from the teachings
herein.
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); Bobbins et al., Diabetes 36: 838-845 ( 1987); Cleland et al., Crit.
Rev.
Therapeutic Drug Carrier 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
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79
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
S 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, andlor 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
l0 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid
substitutions are
preferable.
Polpnucleotide and Polv~ptide ~'_r~gments
The present invention is also directed to polynucleotide fragments of the
IS 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 poiypeptide encoded by the cDNA in a
deposited
clone; is a portion of that shown in SEQ ID NO:X or the complementary strand
20 thereto, or is a portion of a polynucieotide sequence encoding the
polypeptide of SEQ
ID NO:Y. The nucleotide fragments of the invention are preferably at least
about 15
nt, and more preferably at least about 20 nt, still more preferably at least
about 30 nt,
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
25 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 I ) nucleotides, at either terminus or
at both
termini. These nucleotide fragments have uses that include, but are not
limited to, as
30 diagnostic probes and primers as discussed herein. Of course, larger
fragments (e.g.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
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WO 00/63230 PCTIUS00/07677
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-S0. 51-100, 101-150, 151-200, 2CI1-
250,
251-300, 301-350, 351-400, 40150, 451-500, 501-550, 551-600, 651-700, 701-750,
5 751-800, 800-850, 8S 1-900, 90 I -950, 951-1000, I 001-1050, 1051-1100, 1101-
1150,
1151-1200, I 20 I -1250, I 251-1300, 13 01- i 3 50, 13 S 1- I 400, I 40 I -
1450, I 45 I ~- I 500,
1501-1550, 1 SS 1-1600, 16(11 ~-1650, 1651- I 7C)D. I 70 I -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
10 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
15 stringent hybridization conditions or cower stringency conditions are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
In the present inventian, 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-
20 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,
41-60, 61-80, 81-100, 102- I 20, 121-140, 141-160, or 161 to the end of the
coding
25 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 l ) amino acids, at either extreme or at
both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
30 Preferred polypeptide fragments include the secreted protein as well as the
mature form. Further preferred polypeptide fragments include the secreted
protein or
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81
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
S deleted from the carboxy terminus of the secreted protein or mature form.
Furthermore, any combination of the above amino and carboxy terTrrinus
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.
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)
polypeptide of invention protein. Such functional activities include, but are
not
limited to, biological activity, antigenicity Viability 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
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82
of the invention), ability co form multimers with polypeptides of the
invention, and
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
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
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
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
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
chromatography, protein affinity chromatography, and affinity blotting. See
generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-i23. 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
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83
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
NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in ATC_'C'. 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
described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:399$- 4002 ( 19$3)). 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
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8-t
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. 4,631.21 I).
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, IS> 20, 25, 30, 35, 40, 45. 50. S5, 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 welt 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
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
CA 02364209 2001-09-24
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8~
have been shown to be sufficient to raise antibodies capable of binding to, at
the very
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
earner 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 i00 ~g of peptide or
carrier
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 (CH I , CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides. Such fusion
proteins
may facilitate purification and may increase hali~-life in vivo. This has been
shown
for chimeric proteins consisting of the first two domains of the human CD4
CA 02364209 2001-09-24
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86
polypeptide and various domains of the constant regions of the heavy or light
chains
of mammalian immunoglc>bulins. See, e.g., EP 394,827; Traunecker et al.,
Nature,
331:84-86 ( 1988). Enhanced delivery of an antigen across the epithelia)
barrier to the
immune system has been demonstrated for antigens (e.g., insulin) conjugated to
an
FcRn binding partner such as 1gG or Fc fragments (see, e.g., PCT Publications
WO
96/22024 and WO 9910481.3). 1gG Fusion proteins that have a disulfide-linked
dimeric structure due to the 1gG 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;
5,834,252; and 5,83?,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
CA 02364209 2001-09-24
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R7
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,
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,
domains, fragments, etc. of one or more heterologous molecules.
Anti bodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment,
or variant of SEQ ID 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,
polyelonal, monoclonal, multispecific, human, humanized or chimeric
antibodies,
single chain antibodies, Fab fragments, F(ab') fragments, fragments produced
by a
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 immunoglobulin molecules, i.e.,
molecules
that contain an antigen binding site that immunospecifically binds an antigen.
The
immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE,
IgM,
IgD, IgA and IgY), class (e.g., IgG l, IgG2, IgG3, IgG4, IgA 1 and IgA2) or
subclass
of immunoglobulin molecule.
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
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88
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., PC'T
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., 3. 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
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,
CA 02364209 2001-09-24
WO 00/63230 PCT/LJS00/07677
89
ortholog, or homolag of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95°~0, at least 90%, at
least 85%, at
least 80%, at least 75%, at least 70%, at least 65°l0, at least 60%, at
least 55%, 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°h, and less than 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 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, ar 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, 5 X 10-3 M, 10-3 M, 5 X 10-' M,
10~° M, 5
X 10'5 M, 10-5 M, 5 X 106 M, 10-6M, 5 X 10-' M, 10' M, 5 X 10-8 M, 10-8 M, 5 X
10'9
M, 10-9 M, 5 X 10''° M, 10'° M, 5 X 10~" M, 10-" M, 5 X 10-'Z
M,'°''2 M, 5 X 10-"
M, 10''3M,5X 10-'4 M, 10~'4M,5X 10-''M,or 10-'SM.
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 antibady competitively inhibits binding
to the
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 50%.
CA 02364209 2001-09-24
WO 00/63230 PCTI(JS00/07b77
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
5 bind an antigenic epitope disclosed herein. or a portion thereof. The
invention
features both receptor-specific antibodies and iigand-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. Far
10 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%o, at least 90%, at least 85%, at least
80%, at least
15 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
20 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
25 agonists, i.e., potentiate or activate either all ar 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 aganists, 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
30 methods known in the art. See, e.g., PCT publication WO 96/40281; U.S.
Patent No.
5,811,097; Dena et al., Blood 92(6):1981-1988 ( 1998); Chen et al., Cancer
Res.
CA 02364209 2001-09-24
WO 00/63230 PCT/US00/07677
9i
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. tell. Sci. I 11(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): I 153-I 167 (
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.,
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.
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
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
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
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
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 phase clone, and not the method by
which it
is produced.
Methods for producing and screening for specific antibodies using hybridoma
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
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with a polypeptide of the invention or a cell expressing such peptide. Once an
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
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
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including fd and M 13 binding domains expressed from phage with Fab, Fv or
disulfide stabilized Fv antibody domains recombinantly fused to either the
phage
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. PC'T/GB91/Ol 134; PCT publications WO 90102809;
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
92122324;
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
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9~
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); G fillies et al., ( 1989) J.
lmmunol.
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 complernentarity 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 ( I 991 ); Studnicka et
al.,
Protein Engineering 7(6):805-814 (1994): Roguska. et al., PNAS 91:969-973
(1994)),
and chain shuffling (U.S. Patent No. 5,565.332).
Completely human antibodies ate 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
98124893, 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 c:an also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
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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
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
irnmunoglobulin loci by homologous recombination. In particular, homozygous
deletion of the JH region prevents endogenous antibody production. The
modified
embryonic stem cells are expanded and microinjeeted 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 (i995). Fora
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, (..'A) 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
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selected non-human monoclonal antibody, e.~~., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Bioltechnology 12:899-903 ( 1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized
to generate anti-idiotype antibodies that "mimic" poiypeptides of the
invention using
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
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
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
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 poiypeptide of
the
invention, preferably, an antibody that binds to a polypeptide having the
amino acid
sequence of SEQ ID NO:Y .
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
synthesis of overlapping oligonucleotides containing portions of the sequence
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encoding the antibody, annealing and ligadna 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
S particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoalobulin may be chemically
synthesized
or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA
library
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, andlor 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
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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. ~rnd preferably human framework
regions
(see, e.g., Chothia et al., J. Mol. Biol. 278: 4S7-479 ( 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
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 tire 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 314:452-4S4 ( 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. LISA 85:5879-5883 ( 1988); and Ward et al., Nature
334:544-S4 ( 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)).
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Methods of Producing Antibodies
The antibodies of tt~e 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
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 reconnbinant DNA techniques, synthetic techniques, and
in vivo
genetic recombination. The invention. thus, provides repiicable 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
finked 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; PC'f
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
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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 rnay 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
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.SK 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)).
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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
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 fate promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus gename 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.a., see Logan & Shenk, Proc. Natl.
Acad.
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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., giycosylation) and processing
(e.g.,
cleavage) of protein products may be important far the function of the
protein.
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 of the 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 t-2 days in an enriched media, and then are switched to a selective
media.
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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 ( 198 l
)); gpt,
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 I 1(5). I55-215); 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. 150:1 ( 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
CA 02364209 2001-09-24
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10s
cloning, Vol.3. (Academic Press, New York, 19$7)). 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.
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
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106
acids of the polypeptide) of the present invention. For example, antibodies
rnay 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 CVO 93/21232; EP 439,095; Naramura
et
al., lmmunol. Lett. 39:91-99 ( 1994); U.S. Patent 5,474,981; Gillies et al.,
PNAS
89:1428-1432 (1992); Fell et al., J. lmmunol. 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
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 lgM. 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 (199i);
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 lU NO:Y may be fused or conjugated
to
the above antibody portions to increase the in vivo half life of the
polypeptides or for
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107
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. E3iochem. 270:3958-3964
{1995)). In
many cases, the Fc part in <~ 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
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. Ac:ad. 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
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i0R
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,
S 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,?41,900 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
1S 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 iuminol; examples of bioluminescent materials include luciferase,
luciferin,
and aequorin; and examples of suitable radioactive material include 1251,
131I, 11 lin
or 99Tc.
Further, an antibody or fragment thereof rnay 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
2S 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, S-fluorouracii decarbazine), alkylating agents (e.g.,
mechlorethamine,
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1 ()9
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfa~i, dibromomannitol, streptozotocin, mitomycin C.',
and cis-
dichlorodiamine platinurre (11) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, rruthramycin, 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
IS 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas
Ligand
(Takahashi et al., Int. Imrnunol., 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-~ I ("IL-1 "), inter(eukin-2 ("IL-2"), interleukin-6
("IL-6"),
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 Carriers Of Cytotoxic
Agents
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110
In Cancer Therapy: A Review", in Monoclonal Anti bodies'84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis,
Results,
And Future Prospective tJf"fhe'I'herapeutic 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.
lmmunophenotyping
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
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
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the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation andlor 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. 1, 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 ( I % NP-40 or Triton X- 100, I % sodium
deoxycholate, O.I% 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
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, lnc., New York at 10.16.1.
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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), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blr>ckina 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 1251) 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
IS further discussion regarding western blot protocols see, e.g., Ausubel et
al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., lVew
York
at I 0.8.1.
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
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,
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11;
eds, 1994, Current Protocols in Molecular Biology, Vol. I, 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 1251) 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 1251) 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
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.
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1 I -1
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 (CUC) 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 IL-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,
including fragments thereof. Preferred binding affinities include those with a
dissociation constant or Kd less than 5 X 10-2 M, 10-2 M, 5 X 10-3 M, 10-3 M,
5 X 10~'
M,l0~M,5X10-SM,10-SM,SX10'6M,I0'''M,SX 10-'M,10-'M,SX10-8M,
10-g M, 5 X 10'9 M, 10~y M, 5 X 10-'° M, 10-'° M, 5 X 10-" M, 10-
" M, 5 X 10-'2 M, 10-
'2M,SX 10-'~M, 10-'3Iv1,5X 10-'4M, l0''°M,SX 10''SM,and 10-'SM.
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Its
Gene Theranv
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.
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-SOS (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 I I (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 chirneric 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
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); Zi jlstra et al., Nature 342:435-438 ( 1989). In
specific
CA 02364209 2001-09-24
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116
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. C'hem. 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 lysosoma)
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 92106180; WO 92122635; 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
(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al.,
Nature 342:435-438 (1989)).
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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 delivE;r 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: C~lowes 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.
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).
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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., Loeffier 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, rnonocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
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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. EAny stem andlor 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. PC.'T 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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Therapeutic/Prophylactic 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 inventic.,n, 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, ete.
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
phar~rrraceutical
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 poraus, 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 1~idler (eds.), Liss, New Yark, pp. 3_53- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embadiment, 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.), C:RC Pres., Boca Raton, Florida
(1974);
Controlled Drug Bioavailabiiity, Drug Product Design and Performance, Smolen
and
Ball (eds.), Wiley, New Yark ( 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
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acid expression vector and administering it so that it becomes intracellular,
e.g., by
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.,
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
"carrier" refers to a dituent, 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
carriers
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
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pharmaceutical carriers are described in "IZemington's Pharmaceutical
Sciences" by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of
the compound, preferably in purified form, together with a suitable amount of
carrier
so as to provide the form fc~r proper administration to the patient. The
formulation
S 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 cations such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
2S 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
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practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100 mg/kg of the patient's body weight. Y'referably, 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
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, andlor 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
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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 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 imrnunohistoiogical 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 (1:LISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucase
oxidase;
radioisotopes, such as iodine (125I, 1211), 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 ievel indicates that the
subject
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has a particular disease or disorder associated with aberrant expression of
the
polypeptide of interest. Background level can be determined by various methods
including, comparing thc; 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
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 lnc. ( 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 carried 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 (C'f),
whole body
scan such as position emission tomography (PET), magnetic resonance imaging
(MRI), and sonography.
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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
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
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.
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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
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. 'fhe 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.
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Alternatively, streptavidin coated plates can be used in 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
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
poiypeptide 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 (lgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and
any
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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 viva. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide and various
S 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).)
Fusion proteins having disulfide-linked dimeric structures (due to the 1gG)
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.
JO 270:3958-3964 (1995).)
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 parrt thereof. In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
15 example, improved pharrnacokinetic 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
20 purpose of high-throughput screening assays to identify antagonists of h1L-
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.
25 In preferred embodiment , 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.
30 Another peptide tag useful for purification, the "HA" tag, corresponds to
an epitope
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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, ~rnd Protein Pro~ct~,on
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 rnay 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 tic
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 constricts 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. coii and other bacteria. Representative
examples
of appropriate hosts include, but are not limited to, bacterial cells, such as
E. coli,
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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 Sf9 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 QIAGF?N, Inc.; pBluescript vectors, Phagescript vectors,
pNHBA,
pNHl6a, pNHlBA, pNH46A, available from Stratagene Cloning Systems, lnc.; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech,
lnc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
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, pYES2IGS,pPICZ,pGAPZ, pGAPZaIph,
pPIC9, pPIC3.5, pHIL-G2, pHIL-SI,,pPIC3.SK, pPIC9K, and PA08I5 (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 cation 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.
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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-
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 pastoria
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 OZ. 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 pan, to the relatively low affinity of
alcohol
oxidase for O2. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXI ) is
highly active. In the presence of methanol, alcohol oxidase produced from the
AOXl
gene comprises up to approximately 30% of the total soluble protein in Pichia
pastoris. See, Ellis, S.B., et al., ll~lol. Cell. Bial. 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
ADXl
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I ~-I
regulatory sequence is expressed at exceptionally high levels in Pichia yeast
grown in
t:he 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
.1. Cregg, eds. The Humana Press, Tatowa, NJ, 1998. This expression vector
allows
expression and secretion of a protein of the invention by virtue of the strong
ADXl
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory
signal
peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2,
pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAP'l, pGAPZalpha, pPIC9, pPIC3.5,
pHIL-D2, pHIL-SI, pPIC.'.3.5K, 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,
IS including an in-frame ALJG 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 poiynucleotides. For
example,
techniques known in the art may be used to operabty 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
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(see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; U.S. Patent No.
5,733,761, issued March 3l, 1998; 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 Zi jlstra et al., Nature 342:435-438 ( 1.989), 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,
:310:105-11 1 ( 1984)). For example, a polypeptide corresponding to a fragment
of a
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,
amidation, derivatization by known protectingJblocking groups, proteolytic
cleavage,
linkage to an antibody molecule or other cellular iigand, etc. Any of numerous
chemical modifications may be carried aut 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.
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Additions! post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or (=)-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 LLS. Patent NO: 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycollpropylene glycol copolymers, carboxymethylcellulose,
dextran, polyvinyl alcohol and the tike. 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 I 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).
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 3$4, herein incorporated by reference
(coupling PEG
to G-CSF~, see also Malik et al., Exp. Hematol. 20:102$-1035 (1992) (reporting
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pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
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. 'Che amino acid residues having a
free
amino group may include; lysine residues and the N-terminal arruno 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.
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.
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
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1 38
tetramers. In additional embodiments. the multimers of the invention are at
least
dimers, at least trimers, or at least tetrarners.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homorner, 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 describE;d 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
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 andlor 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
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139
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 polypeptid~ 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
(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
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1d~
proteins. Among the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize. Examples of Ieucine 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 rnultimeric 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. Une example is a leucine zipper
derived from
lung surfactant protein 1J (SPD), as described in Hoppe et al. (FEBS Letters
344:191,
(1994)) and in U.S. patent application Ser. No. 081446,922, hereby
incorporated by
reference. Other peptides derived from naturally occurring 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 FlagtB 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
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
CA 02364209 2001-09-24
WO 00/63230 PCT/US00107677
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., tIS 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,
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, potynucleotides 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
liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
2S reference in its entirety).
Uses of th~Eo_ly~~cleotidg~
Each of the potynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes
known techniques.
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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
elected 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 PC'.R mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycler. 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, and preselection by hybridization to construct
chromosome specific-cDNA libraries.
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
Verma et al., "Human Chromosomes: a Manual of Basic T'echniques," 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). Preferred poiynucleotides
correspond to the noncoding regions of the cDNAs because the coding sequences
are
more likely conserved within gene families, thus increasing the chance of
cross
hybridization during chromosomal mapping.
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Once a polynucieotide 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 ane of 50-500 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 palymorphism. if a new polymorphism is identified, this
polymorphic polypeptide can be used for further linkage analysis.
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
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
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subject. In a general embodiment, the kit includes at least one polynucleotide
probe
c;ontaining 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 polymerase 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 mRNA level in a second biological sample).
Preferably, the
polypeptide level or mRNA level in the first biological sample is measured or
estimated and compared to a standard poiypeptide 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
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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 and/or 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,
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
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multiplex hybridization. Smaller probes can be used than with L>NA 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 i5-mer
lowers the melting point (T
m) by 8°-20° C'., vs. 4°-
l6° 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, andlor 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 1., Wiernik, P. H. et al. eds., 161-182
(1985)).
Neoplasias are now believed to result from the qualitative alteration of a
normal
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
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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, andlor 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 Expre.ssion,CRCPress,
Boca
Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et
al.,
Nucleic Acids Research 6. 3073 (1979j; 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
(1988); and Dervan et al., Science 251:1360 ( 1991 ) ) or to the mRNA itself
(antisense
- Okano, J. Neurochem. 56:560 (1991 j; 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 treator prevent disease.
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Polynucleotides of the present invention are also useful in gene therapy. One
goal of gene therapy is tc:> 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 Llnited 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 enaymes, 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
IS 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. Unce 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.
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. (Erlieh, H.,
PCR
Technology, Freeman and Co. (i992).) Once these specific polymorphic loci are
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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
andlor 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 thg;~'olvuentides
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
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 irnmunosorbent
assay
(ELISA) and the radioimmunoassay (R1A>. Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (125I, 1.211), carbon (t4C), sulfur (35S), tritium (3H), indium
(1121n), and
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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,
1311,
112In, 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 99rn~ft. 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).)
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
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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 hemoglohin 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
Iigand
(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-PACrE gels or on molecular sieve gel
filtration
columns using methods well known to those of skill in the art. Polypeptides
can also
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.
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Gene Thera~r Methods
Another aspect of the present invention is to gene therapy methods for
treating
or preventing disorders, diseases and conditions. The gene therapy methods
relate to
the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences
S 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 lnst., 85:207-216
(1993);
Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J_
Irrrmunology 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,
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
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into the cell, including viral sequences, viral particles, liposorne
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 sequerrces 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 pEF11V5,
pcDNA3.1, and pRcICMV2 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
IS thc; expression of poiynucleotide 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 shtick promoters; the albumin
promoter;
the ApoAl promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine 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
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,
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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
S 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 rng/kg body weight to about
50
mglkg 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 mg/kg. 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.
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.
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The constructs may also be delivered with delivery vehicles such as viral
sec;uences, viral particles, liposorne 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. I_iposomal 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.,
Pros.
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
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available materials. Such materials include phosphatidyl, choline,
cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DUPE),
among others. These materials can also be mixed with the DOTMA and DOTAP
starting materials in appropriate ratios. Methods for rnakirtg liposomes using
these
materials are well known in the art.
For example, commercially dioieoylphosphatidyl 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 (SUVs), or large unilamellar vesicles (LUVs), 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.
SUVs are prepared by extended sonication of MLVs to produce a homogeneous
population of unilamellar liposomes. The material to be entrapped is added to
a
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
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then the preformed liposorrres are mixed directly with the DNA. The liposome
and
DIVA 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. US,A, 76:3348 (
1979));
detergent dialysis (Epoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979));
and
reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431
(1980);
Saoka 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
carriers, 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.S. Patent Nos. 5,589,466,
5,693,622,
5.,580,859, 5,703,055, and international publication NO: WO 941~>469 (which
are
herein incorporated by reference) provide methods for delivering DNA-cationic
lipid
complexes to mammals.
In certain embodiments, cells are engineered, ex vrvo 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 Viras, avian
leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
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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, (:iP+E-86, GP+envAml2, and DAN cell lines as described
in Miller, Human Gene Therapy , I:S-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 rnay 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 (I974)). 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);
Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther.,
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1i9
4:759-769 ( 1993); Yang et al., Nature Genet., 7:362-369 ( 1994); ~Nilson et
al.,
Nature , 365:691-692 (1993); and 1J.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 E1 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,
IS 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, IJ.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
A.AV viral particles which contain the polynucieotide 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
ge;nome, and will express the desired gene product.
Another method of gene therapy involves operably associ<rting 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 .tune 24, 1997: International Publication NO: WO
9(i/29411, published September 26, 1996; International Publication NO: WO
94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sei. USA,
8fi:8932-8935 ( 1989); and Zijlstra et al., Nature, 342:43538 ( 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
ar1., 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-1, 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 S ' 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"), gelfoarn sponge depots, other commercially
available
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depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial
solid
(ta.blet 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 poiynucleotide construct can be coated on
the
IS 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 uses 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: I 1277-11281 ( 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
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polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend
upon a number of factors including, for example, the chemical structure 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
poiynucleotide 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
dcaermined 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
~,'~olo~ical 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
a<aivity. Thus, the polynucleotides or polypeptides, or agonists or
antagonists could
be used to treat the associated disease.
~mune Activity
The polynucleotides or polypeptides, 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 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 or some autoimmune
diseases,
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1 (r1
disorders,and/or conditions, acquired (e.g.. by chemotherapy or toxins), or
infectious.
Moreover. a polynucleotides or polypeptides, or agonists or antagonists of the
present
invention can be used as a marker or detector of a particular immune system
disease
or disorder.
A polynucleotides or polypeptides. 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. A polynucleotides or polypeptides,
or
ag;onists 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 treator prevent those diseases, disorders, andlor
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, andlor conditions (e.g. agammaglobulinemia,
dysgammaglobulinemia), ataxia telangiectasia, common variable
immunodeficiency,
Digeorge Syndrome, HIV infection, HTLV-BLV infectian, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe
combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
th.rombocytopenia, or hernoglobinuria.
Moreover, a polynucleotides or polypeptides, 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, a polynucleotides or polypeptides, 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, a polynucleotides or polypeptides, 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.
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16i
A polynucleotides or polypeptides. or agonists or antagonists of the present
invention may also be useful in treating, preventing, andlor diagnosing
autoimmune
diseases, disorders, and/or conditions. Many autoimmune diseases, disorders,
and/or
conditions result from inappropriate 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 a
polynucleotides or
polypeptides, or agonists or antagonists of the present invention that
inhibits an
immune response, particularly the proliferation, differentiation, or
chemotaxis of T
cells, may be an effective therapy in preventing autoimmune diseases,
disorders,
and/or conditions.
Examples of autoimmune diseases. disorders, and/or conditions that can be
treated, prevented, and/or diagnosed or detected by the present invention
include, but
are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid
syndrome,
rheumatoid arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis,
Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia
Gravis,
Neuritis, Ophthalmic, Bullous Pemphigoid, Pemphigus, Poiyendocrinopathies,
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.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or other respiratory problems, may also be treated,
prevented, and/or
diagnosed by polynucleotides or polypeptides, or agonists or antagonists of
the
present invention. Moreover, these molecules can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group incompatibility.
A polynucleotides or polypeptides, or agonists or antagonists of the present
invention may also be used to treat, prevent, andlor diagnose organ rejection
or graft-
versus-host disease (GVHD). Organ rejection occurs by host immune cell
destruction
of the transplanted tissue through an immune response. Similarly, an immune
reaponse is also involved in GVHD, but, in this case, the foreign transplanted
immune
cells destroy the host tissues. The administration of a polynucleotides or
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polypeptides, or agonists or antagonists of the present invention that
inhibits an
immune response, particularly the proliferation, differentiation, or
chemotaxis of T-
ce:lls, may be an effective therapy in preventing organ rejection or GVHD.
Similarly, a polynucleotides or
polypeptides, or agonists or antagonists of the present invention may also be
used to
modulate inflammation. For example, the polypeptide or polynuc:leotide or
agonists
or antagonist may inhibit the proliferation and differentiation of cells
involved in an
inflammatory response. 'These molecules can be used to treat, prevent, and/or
diagnose inflammatory conditions, both chronic and acute conditions, including
chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation
associated with infection (e.g., septic shock, sepsis, or systemic
inflammatory
reaponse syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality,
arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or chemokine
induced
lung injury, inflammatory bowel disease, Crohn's disease, or resulting from
over
production of cytokines (e.g., TNF or IL-l.)
~vneruroli~eraNve Disorders
A polynucleotides or polypeptides, or agonists or antagonists of the invention
can be used to treat, prevent, and/or diagnose hyperproliferative diseases,
disorders,
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
h:yperproliferative disorder.
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
irnmune response. Alternatively, decreasing an immune responses may also be a
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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
ag;onists 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
a~;onists 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, andlor
protein fusions or fragments thereof.
Thus, the present invention provides a method for treating or preventing cell
proliferative diseases, disorders, and/ar 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
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
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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 1.
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
cE;lls either alone, or in combination with or fused to other polynucleotides,
can then
be modulated via an exterrral 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,1. Virology 44:845 (1982); Hocke,
Nature
3.20:275 (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.
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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 thc: disease
site. The
polynucleotides of the present invention may also be administered to disease
sites at
thf: 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
po~lynucleotide 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.
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, andlor conditions. Methods for
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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.
S 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
IS 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
cornbination 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 andlor potent in vivo inhibiting and/or
neutralizing antibodies against poiypeptides or polynucleotides of the present
invention, fragments or regions thereof, for bath immunoassays directed to and
2S 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
with a dissociation constant or Kd less than SX10-6M, 10-6M, 5X 10-'M, 10-'M,
5X10-
$M, 10-8M, 5X10-HM, 10~9M, 5X10'°M, I0'°M, 5X10-"M, 10~"M, SX10-
'ZM, 10~'ZM,
5X.10-'3M, 10~'3M, SX l0~'°M, 1 U~'4M, 5X 10~'SM, and 10~'SM.
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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, throuCh the inhibition of hematopoietic,
tumor-
specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J
Nat1
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
re:Ference)).
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
IS apoptosis of proliferative cells and tissues, for example in the activation
of a death-
domain receptor, such as tumor necrosis factor (TNF) receptor-l, CD95 (Fas/APO-
1),
TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL,) receptor- l 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
acaivation 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;11 i-I 12:23-34 (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).
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
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directed to said polypeptides as described elsewere herein, or indirectly,
such as
activating the expression of proteins known to inhibit metastasis, for example
alpha 4
integrins, (See, e.g., Curr Top I'vlicrobiol lmmunol 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, heteralogous 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 imrnunogenicity and/or antigenicity of praliferating
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
irrrmunogens, or indirectly, such as in activating the expression of proteins
known to
enhance the immune response (e.g. chemakines), 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 arteria-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
arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart
syndrome,
levocardia, tetralogy of fallot, transposition of great vessels, double outlet
right
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ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septa)
defects, such
as aortopulmonary septa) defect, endocardial cushion defects, Lutembacher's
Syndrome, trilogy of Fallot, ventricular heart septa) 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
septa) rupture, heart valve diseases, myocardial diseases, myocardial
ischemia,
pericardial effusion, pericarditis (including constrictive and tuberculous),
praeumopericardium, 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, atria) fibrillation, at.rial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-
ty,pe 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 atria)
tachycardia, ectopic
junctions) 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, mitrai valve prolapse, tricuspid valve
prolapse,
mitral valve insufficiency, mitral valve stenosis, pulmonary atresia,
pulmonary valve
insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
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pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyapathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
repf:rfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm, myocardial inf'arctian 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,
andlor 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 versa 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,
Moyarnoya
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
hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
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(including transient), subclavian steal syndrome, periventricular
leukomalacia,
vascular headache, cluster headache, migraine, and vertebrobasilar
insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
ernbolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein acclusion, carotid artery thrombosis, sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myacardial 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-Ang~genesis Activity
The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad etal., Ce1156:345-355 (19$9). 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 conditians 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. OpthalnZOl. 94:71 S-743 ( I 982); 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/ar diagnosing
an
angiogenesis-related disease and/or disorder, comprising administering to an
individual in need thereof a therapeutically effective amount of a
polynucleotide,
palypeptide, 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 treat or prevent a cancer or
tumor.
Cancers which may be treated, prevented, and/or diagnosed with
polynucleotides,
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polypeptides, antagonists and/or agonists include, but are not limited to
solid tumors,
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;
gliobiastoma;
K;aposi'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; endometryosis; 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.
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For example, within one aspect of the present invention methods are provided
for treating, preventing, and/or diagnosing hypertrophic scars and keloids,
comprising
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 polynucle.otides,
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,
rel:rolental 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 ~t 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
ne:ovascularization (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
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pericorneal vascular plexus c>f the limbos. 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
diseases, disorders, and/or conditions can result in corneal
neovascularization,
including for example, corneal infections (e.g., trachoma, herpes simplex
keratitis,
leishmaniasis and onchocerciasis), imrnunological 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
e~~edrop 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
a:Fter a corneal insult in order to praphylactically prevent corneal
neovascularization.
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In this situation the material cauld 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-
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,
palypeptide, 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 polynueleotide, 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.
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Additionally, diseases, disorders, andlor conditions which can be treated,
prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists
and/or
agonists include, but are not limited to, hernangioma, arthritis, psoriasis,
angiofibroma, atherosclerotic plaques, delayed wound healing, granulations,
hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber
syndrome,
pyogenic granutoma, scleroderrna, trachoma, and vascular adhesions.
Moreover, diseases, disorders, andior 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
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.
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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. >ror example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
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 rnay be utilized in any procedure
wherein a
surgical mesh might be utilized. For example, within one embodiment of the
IS 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 polynucleatide,
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
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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 andlor agonists of the present
invention may also be administered along with other anti-angiogercic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-I, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-I, 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 motybdate complexes include ammonium molybdate and its
hydrates, sodium molyb~date and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(V1) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
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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
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.
2Ei7:17321-17326, 1992); C'hymostatin (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-
31.6, 1992); Thalidomide; Angostatic steroid; AGM-1470;
carboxynaminolmidazole;
and metalloproteinase inhibitors such as BB94.
Diseases at tl2g 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, myorna, lymphoma, endothelioma,
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o:;teoblastoma, osteoclastorna, osteosarcorna, chondrosarcoma, adenoma, breast
cancer, prostate cancer, Kapasi's sarcoma and ovarian cancer); autoimmune
diseases,
disorders, and/or canditions (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 virai infections (such as herpes viruses, pox
viruses and
adenoviruses), inflammation, graft v. host disease, acute graft rejection, and
chronic
graft rejection. in preferred embodiments, the polynucleotides or
polypeptides, and/or
al;onists 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 ar diagnosed by the polynucleotides ar
polypeptides, or
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.,
H:odgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including, but not
limited
ta> sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, 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
c;3rcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
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craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented,
andlor diagnosed by the polynucleotides or pofypeptides, and/or agonists or
antagonists of the inventior>, 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,
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.,
hE:patitis related liver injury, isehemialreperfusion injury, cholestcrsis
(bile duct injury)
and fiver 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 proliferatian 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 heating 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
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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 tc> 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
oh 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, cutis graft, delayed graft,
dermic graft,
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
IS 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, cauld 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, andlor agonists or antagonists of the
invention, could also be used to reduce the side effects of gut toxicity that
result from
radiation, chemotherapy treatment's 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
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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
gl;~nds, 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 poiynucleotides or polypeptides,
and/or
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 polynucteotides or polypeptides, and/or
agonists or
antagonists of the invention, could be used to promote the resurfacing of the
mueosal
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 polynucteotides 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
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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, andlor 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 11
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,
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, andlor 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.
Neurologic,~l 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, andlor agonists or antagonists), include, but
are not
limited to, nervous system injuries, and diseases, disorders, and/or
conditions which
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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
destroyed or injured as a result of infection, for example, by an abscess or
associated
IS 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
di:>order of metabolism including but not limited to, vitamin B12 deficiency,
folic
acid deficiency, Wernieke 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
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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, andlor 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
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 rnay 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: ( L) 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 ar in vivo, e.g., choline
acetyltransferase or
aretylcholinesterase 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.
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Neurol. 70:65-82 ( 1980)) car 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, andlor 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
IS spinal muscular atrophy, progressive bulbar palsy, primary lateral
sclerosis, infantile
and juvenile muscular atrophy, progressive bulbar paralysis of childhood
(Fazio-
Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
~>tfectious 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 andlor T cells, infectious diseases may betreated,
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 po(ynucleotide
or
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polypeptide and/or agonist or antagonist of the present invention. Examples of
viruses, include, but are nc>t 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, F'laviviridae, 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
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,
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inc:lude, but not limited ta, the following Gram-Negative and Gram-positive
bacteria
and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium,
Mycobacterium, Norcardiaj, Cryptococcua neoformans, Aspergillosis, Bacillaceae
(e.g., Anthrax, Clostridium j, 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,
He~licobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,
Mycobacterium leprae, Vibria cholerae, Neisseriaceae (e.g., Acinetobacter,
Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections
(e.g.,
Acainobacillus, 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,
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.
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.
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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 wax, Plasmodium falciparium, Plasmodium malariae and Plasmodium
ovale). These parasites can cause a variety of diseases or symptoms,
including, but
not limited to: Scabies, T'rombiculiasis, 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 usedtotreat,
prevent,
and/or diagnose any of these symptoms or diseases. In specific embodiments,
pcdynucleotides, 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
an.d/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
pcdypeptide or polynucleotide of the present invention can be used as an
antigen in a
vaccine to raise an immune response against infectious disease.
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 276:59-8? (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.
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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 andlor 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-heating wounds includes pressure ulcers, ulcers associated
with
vascular insufficiency, surgical, and traumatic wounds.
IS Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide or polypeptide andlor 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-
Drager syndrome), could all be treated, prevented, and/or diagnosed using the
polynucleotide or polypeptide and/or agonist or antagonist of the present
invention.
f'hemotaxis
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,
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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, and/or 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.
)t3inding 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.
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
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of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using knOWl1 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
campound 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
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 eDNA library created from this RNA is
divided
into pools and used to transfect COS cells or other cells that are not
responsive to the
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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 aganists 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 Fatten, P. A., et al., Curr. Opinion Biotechnal. 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
palypeptides of the invention may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments into a desired
polynucleotide
sequence of the invention molecule by homologous, or site-specific,
recombination.
In another embodiment, polynucleotides and corresponding polypeptides of the
inwention may be alterred by being subjected to random mutagenesis by error-
prone
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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 (1GF-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), fiOA, 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
3~H] thymidine. Both agonist and antagonist compounds may be identified by
this
procedure.
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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 of: (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
of: (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,
any combination or all of contained in the polypeptide sequences of the
invention.
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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, ar
alternatively consist of, any combination or all of the beta pleated sheet
regions in one
of the poiypeptide sequences of the invention.
Targeted Deliver
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 heterologaus 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., L',~NA 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 ki.nase, endonuclease, RNAse, alpha toxin, ricin,
abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed
antiviral protein, alpha-sarcir~ 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 poiypeptide 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 poiypeptides 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
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invention. These methods comprise contacting such an agent with a polypeptide
of the
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
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 1:%, 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.
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
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(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
S example, in Okano, Neurochem., 56:560 ( 1991 ); Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expressic>n, 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,
25 I :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-rnyb 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 vi vo 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 I5. bases of the open reading frame is flanked by
an EcoRl
site on the 5 end and a Hind:(II site on the 3 end. Next, the pair of
oligonucleotides is
heated at 90°C for one minute and then annealed in 2X ligation buffer
(20mM TRIS
HCl pH 7.5, IOmM MgCl2, 10MM 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
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invention. Such a vector would contain a sequence encoding the anti sense
nucleic
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.
IS 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 cclmplementarity to be able to hybridize with the
RNA,
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
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inhibiting translation of mRN.As as well. See generally, Wagner, R., Nature,
372:333-335 ( 1994). Thus, oligonucleotides complementary to either the S~ -
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 S' 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 hut
could be used in accordance with the invention. Whether designed to hybridize
to the
5' -, 3' - or coding region of rnRNA, anti sense 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 1? 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
NO: W088109810, published December IS, 1988) or the blood-brain barrier (see,
e.g., PGT Publication NO: W089110134, 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 oiigonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, ete.
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, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine,
S-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
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5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, I-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine, S-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-S-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5--methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid
(v),
S-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 embadiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
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 (Inoue et ai., Nucl. Acids
Res.,
15:6131-6148 (I987)), or a chimeric RNA-DNA analogue (Inoue 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 oiigonucleotides may be synthesized by the method of Stein et
al.
(Nucl. Acids Res., 16:3209 (:1988)), methylphosphonate oligonucleotides can be
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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., PCI' 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.
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 poiynucleotides of the invention in
vivo.
DNA constructs encoding the ribozyme may be introduced into the cell in the
same
manner as described above f<yr 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 li promoter, so that transfected cells will produce sufficient
quantities of
the ribozyme to destroy endogenous messages and inhibit translation. Since
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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 antagonistlagonist may also be employed to prevent hyper-vascular
diseases, and prevent the proliferation of epithelial lens cells after
extracapsular
cataract surgery. Preventi<m 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.
invention, and/or (b) a ribozyme directed to the polynucleotide of the present
invention.
Other Activities
The polypeptide of tlae 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
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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 f°ibroblast 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 memtrers 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.
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,
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pigmentation, size, and shape (e.g., casmetic 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 andlor 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
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.
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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
S 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 an isolated nucleic acid rnolecuie comprising a nucleotide
sequence which is at least 95~~o 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 I.
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 hybrieiizes does not hybridize under stringent
hybridization
conditions to a nucleic acid molecule having a nucleotide sequence consisting
of only
A residues or of only T residues.
Also preferred is a e:omposition of matter comprising a DNA molecule which
comprises a human cDNA clone identified by a cDNA Clone Identifier in Table I,
which DNA molecule is contained in the material deposited with the American
Type
Culture Collection and given the ATCC Deposit Number shown in Table l for said
cDNA Clone Identifier.
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Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least ~)5'lo 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 1, 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 50 contiguous nucleotides is included in the nucleotide sequence of
the
complete open reading frarrre sequence encoded by said human cDNA clone.
Also preferred is air 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 eDNA 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%n 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 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 ATC_'C' Deposit Number shown for said cDNA clone in
Table
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
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21~
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 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 ATC(~'. 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 ;~ 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 1, 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
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.
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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 <;omposition 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 eDNA
clone in Table I. The nucleic: acid molecules can comprise DNA molecules or
RNA
IS molecules.
Aiso preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about l0 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y wherein ~' 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 I.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a se<iuence of at feast about 30 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 a sequence of at least about 100 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 the complete amino acid sequence of SEQ ID
NO:Y_
Further preferred is an isolated polypeptide comprising an amino acid
S 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 Depasit Number shown for said eDNA 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 A'~fCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
IS at least 95% identical to a sequence of at least abaut 30 contiguous amino
acids in the
amino acid sequence of the secreted portion of the protein encoded by a human
eDNA
clone identified by a cDNA Clone Identifier in Table I 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 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table I.
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.
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
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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 eDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATC'C 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 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 ATC'C' 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 SI~Q 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 C'.lone 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.
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
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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
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
poiypeptide
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 I.
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
selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y
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wherein Y is any integer as defined in Table l; 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 I. 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 pharrx~aceutical 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, marine, 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 2, preferably excluded are one or more polynucleotides
comprising, or alternatively consisting of, a nucleotide sequence referenced
in the
fifth column of Table 2 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 2. 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 2. 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 2
Gene cDNA CloneNT Contig Public Accession Numbers
No. ID
ID SEQ
ID
NO:
X
6 HAP X44 16 779053 AA082324
18 HAGAU43 :?8 844223 AA902665, AI085890, AA843868,
AA890401
20 HE2DY25 30 844237 T94316, T83121, T93917, H10450,
H30532,
H30545, H49083, H49166, N62879,
N79670,
W74616, W94188, W94709, AA127307
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AA 194149, AA 19414b, AA235233,
AA235232,
AA421057, AA768716, AA876662,
AA903009,
AA903180, AA987614, N30489,
N54303, N63992,
N77786, AA642425, AA126824,
AA649251,
AA487432, AA487640, AA758655,
AA758915,
AA778S28, AA.889598, A1073358,
F09058,
_ T80942, T80943, T90382, T93873,
~ AA702576
21 HE2FE89 31 780314 817931, N75829, W67269, W67963,
AA086432,
AA086443, AA 192303, AA 192750,
.AA 196325,
AA 196322, AA253314, AA253484,
AA430209
27 HTOEB55 37 665897 879999, N68522, W00717, W00718,
W80655,
W92741, AA040274, AA040275,
AA040616,
__ AA040617
29 HOGAB51 39 825278 N71250, N71473, W01596
34 HKAFJ47 44 845521 N36923, AA613805, AA832304,
AA879031,
_ AI094705 D82713, AA 170851
4l HWAAY60 51 745917 T62509, T62655, T70365, 831637,
833109,
833206, 835675, 835786, N41705,
N49202,
N54544, N54838, N76247, W4657i,
W46662,
W78930, W79510, W85698, W92370,
W92371,
AA009526, AA009874, AA026397,
AA026453,
AA034488, AA 121798, AA243713,
AA255548,
AA256662, AA461050
49 HMUBY57 59 844800 825219, 830858, 830904, 877058,
AA102309,
AA158754, AA282240, AA282360,
.AA526392,
H89147, AA936465, AA063608,
AA 158755,
AA437393, AA670254, AA434032
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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
~xamnle 1~ Isolation of abSelecte~i,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 Corresponding-Deposited
Plasmid
Lambda Zap pBluescript (pBS)
Uni-Zap XR pBluescript (pBS)
Zap Express pBK
lafmid BA plafmid BA
pSport I pSport 1
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), p131uescript (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 (Aping-Mees, M. A. et al., Strategies 5:58-61 (1992))
are
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commercially available frarn Stratagene Cloning Systems, Inc., 1 1011 N.
Torrey
Pines Road, La Jolla, CA, 92,037. 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 f~rc>rn 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 Sacl and "K" is
for
Kpnl 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 f I 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. U. Box 6009, Gaithersburg, MD 20897. All Sport
vectors
contain an ampicillin resistance gene and may be transformed into E. coli
strain
DH IOB, also available from Life Technologies. (See, for instance, Gruber, C:.
E., et
al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University,
NY) contains an ampicillin resistance gene and can be transformed into E. coli
strain
XL-I Blue. Vector pCR~2.1, which is available from lnvitrogen, 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 polynueleotide of the present
invention
does not comprise the phage vector sequences identified for the particular
clone in
Table l, 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 I . 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
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may include plasmids for more or less than 50 cDNA clones, up to about 500
eDNA
clones.
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 piasmid 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 3'P-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.
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 Harbar 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 ID 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 carried out under routine conditions, for instance, in 25 ul
of reaction
mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture
is
I.5-5 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 I 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
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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.
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 ~' "RACE" protocols which are
well
known in the art. For instance, a method similar to 5' RACE is available for
generating the missing S' 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 oligonucieotide 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 specif c 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.
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Example 2- Isolation of Genomic Clones (:orresponding to a Polvnucleotide
A human genomic fl library (Genomic Systems, lnc.) is screened by PCR
using primers selected for the cDNA sequence corresponding to SEQ 1D NO:X.,
according to the method described in Example 1. (See also, Sambrook.)
Example 3: Tissue Distribution of Polype~tide
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 P3~ using the rediprimeT"' DNA labeling
system (Amersham Life Science), according to manufacturer's instructions.
After
labeling, the probe is purified using CHROMA SPIN-IOOT"" 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 (1M) (Clontech) are examined with the labeled
probe using ExpressHybT"' hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Follawing hybridization and washing,
the
blots are mounted and exposed to film at -70 degree C overnight, and the films
developed according to standard procedures.
F,~xample 4: Chromosomal Manning of t~ he 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 palymerase 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
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gels. Chromosome mapping is determined by the presence of an approximately 100
by PCR fragment in the particular somatic cell hybrid.
~xa~nle S: Bac~g~iat ~,xpression of a Pc~ly~e~~ide
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 Exarnple 1, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHl and XbaI, at the 5' end of the primers in order to clone the amplified
product
into the expression vector. For example, BarnHl 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 (Ampr), a
bacterial origin of replication (ori), an IPTG-regulatable promoter/operator
(PIO), 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 Iigated 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 lacl repressor and also confers kanamycin resistance (Kan~.
Transformants are identified by their ability to grow on LB plates and
ampicillinlkanamycin resistant colonies are selected. Plasmid DNA is isolated
and
confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) 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.6°°) of between
0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of I
mM. IPTG induces by inactivating the lacl repressor, clearing the P/O leading
to
increased gene expression.
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Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 mins at t~000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCI by stirring 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-N~l'A") affinity resin column
(available from
QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with
high affinity and can be purified in a simple one-step procedure (for details
see: The
QlAexpressionist (1995) QIAGEN, Inc., se~pra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCI, 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 5() mM Na-acetate, pH 6 buffer plus 200 mM NaCI.
Alternatively, the protein can be successfully refalded while immobilized on
the Ni-
NTA column. The recommended conditions are as follows: renature using a linear
6M-1M urea gradient in S00 mM NaCI, 20% glycerol, 2U 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 prateins are eluted by the addition
of 250
mM immidazole. lmmidazole 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 TS phage promoter sequence, 4) two lac operator sequences,
5) a
Shine-Delgarno sequence, and 6) the lactose operon repressor gene (laclq). The
origin of replication (oriC) is derived from pUC 19 (LT1, Gaithersburg, MD).
The
promoter sequence and operator sequences are made synthetically.
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2;0
DNA can be inserted into the pHEa by restricting the vector with Ndel and
Xbal, BamHl, Xhol, or Asp71$, 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 l,
using PCR
primers having restriction sites for NdeI (5' primer) and Xbal, 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.
The engineered vector could easily be substituted in the above protocol to
express protein in a bacterial system.
Example 6~ Purification of a P~rueutide from an Inclusion Bodv
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, al( of the following steps are conducted at 4-10 degree
C.
Upon completion of the production phase of the E. toll fermentation, the cell
culture is cooled to 4-10 degree C and the cells harvested by continuous
centrifugation at 15,000 rprn (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, SO 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.
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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 stirring. The refolded diluted protein solutian is kept at 4
degree C
without mixing for 12 houa-s 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 cation exchange resin (e.g., Poros HS-50,
Perceptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1C)00 mM, and 1500 mM NaCI in the same buffer, in a
stepwise manner. T'he 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 poaled and mixed with 4
IS 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 frorrr 0.2 M NaCI, 50 mM sodium
acetate, pH 6.0 to 1.0 M NaC.'1, 50 mM sodium acetate, pH 6.5. Fractions are.
collected under constant A~ monitoring of the effluent. Fractions containing
the
polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypehtide should exhibit greater than 95% purity after the
above refolding and purification steps. Na 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.
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232.
Examine 7: Cloning and Expression of a Pol~~~nt~de in a Baculoviru
Expression S, sv tem
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 Autographa californica nuclear
polyhedrosis virus (AcMNPV ) followed by convenient restriction sites such as
BamHl, 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 Clanked on both sides by viral
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
IS 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 eDNA 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 I % agarase gel using a
commercially available kit ("Geneclean," B10 101 lnc., La Jolla, Ca.). The
fragment
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~3Z
then is digested with appropriate restriction enzymes and again purified on a
I
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" B10 I(:)1 lnc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. toll HB 1 (l l 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.
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., F'roc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
One ug
of BaculoGoldT"' virus DNA and 5 ug of the plasmid are mixed in a sterile well
of a
microtiter plate containing 50 ul of serum-free (race's medium (Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 ul Lipofectin plus 90 ul Grace's
medium are
added, mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture is added drop-wise to Sf9 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 5 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
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2W
culture and baculovirology distributed by Life Technologies lnc.,
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 microcem:rifuge tube containing 200 ul of Grace's medium and
the
suspension containing the recombinant baculovirus is used to infect Sf9 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, Sf9 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 11 medium minus methionine. and
cysteine (available from Life Technologies Inc., Rockville, MD). After 42
hours, 5
uCi of 35S-methionine and ~ uCi 35S-cysteine (available from Amersham) are
added.
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 ofd Pol,~nl~de 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) frorrr Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
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23
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), pBCl2Ml (ATCC 67109),
pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used
include, human Hela, 293, 119 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1,
Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
Alternatively, the polypeptide can be expressed in stable cell tines
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
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 (A'TCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (I_TR) 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 BamHl, Xbal and Asp718, facilitate
the
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236
cloning of the gene of interest. 'The vectors also contain the 3' intron, the
polyadenylation and termirration signal of the rat preproinsulin gene, and the
mouse
DHFR gene under control of the SV40 early promoter.
Specifically, the plasrnid 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 fram a I% agarose
gel.
A polynucleotide of the present invention is amplified according to the
protocol outlined in Example 1. If the naturally occurring 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
vectar can be
modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
The amplified fragment is isolated fram a 1 % agarose gel using a
commercially available kit ("'C~eneclean," BIO 101 lnc., 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
are then ligated with T4 DNA ligase. E. coli HB 101 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 beg 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 t7asks using different concentrations of
methotrexate (50
nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
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237
concentrations of methotrexate are then transferred to new 6-well plates
containing
even higher concentrations of methotrexate ( I 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-PA(:~E and Western blot or by reversed phase HPLC
analysis.
Example 9: Protein F~s~~~
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
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 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified,
using primers that span the 5' 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. 209b46) 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 I , is ligated into this BamI-II
site. Note
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2 ~8
that the polynucleotide is cloned without a stop codon, otherwise a fusion
protein will
not be produced.
If the naturally occurring 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 1gG Fc region:
GGGATCCGGAGCCCAAA'I'CTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTC'.AGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCA'TGATCTCCCGCiACTCCTGAGGTCACATGCGTGGT
GGTGGACGTAAGCCAC:."GAAGACCCI~GAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGT'GTGGTCAGCGTCCr'I'CACCGTCCTGCACCA(lGACT
GGCTGAATGGCAAGGAGTACAAGTGC'AAGGTCTCCAACAAAGCCC'TCCCA
ACCCCCATCGAGAAAAC~CATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCC'fGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGACC"TGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT
GGAGTGGGAGAGCAA'TGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTfC'ITCCTCTACAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCCTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAAC:CACTACACGCAG AAGAGCCTCTCCCTGTC'rCCGG
GTAAATGAGTGCGACC;<C~CCGCGACTCTAGAGGAT (SEQ ID NO:1)
xample 10' Production of an Antibodp_from a Polylre tn ide
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
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239
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonai 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); I-lammerling 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°lo 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 myelvma 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.
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-
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2=1( 1
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
Flab'}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; Morrison e.t 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 }. )
a 11' Pr f ' h-Thr t r a 'n
Assavs
The following protocol produces a supernatant containing a polypeptide to be
tested. This supernatant can then be used in the Screening Assays described in
Examples 13-20.
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/rnl. Add 200 ul 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
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2-i 1
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 .Sml
DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine
S ( 12-604F Biowhittaker))/10% heat inactivated FBS( 14-503F Biowhittaker)11 x
Penstrep(I?-602E Biowhittaker). Let the cells grow overnight.
The next day, mix t.o,gether in a sterile solution basin: 300 ul Lipofectamine
(18324-012 Gibco/BRL) and Sml Optimem 1 (31985070 Gibco/BRL)/96-well plate.
With a small volume mufti-channel pipetter, aliquot approximately tug of an
expression vector containing a polynucieotide 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-~5
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 a12-channel pipetter with tips on every
other
channel, adds the 200u1 of DNA/Lipofectamine/Optimem 1 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 1 x penstrep, ar CHO-5 media ( I 16_6 mg/L of CaCl2 (anhyd); 0.00130
mg/L CuS04-SHzO; 0.050 mglL of Fe(N03)3-9Hz0; 0.417 mg/L of FeS04-7Hz0;
311.80 mg/L of Kcl; 28.64 mg/L of MgCl2; 48.84 mg/L of MgS04; 6995.50 mg/L of
NaCI; 2400.0 mg/L of NaHCO3; 62.50 mg/L of NaH2P04 H20; 71.02 mg/L of
Na2HP04; .4320 mg/L of ZnS04 7Hz0; .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
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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; lOG
mg/L of
Pluronic F-68; O.OlO 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 mglml of L-Arginine-HCL; 7.50
mg/ml
of L-Asparagine-H20; 6.65 mg/ml of I_-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-H20; 31.29 mg/ml of ~,-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.7.5 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-
HZO; 106.97 mg/ml of L-Isoleucine; 1 I 1.45 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 mglml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L--Cryrosine-2Na-2H20; 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/I_, of Folic Acid; 15.60 mg/L of i-Inositol; 3.02
mg/L of
Niacinamide; 3.00 mg/L of I'yridoxal HC-'L; 0.031 mglL 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,.~; 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 rng/L of Sodium Selenite; 20uM of
Ethanolamine; 0.122 mg/h c>f 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 ul for
endotoxin assay in l5ml 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
B adds l .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 2ml deep well. The
supernatants
from each well can then bc; used in the assays described in Examples 13-20.
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2.~3
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.
lFxatn~e 12: CQ,pstructior~ of T ,~S 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 ("ISRE"), located in the promoter of many genes.
The
binding of a protein to these elements alter the expression of the associated
gene.
GAS and ISRE 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 Bells 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.
The Jaks are activated by a wide range of receptors summarized in the Table
below. (Adapted from review by Schidler and Darnel!, 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-3,1L-4, IL-6, IL-7, IL-9,
IL-11, IL-
12, IL-15, Epo, PRL, GH, Ci-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and
(b)
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Class 2 includes 1FN-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 hind 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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2-l~
JAKs STATS GAS(elements) or
I SRE
Ligand yk2 Jakl Jak2 Jak3
IFN family
IFN-a/B + + - - 1,2,3 ISRE
IFN-g + + - 1 GAS (IRF1>Lys6>IFP)
Il-10 + ? ? - 1,3
gp130 family
IL-6 {Pleiotrophic)+ + + ? 1,3 GAS (IRF1>Lys6>IFP)
II-11(Pleiotrophic)? + ? '~ 1,3
OnM(Pleiotrophic)? + + '? 1,3
LIF(Pleiotrophic)? + + ? 1,3
CNTF(Pleiotrophic)-/+ + + '? 1,3
G-CSF(Pleiotrophic)? + ? ? 1,3
IL-12(Pleiotrophic)+ - + + 1,3
g-C family
IL-2 (lymphocytes)- + - + 1,3,5 GAS
IL-4 (lymph/myeloid)- + - + 6 GAS (IRF1 = IFP Ly6)(IgH)
IL-7 (lymphocytes)- + - + 5 GAS
IL-9 (lymphocytes)- + - + S GAS
IL-13 {lymphocyte)- + ? ? 6 GAS
IL-IS ? + ? + 5 GAS
~;p140 family
IL-3 (myeloid) - - + - 5 GAS (IRF1>IFPLy6)
IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid)- - + - 5 GAS
Growth hormone
famil~r
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRFI=IFPLy6)
Receptor Txrosine
Kinases
EGF ? + + - 1,3 GAS (IRF1)
PDGF ? + + - 1,3
CSF-I ? + + - 1,3 GAS (not IRFI)
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To construct a synthetic GAS containing promoter element, which is used in
the Biological Assays described in Examples 13-~ 14, a PCR based strategy is
employed to generate a GAS-SV40 promoter sequence. The 5' primer contains four
tandem copies of the CAS binding site found in the 1RF1 promoter and
previously
demonstrated to bind STATs upon induction with a range of cytokines (Rothman
et
al_, Immunity 1:457-468 ( 1994).), although other GAS or ISRE elements can be
used
instead. The 5' primer also contains l8bp of sequence complementary to the.
SV40
early promoter sequence and is flanked with an XhoI site. The sequence of the
5'
primer is:
5':GCGCCTCGAGATTTC(_'CCGAAATCTAGATTTCCCCGAAATGATTTCCCC
GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID N0:3)
The downstream primer is complementary to the SV40 promoter and is
flanked with a Hind III site: 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
(SEQ ID N0:4)
IS PCR amplification is performed using the SV40 promoter template present in
the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.)
Sequencing
with forward and reverse primers confirms that the insert contains the
following
sequence:
5':CTCGAGATTTCCCCGAAATCI'AGATTTCCCCGAAATGATTTCCCCGAAA
TGATTTCCCCGAAATATCTGCCATCT'C.'AATTAGTCAGCAACCATAGTCCCG
CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT
CCGCCCCATGGCTGACTAAT'ITT>ZTTATTTATGCAGAGGCCGAGGCCGCC
TCGGCCTCTGAGCTATT<JCAGAAGTAGTGAGGAGGCTTI'TTTGGAGGCCT
AGGCITITGCAAAAAGCTT:3' (SEQ ID NO:S)
With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2
reporter construct is next engineered. Here, the reporter molecule is a
secreted
alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can
be
instead of SEAP, in this or in any of the other Examples. Well known reporter
molecules that can be used instead of SEAP include chloramphenicol
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2-t~
acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase,
green
fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is
subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIIl
and
Xhol, effectively replacing the SV40 promoter with the amplified GAS:SV40
promoter element, to create the GAS-SEAP vector. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred for
mammalian
expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-
SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using
SaII and Notl, and inserted into a backbone vector containing the neomycin
resistance
gene, such as pGFP-I (Clontech), using these restriction sites in the multiple
cloning
site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into
mammalian cells, this vector can then be used as a reporter molecule for GAS
binding
as described in Examples 13-14.
Other constructs can be made using the above description and replacing GAS
with a different promoter sequence. For example, construction of reporter
molecules
containing NF'K-B and EGR promoter sequences are described in Examples 15 and
16. However, many other promoters can be substituted using the protocols
described
in these Examples. For instance, SRE, 1L-2, NEAT, or Osteocalcin promoters can
be
substituted, alone or in combination (e.g., GASINF-KB/EGR, GAS/NF-KB, Il-
2/NFAT, or NF-KB/GAS).. Similarly, other cell lines can be used to test
reporter
construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-
cell),
Saos-2 (osteoblast), HUVA(:: (aortic), or Cardiomyocyte.
Exa ple 13: High-Througtit:ut Screening Assay for T-cell Activit~r.
The following protocol is used to assess T-cell activity by identifying
factors,
and determining whether supernate containing a polypeptide of the invention
proliferates and/or differentiates T-cells. T-cell activity is assessed using
the
GAS/SEAP/Neo construct produced in Example 12. Thus, factors that increase
SEAP
activity indicate the ability to activate the Jaks-STATS signal transduction
pathway.
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2-18
The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152),
although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC
Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are I;ymphoblastic CD4+ Th 1 helper cells. In order to generate
stable cell lines, approximately 2 million Jurkat cells are transfected with
the GAS-
SEAP/neo vector using DMR1E-C (Life Technologies)(transfection procedure
described below). The transfected cells are seeded to a density of
approximately
20,000 cells per well and transfectants resistant to 1 mg/mi genticin
selected.
Resistant colonies are expanded and then tested for their response to
increasing
concentrations of interferon gamma. The dose response of a selected clone is
demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 welts
containing 200 ul of cells. Thus, it is either scaled up, or performed in
multiple to
generate sufficient cells for multiple 96 well plates. Jurkat cells are
maintained in
RPMI + 10% serum with 1 %QPen-Strep. Combine 2.5 mls of OP'FI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a'r25 flask. Add 2.5 ml OPTI~-MEM
containing 50 ul of DMRIIC and incubate at room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required
number of cells ( 10' per transfection), and resuspend in OPTI-MEM to a final
concentration of 10' cells/ml. Then add lml of I x 10' cells in OPTI-MEM to
T25
flask and incubate at 37 degrees C for 6 hrs. After the incubation, add 10 ml
of RPMI
+ 15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPM1 + 10%
serum, I mg/ml Genticin, and 1% Pen-Strep. These cells are treated with
supernatants containing polypeptides of the invention andlor induced
polypeptides of
the invention as produced by the protocol described in Example I 1.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh RPM1 + 10% serum to a density of 500,000 cells per ml..
The
exact number of cells required will depend on the number of supernatants being
screened. For one 96 well plate, approximately 10 million cells (for 10
plates, 100
million cells) are required.
CA 02364209 2001-09-24
DEMANDES OU BREVETS VOLUMlNEUX
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CECI EST LE TOME ~ DE Z
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