Note: Descriptions are shown in the official language in which they were submitted.
WO 00/55177 PCT/US00/06058
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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
reticulum (ER). The ER separates the membrane-bounded proteins from all other
types of proteins. Once localized to the ER, both groups of proteins can be
further
directed to another organelle called the Golgi apparatus. Here, the Golgi
distributes
the proteins to vesicles, including secretory vesicles, the cell membrane,
lysosomes,
and the other organelles.
Proteins targeted to the ER by a signal sequence can be released into the
extracellular space as a secreted protein. For example, vesicles containing
secreted
proteins can fuse with the cell membrane and release their contents into the
extracellular space - a process called exocytosis. Exocytosis can occur
constitutively
or after receipt of a triggering signal. In the latter case, the proteins are
stored in
secretory vesicles (or secretory granules) until exocytosis is triggered.
Similarly,
proteins residing on the cell membrane can also be secreted into the
extracellular
space by proteolytic cleavage of a "linker" holding the protein to the
membrane.
Despite the great progress made in recent years, only a small number of genes
encoding human secreted proteins have been identified. These secreted proteins
include the commercially valuable human insulin, interferon, Factor VIII,
human
growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of
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the pervasive role of secreted proteins in human physiology, a need exists for
identifying and characterizing novel human secreted proteins and the genes
that
encode them. This knowledge will allow one to detect, to treat, and to prevent
medical disorders by using secreted proteins or the genes that encode them.
Summary of the Invention
The present invention relates to novel polynucleotides and the encoded
polypeptides. Moreover, the present invention relates to vectors, host cells,
antibodies, and recombinant and synthetic methods for producing the
polypeptides
and polynucleotides. Also provided are diagnostic methods for detecting
disorders
and conditions related to the polypeptides and polynucleotides, and
therapeutic
methods for treating such disorders and conditions. The invention further
relates to
screening methods for identifying binding partners of the polypeptides.
Detailed Description
Deiiinitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide. The
term "isolated" does not refer to genomic or cDNA libraries, whole cell total
or
mRNA preparations, genomic DNA preparations (including those separated by
electrophoresis and transferred onto blots), sheared whole cell genomic DNA
preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
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3
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 extraeellular space, the secreted protein can undergo extracellular
processing
to produce a "mature" protein. Release into the extracellular space can occur
by many
mechanisms, including exocytosis and proteolytic cleavage.
In specific embodiments, the polynucleotides of the invention are at least 15,
at least 30, at least 50, at least 100, at least 125, at least 500, or at
least 1000
continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb,
50 kb, 15
kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further
embodiment,
polynucleotides of the invention comprise a portion of the coding sequences,
as
disclosed herein, but do not comprise all or a portion of any intron. In
another
embodiment, the polynucleotides comprising coding sequences do not contain
coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest
in the
genome). In other embodiments, the polynucleotides of the invention do not
contain
the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,
4, 3, 2, or
1 genomic flanking gene(s).
As used herein, a "polynucleotide" refers to a molecule having a nucleic acid
sequence contained in SEQ ID NO:X or the cDNA contained within the clone
deposited with the ATCC. For example, the polynucleotide can contain the
nucleotide sequence of the full length cDNA sequence, including the 5' and 3'
untranslated sequences, the coding region, with or without the signal
sequence, the
secreted protein coding region, as well as fragments, epitopes, domains, and
variants
of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers
to a
molecule having the translated amino acid sequence generated from the
polynucleatide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:X
was often generated by overlapping sequences contained in multiple clones
(contig
analysis). A representative clone containing all or most of the sequence for
SEQ ID
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
shown in Table l, each clone is identified by a cDNA Clone ID (Identifier) and
the
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4
ATCC Deposit Number. The ATCC is located at 10801 University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of the deposit
of
microorganisms for purposes of patent procedure.
A "polynucleotide" of the present invention also includes those
polynucleotides capable of hybridizing. under stringent hybridization
conditions, to
sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within
the clone deposited with the ATCC. "Stringent hybridization conditions" refers
to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x
SSC
(750 mM NaCl, 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 NaH~PO~; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE,
0.1 % SDS. In addition, to achieve even lower stringency, washes performed
following stringent hybridization can be done at higher salt concentrations
(e.g. 5X
SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
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Of course, a polynucleotide which hybridizes only to polyA+ sequences (such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
complementary stretch of T (or U) residues, would not be included in the
definition of
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically
any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA or modified RNA or DNA. For example, polynucleotides can be composed of
single- and double-stranded DNA, DNA that is a mixture of single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or a mixture
of single-
and double-stranded regions. In addition, the polynucleotide can be composed
of
triple-stranded regions comprising RNA or DNA or both RNA and DNA. A
polynucleotide may also contain one or more modified bases or DNA or RNA
backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications can be made to DNA and RNA; thus, "polynucleotide" embraces
chemically, enzymatically, or metabolically modified forms.
The polypeptide of the present invention can be composed of amino acids
joined to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and may contain amino acids other than the 20 gene-encoded amino
acids.
The polypeptides may be modified by either natural processes, such as
posttranslational processing, or by chemical modification techniques which are
well
known in the art. Such modifications are well described in basic texts and in
more
detailed monographs, as well as in a voluminous research literature.
Modifications
can occur anywhere in a polypeptide, including the peptide backbone, the amino
acid
side-chains and the amino or carboxyl termini. It will be appreciated that the
same
type of modification may be present in the same or varying degrees at several
sites in
a given polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched , for example, as a result of
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ubiquitination, and they may be cyclic, with or without branching. Cyclic,
branched,
and branched cyclic polypeptides may result from posttranslation natural
processes or
may be made by synthetic methods. Modifications include acetylation,
acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation, formation
of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation,
gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, pegylation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES,
2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York ( 1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 ( 1983); Seifter et al.,
Meth
Enzymol 182:626-646 ( 1990); Rattan et al., Ann NY Acad Sci 663:48-62 (
1992).)
"SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ ID NO:Y"
refers to a polypeptide sequence, both sequences identified by an integer
specified in
Table 1.
"A polypeptide having biological activity" refers to polypeptides exhibiting
activity similar, but not necessarily identical to, an activity of a
polypeptide of the
present invention, including mature forms, as measured in a particular
biological
assay, with or without dose dependency. In the case where dose dependency does
exist, it need not be identical to that of the polypeptide, but rather
substantially similar
to the dose-dependence in a given activity as compared to the polypeptide of
the
present invention (i.e., the candidate polypeptide will exhibit greater
activity or not
more than about 25-fold less and, preferably, not more than about tenfold less
activity, and most preferably, not more than about three-fold less activity
relative to
the polypeptide of the present invention.)
Many proteins (and translated DNA sequences) contain regions where the
amino acid composition is highly biased toward a small subset of the available
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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
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 BLASTX program masked amino acids in that region due
to
the detection of a low 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 ), and/or the deposited clone is
(are) the
definitive embodiments) of the present invention, and should not be construed
as
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limiting the present invention to the partial sequence shown in an alignment,
unless
specifically noted otherwise herein.
Polynucleotides and Poly~eptides 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: Soares fetal liver spleen INFLS and to a lesser extent in Soares
placenta
Nb2HP, Hepatocellular Tumor; re-excision, Human Placenta, Human Osteoclastoma,
Soares_multiple_sclerosis_2NbHMSP, Soares_placenta_8to9weeks 2NbHP8to9W,
Human Amygdala, Soares_fetal lung_NbHLI9W, Adrenal Gland,normal, Soares
adult brain N2b4HB55Y, Hepatocellular Tumor, Human Stomach;re-excision,
Salivary Gland, Lib 2, Human Activated T-Cells, Pancreas Islet Cell Tumor,
Gessler
Wilms tumor, Human adult lung 3' directed MboI cDNA, Fetal Heart,
NCI CGAP_AA1, NCI_CGAP Br2, NCI CGAP_LuS, NCI CGAP_KidS, Nine
Week Old Early Stage Human.
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 1191 of SEQ ID
NO:11, b
is an integer of 15 to 1205, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:1 l, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
It has been discovered that this gene is expressed primarily in the following
tissues: Synovial Fibroblasts (Ill/TNF), subt and to a lesser extent in
Soares multiple sclerosis_2NbHMSP, 12 Week Old Early Stage Human, II,
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9
Soares_pregnant uterus NbHPU, Human Amygdala, Human Fetal Brain, normalized
50021F, normalized A5002F, Human White Fat. Human Frontal Cortex,
Schizophrenia, Human Osteoclastoma, re-excision, Ulcerative Colitis,
Soares NhHMPu S l, Human Thymus Stromal Cells, Bone Marrow Stromal Cell,
untreated, H. Frontal cortex,epileptic:re-excision, 12 Week Old Early Stage
Human,
Human Eosinophils, Human Bone Marrow, treated, Nine Week Old Early Stage
Human.
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 1830 of SEQ ID
N0:12, b
is an integer of 15 to 1844, 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
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
It has been discovered that this gene is expressed primarily in Human
Amygdala.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
62 as residues: Asn-34 to Gly-39.
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 1 to 1256 of SEQ ID
N0:13, b
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is an integer of 15 to 1270, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:13, and where b is greater than or
equal to a
+ 14.
5 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.
bbs1129340 (all information available through the recited accession number is
10 incorporated herein by reference) which is described therein as "X-linked
retinopathy
protein." A partial alignment demonstrating the observed homology is shown
immediately below.
>bbs~129340 X-linked retinopathy protein (C-terminal, clone XEH.8c)
1S [human,
Peptide Partial, 100 aa] [Homo Sapiens] >pir~A45010~A46010 X-
linked
retinopathy protein (C-terminal, clone XEH.8c) - human
(fragment)
Length = 100
Minus Strand HSPs:
Score = 168 (59.1 bits), Expect = 2.4e-11, P = 2.4e-11
2S Identities = 39/71 (54%), Positives = 47/71 (66%), Frame = -2
Query: 1236 FFFFFETESHPVAQAGVQWHDLGSLQSLPPRFKRFPNLSLPSS*QYKCAHHNRVIFVFLV
1057
FFFFFETES VA+AGVQW DLGSL+S PP P + + HH ++IFVFLV
3O Sbjct: 2 FFFFFETESCSVAEAGVQWCDLGSLKSPPPGSSDSPASASRVAGITGMHHHTQLIFVFLV
61
Query: 1056 ETGFSHVGQAD 1024
3S ETG SH+ +D
Sbjct: 62 ETG-SHMQLSD 71
The segment of bbs1129340 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 109. Based on the structural similarity these homologous
40 polypeptides are expected to share at least some biological activities.
Such activities
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 110 which correspond to the Query
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sequence in the alignment shown above (gaps introduced in the sequence by the
computer are. of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: KMH2, L428. When tested against both Jurkat T-cells and U937 Myeloid
cell
lines, supernatants removed from cells containing this gene activated the GAS
assay.
Thus, it is likely that this gene activates both T-cells and myeloid cells.
and to a lesser
extent other immune cells, through the Jak-STAT signal transduction pathway.
The
gamma activating sequence (GAS) is a promoter element found upstream of many
genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a
large, signal transduction pathway involved in the differentiation and
proliferation of
cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding
of the
GAS element, can be used to indicate proteins involved in the proliferation
and
differentiation of cells.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
63 as residues: Leu-33 to Asp-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 N0:14 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
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 1223 of SEQ ID
N0:14, b
is an integer of 15 to 1237, 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 GENE NO: 5
It has been discovered that this gene is expressed primarily in L428 cells,
which are derived from Hodgkin's lymphoma cells.
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?
related to SEQ ID N0:15 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 2168 of SEQ ID
N0:15, b
is an integer of 15 to 2182, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:15, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
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.
gi11872200 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "MYB PROTO-
ONCOGENE PROTEIN." A partial alignment demonstrating the observed homology
is shown immediately below.
>gi~1872200 alternatively spliced product using exon 13A [Homo Sapiens]
>sp~P78525~P78525 MYB PROTO-ONCOGENE PROTEIN (C-MYB).
Length = 666
Plus Strand HSPs:
Score = 265 (93.3 bits), Expect = 1.1e-19, P = 1.1e-19
Identities = 61195 (64%), Positives = 64/95 (67%), Frame = +3
Query: 843 TESCSVAQA--GVQWHDLSSLQAPPPGFMPFSCLSLPSSWDYRLPLPYLANFFWLVEMG
1016
T++ VA A GVQWHD SLQ PPGF FSCLSLP SWDYR P P ANF LVE G
Sbjct: 557 TQTSPVADAPTGVQWHDFGSLQPLPPGFKRFSCLSLPRSWDYRHPPPRPANF-EFLVETG
615
Query: 1017 FHHISQDGLNLLTS*SARL-ASQSAGITGVSHLARP 1121
F H+ Q GL LLTS ASQSA ITGVSH ARP
Sbjct: 616 FLHVGQAGLELLTSGDLPASASQSARITGVSHRARP 651
The segment of gi11872200 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 111. Based on the structural similarity these homologous
polypeptides are expected to share at least some biological activities. Such
activities
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13
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 112 which correspond to the Query
sequence in the alignment shown above (gaps introduced in the sequence by the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Human Adult Pulmonary.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:16 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 1571 of SEQ ID
N0:16, b
is an integer of 15 to 1585, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO: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 the following
tissues: Soares fetal liver spleen 1NFLS and to a lesser extent in Soares
placenta
Nb2HP, Human Endometrial Tumor, Keratinocyte, Primary Dendritic Cells, lib 1,
Stratagene placenta (#937225), Stratagene NT2 neuronal precursor 937230,
Endothelial-induced, Activated T-cell( 12h)/Thiouridine-re-excision, Amniotic
Cells -
TNF induced, Breast Lymph node cDNA library, Amniotic Cells - Primary Culture,
H. Lymph node breast Cancer, Macrophage-oxLDL, Stratagene NT2 neuronal
precursor 937230, Soares breast 2NbHBst, Macrophage (GM-CSF treated),
Macrophage-oxLDL; re-excision, Human Placenta, Soares melanocyte 2NbHM,
human tonsils, Endothelial cells-control, Monocyte activated, Human Pancreatic
Langerhans, Normal trachea, Tongue Normal, Prostate, A 1-CELL LINE, L 1 Cell
line,
WO 00/55177 cA 02361277 2001-08-28 PCT/US00/06058
14
Stratagene neuroepithelium (#937231 ). Human Umbilical Vein Endothelial Cells,
fract. A, Aorta endothelial cells + TNF-a. Human adult small intestine.re-
excision,
Hepatocellular Tumor, pBMC stimulated w/ poly I/C, NTERA2 + retinoic acid, 14
days, Human Stomach;re-excision, Healing groin wound, 6.5 hours post incision,
NCI CLAP Co3, NCI_CGAP GC2. NCI CGAP_Prl, Human Umbilical Vein;
Reexcision, Prostate BPH, Stratagene colon (#937204), Stratagene NT2 neuronal
precursor 937230. Human Thymus. Human Fetal Dura Mater, Human Uterine
Cancer, Merkel Cells, Human Hypothalmus,Schizophrenia, Human Pancreas Tumor;
Reexcision, Liver, Hepatoma, Soares adult brain N2b5HB55Y, Epithelial-TNFa and
INF induced, Human Testes Tumor, re-excision, Stratagene colon (#937204),
Human
Whole Six Week Old Embryo, Stratagene liver (#937224), PC3 Prostate cell line,
Soares total fetus Nb2HF8 9w, Fetal Heart, Resting T-Cell Library,II, Colon
Carcinoma, NCI CGAP_GCBI, Breast Lymph node cDNA library, H Macrophage
(GM-CSF treated), re-excision, Dendritic cells, pooled, Primary Dendritic
cells,frac 2,
Human Fetal Lung III, Human Placenta, Bone marrow, Human Fetal Heart, Human
Adult Pulmonary;re-excision, Human Bone Marrow, treated, Human 8 Week Whole
Embryo, Soares_fetal heart NbHH 19W.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:17 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 1874 of SEQ ID
N0:17, b
is an integer of 15 to 1888, 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
WO 00/55177 CA 02361277 2001-08-28 PCTJUS00/06058
example, the sequence accessible through the following database accession no.
gi1288145 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "put. ORF
[Homo
Sapiens]." A partial alignment demonstrating the observed homology is shown
5 immediately below.
>gi~288145 put. ORF [Homo Sapiens] >pir~I38022~I38022 hypothetical protein
human >sp~Q29976~Q29976 MAHLAVU HEPATOCELLULAR CARCINOMA HHC(M)
1~ DNA.
Length = 196
Minus Strand HSPs:
15 Score = 267 (94.0 bits), Expect = 4.5e-22, P = 4.5e-22
Identities = 55/80 (68%), Positives = 60/80 (75%), Frame = -2
Query: 245 HLRSGVQDQLGQHG*SPSLLKVQKLAWHGGMHL*FQLLGRLRQENCLSPGGEGCSEPRSH
66
ZO HLRSGVQD GQHG PSLLK+Q+LA HGG L QLL RLRQEN L+ GG GCSEP+SH
Sbjct: 3 HLRSGVQDYPGQHGKIPSLLKIQELAGHGGRCLQSQLLRRLRQENHLNSGGRGCSEPKSH
62
Query: 65 RCTPAWATE*DSVTK*NINK 6
ZS C PAW TE DSV+K N K
Sbjct: 63 LCIPAWVTEGDSVSKQNKTK 82
The segment of gi1288145 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 113. Preferred polypeptides of the invention comprise
30 polypeptides having the amino acid sequences) set out as SEQ ID NO. 114
which
correspond to the Query sequence in the alignment shown above (gaps introduced
in
the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in Human Adult
Pulmonary;re-excision.
35 Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:18 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
40 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 1366 of SEQ ID
N0:18, b
WO 00/55177 PCT/US00/06058
CA 02361277 2001-08-28
16
is an integer of 15 to 1380, 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
It has been discovered that this gene is expressed primarily in the following
tissues: Amniotic Cells - Primary Culture and to a lesser extent in breast
lymph node
CDNA library, Smooth muscle, control; re-excision, Myoloid Progenitor Cell
Line,
Macrophage-oxLDL, PERM TF274, Hemangiopericytoma, NCI CGAP_PrS,
NCI CLAP Pr22, Human Testes, Reexcision, Soaves placenta Nb2HP.
When tested against fibroblast cell lines, supernatants removed from cells
containing this gene activated the EGR1 assay. Thus, it is likely that this
gene
activates fibroblast cells through a signal transduction pathway, Early growth
response 1 (EGR 1 ) is a promoter associated with certain genes that induces
various
tissues and cell types upon activation, leading the cells to undergo
differentiation
and proliferation.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
68 as residues: Trp-10 to Lys-18, Val-32 to Cys-38, Asp-41 to Thr-47.
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
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 1034 of SEQ ID
N0:19, b
is an integer of 15 to 1048, 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
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
17
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.
gnlIPIDId103S484 (all information available through the recited accession
number is
S incorporated herein by reference) which is described therein as "KIAA0783
protein."
A partial alignment demonstrating the observed homology is shown immediately
below.
>gnl~PIDId1035484
(AB018326)
KIIvA0783
protein
[Homo
sapiens]
> sp~D10354841D1035484 KIAA0783 PROTEIN.
L ength = 888
Plus StrandHSPs:
IS Score = (686.4 bits), Expect = 4.5e-265, Sum P(2)
1950 = 4.5e-265
Identi ties 374/393 (950), Positives = 374/393 (95%),
= Frame = +1
Query: 361 AYCKQHADRLDRKWKRKNYLALQSYCKMSLQEREKQLSPEAQARINARLQQYRAKAELAR
540
2O AYCKQHADRLDRKWKRKD1YLALQSYCKMSLQEREKQLSPEAQARINARLQQYRAKAELAR
Sbjct: 493 AYCKQHADRLDRKWKRKi~TYLALQSYCKMSLQEREKQLSPEAQARINARLQQYRAKAELAR
552
Query: 541 STRPQAWVPREKLPRPLTSSASAIRKLMRKAELMGISTDIFPVDNSDTSSSVDGRRKHKQ
2S 720
STRPQAWVPREKLPRPLTSSASAIRKLMRKAELMGISTDIFPVDNSDTSSSVDGRRKHKQ
Sbjct: 553 STRPQAWVPREKLPRPLTSSASAIRKLMRKAELMGISTDIFPVDNSDTSSSVDGRRKHKQ
612
3O Query: 721 PALTADFVNYYFERNMRMIQIQENMAEQKNIKDKLENEQEKLHVEYNKLCESLEELQNLN
900
PALTADFVNYYFERNMRIMIQIQENMAEQKNIKDKLENEQEKLHVEYNKLCESLEELQNLN
Sbjct: 613 PALTADFVNYYFERNMRT_dIQIQENMAEQKNIKDKLENEQEKLHVEYNKLCESLEELQNLN
672
3S
Query: 901 GKLRSEGQGIWALLGRITGQKLNIPAILRAPKERKPSKKEGGTQKTSTLPAVLYSCGICK
1080
GKLRSEGQGIWALLGRITGQKLNIPAILRAPKERKPSKKEGGTQKTSTLPAVLYSCGICK
Sbjct: 673 GKLRSEGQGIWALLGRITGQKLNIPAILRAPKERKPSKKEGGTQKTSTLPAVLYSCGICK
4O 732
Query: 1081 KNHDQHLLLLCDTCKLHYHLGCLDPPLTRMPRKTKNSYWQCSECDQAGSSDMEADMAMET
1260
KNHDQHLLLLCDTCKLHYHLGCLDPPLTRMPRKTKNSYWQCSECDQAGSSDMEADMAMET
4S Sbjct: 733 KNHDQHLLLLCDTCKLHYHLGCLDPPLTRMPRKTKNSYWQCSECDQAGSSDMEADMAMET
792
Query: 1261 LPDGTKRSRRQIKEPVKFVPQDVPPEPKKIPIRNTRTRGRKRSFXXXXXXXXXXXXXXXX
1440
SO LPDGTKRSRRQIKEPVKFVPQDVPPEPKKIPIRNTRTRGRKRSF
Sbjct: 793 LPDGTKRSRRQIKEPVKFVPQDVPPEPKKIPIRNTRTRGRKRSFVPEEEKHEERVPRERR
852
Query: 1441 XXXSVLQKKPKAEDLRTECATCKGTGDNENLVR 1539
SS SVLQKKPKAEDLRTECATCKGTGDNENLVR
Sbjct: 853 QRQSVLQKKPKAEDLRTECATCKGTGDNENLVR 885
WO 00/55177 cA 02361277 2001-08-28 PCT/US00/06058
lh
Score = (221.8 bits), Expect = 4.5e-265, Sum P(2)
630 = 4.5e-265
Identi ties= 114/114 (1000), Positives = 114/114 (1000),
Frame = +2
Query: 17 KCGVSPSCELCPNQDGIFKETDAGRWVHIVCALYVPGVAFGDIDKLRPVTLTEMNYSKYG
S 196
KCGVSPSCELCPNQDGIFKETDAGRWVIIIVCALYVPGVAFGDIDKLRPVTLTEP4T1YSKYG
Sbjct: 378 KCGVSPSCELCPNQDGIFKETDAGRPTVHIVCALYVPGVAFGDIDKLRPVTLTEMNYSKYG
437
lO Query: 197 AKECSFCEDPRFARTGVCISCDAGMCRAYFHVTCAQKEGLLSEAAAEEDIADPF
358
AKECSFCEDPRFARTGVCISCDAGMCRAYFHVTCAQKEGLLSEAAAEEDIADPF
Sbjct: 438 AKECSFCEDPRFARTGVCISCDAGMCRAYFHVTCAQKEGLLSEAAAEEDIADPF
491
Score = (55.3 bits), Expect = 1.4e-75, Sum P(2) =
157 1.4e-75
1S Identi ties= 28/79 (350), Positives = 41/79 (51%), Frame
= +1
Query: 1495CATCKGTGDNENLVRCDECRLCYHFGCLDPPLKKSPKQTGYG-WICQECDSSSSKEDENE
1671
C CK D L+ CD C+L YH GCLDPPL + P++T W C ECD
+ S + E +
2O Sbjct: 728 CGICKKNHDQHLLLLCDTCKLHYHLGCLDPPLTRMPRKTKNSYWQCSECDQAGSSDMEAD
787
Query: 1672AERKNISQELNMEQKNPKK 1728
+ + ++ K+
2S Sbjct: 788 MAMETLPDGTKRSRRQIKE 806
The segment of gnlIPIDId103S484 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 11S,SEQ ID NO. 117,SEQ ID NO. 119. Preferred
polypeptides of the invention comprise polypeptides having the amino acid
30 sequences) set out as SEQ ID NO. 116,SEQ ID NO. 118,SEQ ID NO. 120 which
correspond to the Query sequence in the alignment shown above (gaps introduced
in
the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Snares fetal_liver_spleen_1NFLS_S1, NCI_CGAP Prl2 and to a lesser
3S extent in Morton Fetal Cochlea, Soares_pregnant uterus_NbHPU, Stratagene
pancreas (#937208), HEL cell line, Human Synovium, Human Prostate Cancer,
Stage
C fraction, Amniotic Cells - Primary Culture, Healing groin wound, 6.S hours
post
incision, Soares_NhHMPu S l, Synovial hypoxia, NTERA2, control, Human adult
(K.Okubo), NCI CLAP Lu6, NCI CGAP_GCB 1, NCI CLAP Pr22, Colon
40 Carcinoma, Colon Normal II, Human Placenta, Nine Week Old Early Stage
Human,
Snares infant brain 1NIB.
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
45 the present invention. Preferably, such related polynucleotides are
specifically
WO 00/55177 CA 02361277 2001-08-28 PCT/US00/06058
19
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 1868 of SEQ ID
N0:20, b
is an integer of 15 to 1882, 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.
bbsf 129340 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "X-linked
retinopathy
protein." A partial alignment demonstrating the observed homology is shown
immediately below.
>bbs~129340 X-linked retinopathy protein (C-terminal, clone XEH.8c}
[human,
Peptide Partial, 100 aa] [Homo Sapiens] >pir~A46010~A46010 X-
linked
(fragment)
retinopathy protein (C-terminal, clone XEH.8c) - human
Length = 100
Plus Strand HSPs:
Score = 99 (34.8 bits), Expect = 2.3e-12, Sum P(3) = 2.3e-12
Identities = 19/26 (730), Positives = 22/26 (84%), Frame = +2
Query: 44 ETESHSWQAGVQWRNLGSLQPPPPG 121
ETES SV +AGVQW +LGSL+ PPPG
Sbjct: 7 ETESCSVAEAGVQWCDLGSLKSPPPG 32
Score = 61 (21.5 bits), Expect = 2.3e-12, Sum P(3) = 2.3e-12
Identities = 13/17 (76%), Positives = 14/17 (82%), Frame = +3
Query: 120 GSSDSPASXF*VAGVTG 170
GSSDSPAS VAG+TG
Sbjct: 32 GSSDSPASASRVAGITG 48
Score = 60 (21.1 bits), Expect = 3.7e-08, Sum P(2) = 3.7e-08
Identities = 14/26 (530), Positives = 16/26 (61%), Frame = +1
Query: 265 DLPTSASPSAGITGVSHCARLTVSFL 342
D P SAS AGITG+ H +L FL
Sbjct: 35 DSPASASRVAGITGMHHHTQLIFVFL 60
WO 00/55177 CA 02361277 2001-08-28 PCT/US00/06058
Score = 55 (19.4 bits), Expect = ~.3e-12, Sum P(3) = 2.3e-12
Identities = 11/16 (680), Positives = 12/16 (75%), Frame = +1
Query: 178 HHTRLIFVFLVGMRFH 225
HHT+LIFVFLV H
Sbjct: 51 HHTQLIFVFLVETGSH 66
The segment of bbs1129340 that is shown as "Sbjct" above is set out as
10 sequences) SEQ ID NO. 121,SEQ ID NO. 123,SEQ ID NO. 125,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 and
described elsewhere herein. Assays for determining such activities are also
known in
the art some of which have been described elsewhere herein. Preferred
polypeptides
15 of the invention comprise polypeptides having the amino acid sequences) set
out as
SEQ ID NO. 122,SEQ ID NO. 124,SEQ ID NO. 126,SEQ ID NO. 128 which
correspond to the Query sequence in the alignment shown above (gaps introduced
in
the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in Human Adult
20 Retina.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
70 as residues: Pro-52 to Cys-57.
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
general formula of a-b, where a is any integer between 1 to 1231 of SEQ ID
N0:21, b
is an integer of 15 to 1245, 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
W O- 00/55177 PCT/US00/06058
CA 02361277 2001-08-28
21
It has been discovered that this gene is expressed primarily in Human
Amy gdala.
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 796 of SEQ ID
N0:22, b
is an integer of 15 to 810, where both a and b correspond to the positions of
nucleotide residues shown in 5EQ ID N0:22, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
It has been discovered that this gene is expressed primarily in the following
tissues: Human Amygdala.
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 1661 of SEQ ID
N0:23, b
is an integer of 15 to 1675, 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 the following
tissues: Soares NhHMPu_S l, Human Amygdala.
WO 00/55177 cA 02361277 2001-08-28 PCT/US00/06058
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
73 as residues: Pro-20 to Cys-30.
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 axe
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 1055 of SEQ ID
N0:24, b
is an integer of 15 to 1069, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID 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 the following
tissues: Human Fetal Brain, Infant brain, Bento Soares and to a lesser extent
in Soares
infant brain 1NIB, Soares adult brain N2b4HB55Y, H. Kidney Cortex, subtracted,
re-
rescue, random primed, Frontal Lobe. Dementia, Human Cerebellum, subtracted,
Human Amygdala;re-excision, NCI CGAP_GC3, Stratagene NT2 neuronal precursor
937230, normalized infant brain cDNA, Human Hypothalamus,schizophrenia, re-
excision, NTERA2 + retinoic acid, 14 days, Human Manic Depression Tissue,
Brain
frontal cortex, Endothelial cells-control, Human Amygdala, Smooth
muscle,control.
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 1461 of SEQ ID
N0:25, b
is an integer of 15 to 1475, where both a and b correspond to the positions of
WO 00155177 CA 02361277 2001-08-28 PCT/US00106058
73
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: 16
It has been discovered that this Gene is expressed primarily in the following
tissues: Glioblastoma, Endothelial cells-control. Human Amygdala, Soares
infant
brain 1NIB.
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 integer between 1 to 917 of SEQ ID
N0:26, b
is an integer of 15 to 931, 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
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.
gi12197085 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "ORF2-like
protein."
A partial alignment demonstrating the observed homology is shown immediately
below.
>gi~2197085 (AF003535) ORF2-like protein [Homo Sapiens]
Length = 573
Plus Strand HSPs:
Score = 187 (65.8 bits), Expect = 6.1e-24, Sum P(3) = 6.1e-24
3S Identities = 34/48 (70%), Positives = 39/48 (81%), Frame = +2
WO- 00/55177 PCT/US00106058
CA 02361277 2001-08-28
7-1
Query: 605 KRSIHHDQVRFIPGXQGGIFNINKSINVINHTSKVKGKNHMIITTDAKK 748
K+ IHHDQV FIPG QGG7FNI KSINVI H ++ K KNHMII+ DA+K
Sbjct: 70 KKLIHHDQVGFIPGMQGWFNIRKSINVIQHINRAKDKNHMIISIDAEK 117
Score = 81 (28.5 bits), Expect = 6.1e-24, Sum P(3) = 6.1e-24
Identities = 15/23 (65%), Positives = 18/23 (78%), Frame = +3
Query: 459 LPNSFYDVGITLIPKRDKDTTRK 527
lO LPNSFY+ I LIPK +DTT+K
Sbjct: 19 LPNSFYEASIILIPKPGRDTTKK 41
Score = 77 (27.1 bits), Expect = 5.1e-24, Sum P(3) = 6.1e-24
Identities = 17/26 (65%), Positives = 20/26 (760), Frame = +1
Query: 514 TLQENYRSISLMNXYAKILXKILTNQ 591
T +EN+R ISLMN AKIL KIL N+
Sbjct: 39 TKKENFRPISLMNIDAKILNKILANR 64
The segment of gil2197085 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 129,SEQ ID NO. 131,SEQ ID NO. 133. Preferred
polypeptides of the invention comprise polypeptides having the amino acid
sequences) set out as SEQ ID NO. 130,SEQ ID NO. 132,SEQ ID NO. 134 which
correspond to the Query sequence in the alignment shown above (gaps introduced
in
the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in Human
Amygdala.
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
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 897 of SEQ ID
N0:27, b
is an integer of 15 to 91 l, where both 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
WO 00/55177 PCT/US00/06058
CA 02361277 2001-08-28
>j
It has been discovered that this gene is expressed primarily in the following
tissues: Human Amygdala;re-excision. Endothelial cells-control.
Many polynucleotide sequences. such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:28 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 integer between 1 to 1683 of SEQ ID
N0:28, b
is an integer of 15 to 1697, 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.
1 S FEATURES OF PROTEIN ENCODED BY GENE NO: 19
It has been discovered that this gene is expressed primarily in Human
Amygdala;re-excision.
Many polynucleotide sequences. such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:29 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 1128 of SEQ ID
N0:29, b
is an integer of 15 to 1142, 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
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
WO.00/55177 PCT/US00/06058
CA 02361277 2001-08-28
76
example, the sequence accessible through the following database accession no.
gi1347314 (all information available through the recited accession number is
incorporated herein by reference) which is described therein as "hnRNP H [Homo
Sapiens]." A partial alignment demonstrating the observed homology is shown
S immediately below.
>gi~347314 hnRNP H [Homo Sapiens] >pir~I39358~I39358 49K protein - human
>sp~P31943~ROH1 HUMAN HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN H
(HNRNP H).
Length = 449
Plus Strand HSPS:
Score = 464 (163.3 bits), Expect = 6.8e-78, Sum P(3) = 6.8e-78
IS Identities = 88/113 (770), Positives = 99/113 (870), Frame = +2
Query: 140 QAFKSNGIEMDL~7VMKHNGPND---ASDGTVRLRGLPFGCSKEEIVQFFQGLEIVPNGITL
310
+ FKSN +EMDtnrV+KH GPN A+DG VRLRGLPFGCSKEEIVQFF GLEIVPNGITL
2O Sbjct: 84 EVFKSNNVEMDL~iVLKHTGPNSPDTANDGFVRLRGLPFGCSKEEIVQFFSGLEIVPNGITL
143
Query: 311 TMDYQGRSTGEAFVQFASKEIAENALGKHKERIGHRYIEIFRSSRSEIKGFYD 469
+D+QGRSTGEAFVQFAS+EIAE AL KHKERIGHRYIEIF+SSR+E++ YD
2S Sbjct: 144 PVDFQGRSTGEAFVQFASQEIAEKALKKHKERIGHRYIEIFKSSRAEVRTHYD 196
The segment of gi1347314 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 135. Based on the structural similarity these homologous
30 polypeptides are expected to share at least some biological activities.
Such activities
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 136 which correspond to the Query
3S sequence in the alignment shown above (gaps introduced in the sequence by
the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Soares fetal liver spleen 1NFLS and to a lesser extent in Nine Week
Old
Early Stage Human, Stromal cell TF274, Stratagene colon (#937204),
Osteoblasts,
40 Human 8 Week Whole Embryo, Resting T-Cell Library,II, Primacy Dendritic
Cells,
lib I, Soares_NhHMPu_S1, Stratagene muscle 937209, Stratagene HeLa cell s3
WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
77
937216, Human Ovarian Cancer Reexcision, Macrophage (GM-CSF treated), 12
Week Old Early Stage Human, Stratagene colon (#937204), Stratagene fibroblast
(#937212), Smooth muscle,control, Human Testes, Soares_NhHMPu_S1, Stratagene
HeLa cell s3 937216, Soares_parathyroid_tumoyNbHPA,
Soaves_multiple_sclerosis_2NbHMSP. SoaresNhHMPu_S1, Soaves infant brain
1NIB, H. Kidney Pyramid, subtracted. Human Bone Marrow, Human B Cell 8866,
Human Fibrosarcoma. Al-CELL LINE, Human White Adipose, Healing Abdomen
wound;70&90 min post incision, Human stomach, Frontal lobe,dementia;re-
excision,
Activated T-cells, Human Adult Heart:re-excision, Smooth Muscle- HASTE
normalized, Stomach cancer (human):re-excision, pBMC stimulated w/ poly I/C,
NTERA2 + retinoic acid, 14 days, Glioblastoma, Human Stomach;re-excision,
Human Frontal Cortex, Schizophrenia, Jurkat T-Cell, S phase, Spinal Cord, re-
excision, Temporal cortex-Alzheizmer; subtracted, Human Brain, Striatum, 12
Week
Old Early Stage Human, II, Human Jurkat membrane bound polysomes, Human
Uterine Cancer, T-Cell PHA 24 hrs, Human Hippocampus, Ulcerative Colitis,
Hemangiopericytoma, PC3 Prostate cell line, T Cell helper I,
Soaves_placenta_8to9weeks_2NbHP8to9W, Colon Normal II, Dendritic cells,
pooled,
Human Fetal Lung III, Soaves_multiple_sclerosis_2NbHMSP, Human Placenta,
human tonsils, Human Adult Pulmonary;re-excision, Anergic T-cell, Human
Amygdala, Monocyte activated, Neutrophils IL-1 and LPS induced, Hodgkin's
Lymphoma II, Stratagene endothelial cell 937223, Keratinocyte, Soaves placenta
Nb2HP.
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 2308 of SEQ ID
N0:30, b
is an integer of 15 to 2322, where both a and b correspond to the positions of
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
28
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
It has been discovered that this gene is expressed primarily in Human Adult
Heart;re-excision.
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 polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 644 of SEQ ID
N0:31, b
is an integer of 15 to 658, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:31, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 22
It has been discovered that this gene is expressed primarily in the following
tissues: Soares total fetus Nb2HF8 9w, Epithelial-TNFa and INF induced.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
. related to SEQ ID N0:32 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 polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1423 of SEQ ID
N0:32, b
is an integer of 15 to 1437, 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.
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
~ c~
FEATURES OF PROTEIN ENCODED BY GENE NO: 23
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.
15
gnlIPIDIe1249651 (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as an Arabidopsis
putative protein. A partial alignment demonstrating the observed homology is
shown
immediately below.
>gnl~PID~e1249651 (AL021711) putative protein [Arabidopsis thaliana]
>gnl~PID~e1248930 (AL021687) putative protein [Arabidopsis
thaliana] >sp~050052~050052 HYPOTHETICAL 160.5 KD PROTEIN.
Length = 1405
Minus Strand HSPs:
Score = (277.0 bits), Expect = 1.5e-76, P = 1.5e-76
787
Identi ties= 138/203 (670), Positives = 165/203 (81%),
Frame = -2
Query: 1742SSKTFKPKKNIPEGSHQYELLKHAEATLGSGNLRQAVMLPEGEDLNEWIAVNTVDFFNQI
1563
+ KTF+PKK+ P G+ EL KH +ATLGSGNLR+AV LP GEDLNEW+AVNTVDFFNQ+
Sbjct: 1199NQKTFRPKKSAPSGTKGAELRKHIDATLGSGNLREAVKLPPGEDLNEWLAVNTVDFFNQV
1258
Query: 1562NMLYGTITEFCTEASCPVMSAGPRYEYHWADGTNIKKPIKCSAPKYIDYLMTWVQDQLDD
1383
N+L+GT+TEFCT +C M+AGP+YEY WADG IKKPI+ SAPKY++YLM
W++ QLDD
3O Sbjct: 1259NLLFGTLTEFCTPENCSTMTAGPKYEYRWADGVQIKKPIEVSAPKYVEYLMDWIETQLDD
1318
Query: 1382ETLFPSKIGVPFPKNFMSVAKTILKRLFRVYAHIYHQHFDSVMQLQEEAHLNTSFKHFIF
1203
3S ET+FP K+G FP NF V KTI KRLFRVYAHIYH HF ++ L+EEAHLNT
FKHFI
Sbjct: 1319ETIFPQKLGAAFPPNFKEVVKTIFKRLFRVYAHIYHSHFQKIVSLKEEAHLNTCFKHFIL
1378
Query: 1202FVQEFNLIDRRELAPLQELIEKL 1134
4O F EF LID++ELAPLQELIE +
Sbjct: 1379FTHEFVLIDKKELAPLQELIESI 1401
The segment of gnIIPIDIe 1249651 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 137. Preferred polypeptides of the invention comprise
4~ polypeptides having the amino acid sequences) set out as SEQ ID NO. 138
which
correspond to the Query sequence in the alignment shown above (gaps introduced
in
the sequence by the computer are, of course, removed).
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
It has been discovered that this gene is expressed primarily in the following
tissues: Primary Dendritic Cells, lib I and to a lesser extent in Human
Osteoclastoma,
re-excision, NCI CGAP_GCB l, Human Fetal Heart, Differential (Fetal-Specific),
B
Cell lymphoma, Dendritic cells, pooled. NCI_CGAP Br2, Soares_I~IhHMPu_Sl,
5 Activated T-Cell ( l2hs)/Thiouridine labelledEco, Human Osteoclastoma,
Spleen,
Chronic lymphocytic leukemia, Osteoblasts, Human Prostate,differential
expression,
Kidney medulla, pBMC stimulated w/ poly I/C, Jurkat T-Cell, S phase, Synovial
Fibroblasts (Ill/TNF), subt, Human Adult Small Intestine, Human Prostate, T-
Cell
PHA 16 hrs, NCI_CGAP LuS, NCI CLAP GCB 1, NCI_CGAP_Kid3,
10 NCI CLAP Brn23, Monocyte activated; re-excision, Human Pancreas Tumor, T-
Cell PHA 24 hrs, Human Heart, Human Thymus Stromal Cells, Epithelial-TNFa and
INF induced, T Cell helper I, Human T-Cell Lymphoma, Human Testes Tumor,
Colon Tumor II, Soares melanocyte 2NbHM, Soares_fetal_lung NbHLI9W, Primary
Dendritic cells,frac 2, Human Placenta, human tonsils, Human Adult
Pulmonary;re-
15 excision, Human adult (K.Okubo), HUMAN B CELL LYMPHOMA. Activated T-
cell( 12h)/Thiouridine-re-excision, Bone Marrow Cell Line (RS4;11 ), Hodgkin's
Lymphoma II, NCI CGAP_GCB 1.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
20 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. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
25 general formula of a-b, where a is any integer between 1 to 1954 of SEQ ID
N0:33, b
is an integer of 15 to 1968, 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.
30 FEATURES OF PROTEIN ENCODED BY GENE NO: 24
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
31
example, the sequence accessible through the following database accession no.
gnlIPIDIe206544 (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "pval
[Plasmodium
vivax]." A partial alignment demonstrating the observed homology is shown
immediately below.
>gnl~PID~e206544 peal [Plasmodium vivax] >sp~Q26195~Q26195 PVA1 GENE.
Length = 200
Minus Strand HSPs:
Score = 169 (59.5 bits), Expect = 4.3e-12, P = 4.3e-12
Identities = 40/74 (540), Positives = 45/74 (600), Frame = -1
IS Query: 715 DRVWLCHPGWSAWQSWLTAASTSW-GQAILLSLPPSSL~7DYRHMPPQSTTFFYL*RG-GF
542
DRV LCHPGWSAWQS T AST Q+ L LP SSw7DYR +pp F + R
Sbjct: 114 DRVLLCHPGWSAWQSLFTVASTFLVKQSSCLGLP-SS64DYRRIPPHLANFSFFCRNKSL
172
Query: 541 AM*PRLVSSS*AQAI 497
M PRL+ +S Q I
Sbjct: 173 TMLPRLILNSWPQVI 187
The segment of gnlIPIDIe206544 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 139. Based on the structural similarity these homologous
polypeptides are expected to share at least some biological activities. Such
activities
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 140 which correspond to the Query
sequence in the alignment shown above (gaps introduced in the sequence by the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Soares placenta Nb2HP and to a lesser extent in Epithelial-TNFa and
INF
induced, Activated T-Cell labeled with 4-thioluri, Human Fetal Bone, Human
Heart,
Stratagene lung (#937210), Primary Dendritie cells,frac 2.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:34 and may have been publicly available prior to
conception of
CA 02361277 2001-08-28 pC'T/US00/06058
WO 00/55177
3~
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 740 of SEQ ID
N0:34, b
is an integer of 15 to 754, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID 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 is expressed primarily in the following
tissues: Human Endometrial Tumor and to a lesser extent in Soares infant brain
1NIB,
Soares melanocyte 2NbHM, Soares fetal liver spleen 1NFLS, Salivary Gland, Lib
2,
H. Kidney Medulla, re-excision, Human Adrenal Gland Tumor, Human Placenta,
Soares_pregnant uterus_NbHPU, Human T-cell lymphoma;re-excision, Stratagene
ovarian cancer (#937219), Synovial Fibroblasts (Il1/TNF), subt, NCI_CGAP_GCB
l,
Soares fetal liver_spleen_1NFLS_Sl, Human Thymus, Stratagene fetal spleen
(#937205), Human Pancreas Tumor, Human Heart, Rejected Kidney, lib 4,
Hepatocellular Tumor; re-excision, Human Substantia Nigra, Early Stage Human
Brain, Pancreatic Islet, Soares_fetal heart NbHH 19W,
Soares multiple sclerosis 2NbHMSP, Endothelial-induced, Human 8 Week Whole
Embryo, Soares placenta Nb2HP, Messangial cell, frac 1, Human epithelioid
sarcoma, Human Colon, subtraction, Human Umbilical Vein Endothelial Cells,
fract.
A, Adipocytes;re-excision, Soares retina N2b4HR, Human adult small
intestine,re-
excision, Smooth Muscle- HASTE normalized, Human Colon Cancer;re-excision,
Synovial IL-1/TNF stimulated, Stratagene fetal retina 937202, Synovial hypoxia-
RSF
subtracted, Glioblastoma, Human Stomach;re-excision, Synovial hypoxia, Human
Manic Depression Tissue, KMH2, Spinal Cord, re-excision, Fetal Liver,
subtraction
II, Human Prostate, Human Bone Marrow, re-excision, Human Uterine Cancer,
Stromal cell TF274, Human Rhabdomyosarcoma, Ulcerative Colitis, Human Thymus
Stromal Cells, Epithelial-TNFa and INF induced, Bone Marrow Stromal Cell,
untreated, Human T-Cell Lymphoma, Soares breast 3NbHBst, Human Testes Tumor,
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
33
Human Testes, Reexcision, Human Adult Puhnonary;re-excision, Endothelial cells-
control, Colon Normal III, Human Osteoclastoma, Human Mierovascular
Endothelial
Cells, fract. A, Keratinocyte, Nine Week Old Early Stage Human.
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 1310 of SEQ ID
N0:35, b
is an integer of 15 to 1324, 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 the following
tissues: Soares_multiple_sclerosis_2NbHMSP and to a lesser extent in Human
Lung
Cancer;re-excision, Healing groin wound; 7.5 hours post incision, L428,
Stratagene
neuroepithelium NT2RAMI 937234, Human Adipose,
Soares_senescent fibroblasts NbHSF, HUMAN B CELL LYMPHOMA, Soares
placenta Nb2HP.
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
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 1085 of SEQ ID
N0:36, b
is an integer of 15 to 1099, where both a and b correspond to the positions of
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
3-1
nucleotide residues shown in SEQ ID 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: NCI_CGAP_GCB 1 and to a lesser extent in
Soares_fetal_liver_spleen_1NFLS_Sl, Human Tonsil, Lib 3, KMH2, T cell helper
II,
Hodgkin's Lymphoma II, Human 8 Week Whole Embryo, Soares infant brain 1NIB.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
86 as residues: Val-34 to Glu-39.
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 1365 of SEQ ID
N0:37, b
is an integer of 15 to 1379, 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 PROTEIN ENCODED BY GENE NO: 28
It has been discovered that this gene is expressed primarily in the following
tissues: Soares infant brain 1NIB and to a lesser extent in Morton Fetal
Cochlea,
NCI CGAP_GCB l, Soares fetal liver spleen 1NFLS, Soares_NhHMPu S l, Aorta
endothelial cells + TNF-a, Healing groin wound, 6.5 hours post incision, KMH2,
Apoptotic T-cell, Synovial Fibroblasts (control), Human Liver, normal,
NCI CGAP_Ew l, NCI CGAP_GC3, NCI_CGAP GC4, NCI CGAP_GCB 1, Colon
Tumor II, NCI CLAP Br2, Soares melanocyte 2NbHM, Bone marrow, Human
Adult Pulmonary;re-excision, T cell helper II, Hodgkin's Lymphoma II.
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
87 as residues: Thr-22 to Lys-30.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
5 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
10 general formula of a-b, where a is any integer between 1 to 567 of SEQ ID
N0:38, b
is an integer of 15 to 581, 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.
15 FEATURES OF PROTEIN ENCODED BY GENE NO: 29
It has been discovered that this gene is expressed primarily in the following
tissues: Nine Week Old Early Stage Human and to a lesser extent in
NCI CGAP_GCB l, Soares NhHMPu_S l, Soares melanocyte 2NbHM, HPAS
(human pancreas, subtracted), Human Pituitary, re-excision, Human Primary
Breast
20 Cancer;re-excision, human colon cancer, HSA 172 Cells, Raji Cells,
cyclohexamide
treated, Human Osteosarcoma, KMH2, Breast Cancer Cell line, angiogenic, L428,
Apoptotic T-cell, Human Primary Breast Cancer Reexcision,
Soares_pregnant uterus_NbHPU, Stratagene neuroepithelium (#937231 ), Human
Whole Six Week Old Embryo, Soares_parathyroid_tumor NbHPA, Human Synovial
25 Sarcoma, Human Microvascular Endothelial Cells, fract. A, Activated T-
cell(12h)/Thiouridine-re-excision, Soares infant brain 1NIB.
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
30 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
WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
36
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 1270 of SEQ ID
N0:39, b
is an integer of 15 to 1284, 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
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.
gnIIPIDlel283869 (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "neuropathy
target
esterase." A partial alignment demonstrating the observed homology is shown
immediately below.
>gnl~PID~e1283869 (AJ004832) neuropathy target esterase [Homo sapiens]
Length = 1327
Plus Strand HSPs:
Score = 955 (336.2 bits), Expect = 1.9e-95, P = 1.9e-95
Identities = 181/181 (1000), Positives = 181/181 (100%), Frame = +3
Query: 3 DMAEIQSRLAYVSCVRQLEWKSSSYCEYLRPPIDCFKTMDFGKFDQIYDVGYQYGKAVF
182
DMAEIQSRLAYVSCVRQLEWKSSSYCEYLRPPIDCFKTMDFGKFDQIYDVGYQYGKAVF
Sbjct: 1147 DMAEIQSRLAWSCVRQLEWKSSSYCEYLRPPIDCFKTMDFGKFDQIYDVGYQYGKAVF
1206
3O Query: 183 GGWSRGNVIEKMLTDRRSTDLNESRRADVLAFPSSGFTDLAEIVSRIEPPTSYVSDGCAD
362
GGWSRGNVIEKMLTDRRSTDLNESRRADVLAFPSSGFTDLAEIVSRIEPPTSYVSDGCAD
Sbjct: 1207 GGWSRGNVIEKMLTDRRSTDLNESRRADVLAFPSSGFTDLAEIVSRIEPPTSWSDGCAD
1266
Query: 363 GEESDCLTEYEEDAGPDCSRDEGGSPEGASPSTASEMEEEKSILRQRRCLPQEPPGSATD
542
GEESDCLTEYEEDAGPDCSRDEGGSPEGASPSTASEMEEEKSILRQRRCLPQEPPGSATD
Sbjct: 1267 GEESDCLTEYEEDAGPDCSRDEGGSPEGASPSTASEMEEEKSILRQRRCLPQEPPGSATD
1326
Query: 543 A 545
A
Sbjct: 1327 A 1327
The segment of gnIIPIDlel283869 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 141. Based on the structural similarity these homologous
CA 02361277 2001-08-28
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37
polypeptides are expected to share at least some biological activities. Such
activities
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 142 which correspond to the Query
sequence in the alignment shown above (gaps introduced in the sequence by the
computer are, of course, removedj.
It has been discovered that this gene is expressed primarily in the following
tissues: Soares infant brain 1NIB and to a lesser extent in Primary Dendritic
Cells, lib
l, Human adult testis, large inserts, Human Adult Testes, Large Inserts,
Reexcision,
Soares breast 2NbHBst, Soares fetal liver spleen 1NFLS, breast lymph node CDNA
library, NCI CGAP_GC4, HEL cell line, Stratagene HeLa cell s3 937216,
Macrophage (GM-CSF treated), Smooth muscle, serum induced,re-exc, Human
Eosinophils, Monocyte activated, Bone Marrow Cell Line (RS4;11 ), Nine Week
Old
Early Stage Human, Human frontal cortex, CHME Cell Line,untreated, Larynx
Tumour, NCI CLAP Lu 1, NCI CLAP GCB 1, NCI_CGAP Pr23, Ku 812F
Basophils Line, HL-60, PMA 4H, Human Adult Pulmonary, eosinophil-ILS induced,
Human Skin Tumor, Healing groin wound - zero hr post-incision (control),
Soares
adult brain N2b4HB55Y, NTERA2 + retinoic acid, 14 days, Glioblastoma, Human
Adipose Tissue, re-excision, Human Colon; re-excision, Myoloid Progenitor Cell
Line, Spleen metastic melanoma, Human Infant Brain, Human Thymus, Human
Brain, Striatum, Human Fetal Dura Mater, Spinal cord, PERM TF274, Epithelial-
TNFa and INF induced, Human Thymus, Hemangiopericytoma, H. Frontal
cortex,epileptic;re-excision, Pancreas Islet Cell Tumor, Human T-Cell
Lymphoma,
Primary Dendritic cells,frac 2, Hodgkin's Lymphoma II, Human 8 Week Whole
Embryo, Human Cerebellum.
The tissue distribution in infant brain tissue suggests that the protein
product
of this clone is useful for the detection/treatment of neurodegenerative
disease states
and behavioural disorders such as Alzheimers Disease, Parkinsons Disease,
Huntingtons Disease, Tourette Syndrome, schizophrenia, mania, dementia,
paranoia,
obsessive compulsive disorder, panic disorder, learning disabilities, ALS,
psychoses,
autism, and altered behaviors, including disorders in feeding, sleep patterns,
balance,
CA 02361277 2001-08-28
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3h
and perception. In addition, the gene or gene product may also play a role in
the
treatment and/or detection of developmental disorders associated with the
developing
embryo, or sexually-linked disorders. 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 N0:40 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 778 of SEQ ID
N0:40, b
is an integer of 15 to 792, 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 the following
tissues: Soares infant brain INIB 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,
Soares_parathyroid tumoyNbHPA.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
90 as residues: Ser-20 to Asp-25.
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 polynucleotides comprising a nucleotide sequence described by
the
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
39
general formula of a-b, where a is any integer between 1 to 1337 of SEQ ID
N0:41, b
is an integer of 1 ~ to 1351, 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 the following
tissues: Soares_fetal heart NbHHI9W, Stratagene endothelial cell 937223,
Stratagene lung (#937210), Stratagene muscle 937209, Human B-cell lymphoma and
to a lesser extent in Human Fetal Lung. Human colon carcinoma (HCC) cell line,
remake, Soares retina N2b4HR, HEL cell line, Stromal-Osteoclastoma, Human
Manic
Depression Tissue, Prostate BPH, Soaves multiple_sclerosis_2NbHMSP, T-Cell PHA
16 hrs, human ovarian cancer, Human Fetal Dura Mater, Rejected Kidney, lib 4,
Soares_NhHMPu S1, H. Frontal cortex,epileptic;re-excision, Pancreas Islet Cell
Tumor, Human T-Cell Lymphoma, Human Placenta, Dendritic cells, pooled, Human
Fetal Lung III, Human Adult Pulmonary;re-excision, Human Microvascular
Endothelial Cells, fract. A, CD34 positive cells (Cord Blood), T cell helper
II, Nine
Week Old Early Stage Human, Soaves placenta Nb2HP.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
91 as residues: Glu-45 to Thr-63.
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 2798 of SEQ ID
N0:42, b
is an integer of 15 to 2812, 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.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 33
It has been discovered that this gene is expressed primarily in the following
tissues: NCI_CGAP_Kid6, Soares_fetal_heart_NbHH 19W and to a lesser extent in
Human Epididymus, NCI_CGAP_Kid3. Colon Tumor II, Human Fetal Lung III,
5 HUMAN B CELL LYMPHOMA, Human Testes, Smooth Muscle Serum Treated,
Norm, human colon cancer, Aorta endothelial cells + TNF-a, NCI CGAP_Co9,
NCI CGAP_GC4, Human Lung Cancer;re-excision, Human Osteosarcoma, Jurkat T-
Cell, S phase, Soares_fetal lung NbHLI9W, Soares_senescent fibroblasts_NbHSF,
Human Umbilical Vein Endothelial Cells, uninduced, Soares breast 2NbHBst,
10 NCI CGAP_KidS, NCI_CGAP_Kid6, Human Adult Pulmonary;re-excision,
Activated T-Cell (l2hs)/Thiouridine IabelIedEco.
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
15 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 1131 of SEQ ID
N0:43, b
20 is an integer of 15 to 1145, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 34
25 It has been discovered that this gene is expressed primarily in the
following
tissues: Human Osteoclastoma and to a lesser extent in Soares infant brain
1NIB,
Soares placenta Nb2HP, Morton Fetal Cochlea, STRATAGENE Human skeletal
muscle cDNA library, cat. #936215., Colon Carcinoma, NCI CGAP_KidS, Primary
Dendritic Cells, lib l, Soares_parathyroid_tumor NbHPA, Myoloid Progenitor
Cell
30 Line, Stratagene muscle 937209, Spinal Cord, re-excision, Mo7e Cell Line GM-
CSF
treated (lng/ml), TF-1 Cell Line GM-CSF Treated, Clontech human aorta polyA+
mRNA (#6572), Human Ovarian Cancer Reexcision, Bone Marrow Stromal Cell,
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
41
untreated, Colon Tumor. Soares_pregnant_uterus_NbHPU, Human Adult
Pulmonary;re-excision, Colon Normal III. NCI_CGAP LuS, NCI CGAP_GCB 1,
Human Astrocyte, Soares ovary tumor NbHOT, Osteoclastoma-normalized A, Larynx
Normal, Human OB HOS treated (1 nM E2) fraction I,
Soares_multiple_sclerosis_2NbHMSP, Soares_placenta_8to9weeks_2NbHP8to9W,
Human Adult Retina, Human Pituitary, subtracted, HUMAN STOMACH, Human
Lung, Morton Fetal, STROMAL -OSTEOCLASTOMA, NTERA2 + retinoic acid, 14
days, H. Kidney Cortex, subtracted, KMH2, Human Prostate, Human promyelocyte,
normalized infant brain cDNA, Normalized infant brain, Bento Soares,
STRATAGENE Human skeletal muscle cDNA library, cat. #936215., Stratagene
NT2 neuronal precursor 937230, Macrophage-oxLDL, Human Adrenal Gland Tumor,
NTERA2, control, Gessler Wilms tumor, Soares_parathyroid_tumor NbHPA,
Stratagene lung (#937210), Pancreas Islet Cell Tumor, 12 Week Old Early Stage
Human, Colon Tumor II, Primary Dendritic cells,frac 2, NCI CGAP_GCB l, Human
fetal brain (TFujiwara), NCI_CGAP AA1, NCI CGAP_CoB, NCI_CGAP Co9,
NCI CLAP Pr2, NCI CGAP_GCB1, NCI CGAP_Prl2,
Soares total_fetus Nb2HF8 9w, Stratagene lung carcinoma 937218, Monocyte
activated, Human Testes, NCI_CGAP Pr2, Hodgkin's Lymphoma II, Human 8 Week
Whole Embryo, Nine Week Old Early Stage Human,
Soares total_fetus Nb2HF8 9w.
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 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 1495 of SEQ ID
N0:44, b
is an integer of 15 to 1509, 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.
WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
FEATURES OF PROTEIN ENCODED BY GENE NO: 35
It has been discovered that this Gene is expressed primarily in the following
tissues: Soares placenta Nb2HP and to a lesser extent in Soares retina N2b4HR,
NCI_CGAP_GCB l, Stratagene ovarian cancer (#937219), Human Chondrosarcoma,
Stratagene lung (#937210), Human T-Cell Lymphoma, Human Placenta, Morton
Fetal Cochlea, HSC172 cells, Stratagene fetal retina 937202, Human adult small
intestine,re-excision, NTERA2 teratocarcinoma cell line+retinoic acid (14
days),
Human endometrial stromal cells-treated with estradiol, H Female Bladder,
Adult,
KMH2, Soares total_fetus_Nb2HF8 9w, Mo7e Cell Line GM-CSF treated (lng/ml),
Apoptotic T-cell, Human Uterine Cancer, Soares NFL T_GBC_S l, Human
Activated T-Cells, re-excision, Human Thymus Stromal Cells, 12 Week Old Early
Stage Human. Human fetal brain (TFujiwara), NCI_CGAP_Kid3, NCI CGAP_Brl.l,
Stratagene hNT neuron (#937233), Human Testes Tumor, Human Adult
Pulmonary;re-excision, Human 8 Week Whole Embryo, Primary Dendritic Cells, lib
l, Soares infant brain 1NIB.
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 1843 of SEQ ID
N0:45, b
is an integer of 15 to 1857, 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: Human Adult Pulmonary;re-excision, Human Endometrial Tumor.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
CA 02361277 2001-08-28
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=13
related to SEQ ID N0:46 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 1796 of SEQ ID
N0:46, b
is an integer of 15 to 1810, 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 the following
tissues: Human Adult Pulmonary;re-excision, Human Adult Pulmonary and to a
lesser extent in Human Fetal Lung III, Kidney Pyramids, Soares ovary tumor
NbHOT, Human Tonsil, Lib 3, Human Endometrial Tumor.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
96 as residues: Pro-28 to Ser-35.
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 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 1865 of SEQ ID
N0:47, b
is an integer of 15 to 1879, 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
CA 02361277 2001-08-28
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4-l
It has been discovered that this gene is expressed primarily in the following
tissues: Human Adult Pulmonary;re-excision, Soares_pregnant uterus NbHPU,
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 N0:48 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 542 of SEQ ID
N0:48, b
is an integer of 15 to 556, where both a and b correspond to the positions of
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
It has been discovered that this gene is expressed primarily in the following
tissues: Human Primary Breast Cancer;re-excision, Soares breast 3NbHBst and to
a
lesser extent in Namalwa Cells, Amniotic Cells - Primary Culture, NTERA2 +
retinoic acid, 14 days, Human Whole Six Week Old Embryo, Soares fetal liver
spleen
1 NFLS .
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:49 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 593 of SEQ ID
N0:49, b
is an integer of 15 to 607, 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.
WO-00/55177 CA 02361277 2001-08-28
PCT/US00/06058
-~
FEATURES OF PROTEIN ENCODED BY GENE NO: 40
It has been discovered that this gene is expressed primarily in the following
tissues: Amniotic Cells - Primary Culture.
Many polynucleotide sequences. such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:50 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. 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 582 of SEQ ID
N0:50, b
is an integer of 15 to 596, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:50, 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: Amniotic Cells - Primary Culture, 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 N0:51 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 270 of SEQ ID
N0:51, b
is an integer of 15 to 284, 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
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
46
It has been discovered that this gene is expressed primarily in the following
tissues: Smooth muscle, control; re-excision, Smooth muscle-ILb induced, HSA
172
Cells, Amniotic Cells - Primary Culture, Spleen metastic melanoma, H. Kidney
Medulla, re-excision, CD34 depleted Buffy Coat (Cord Blood), Stromal cell
TF274,
Spinal cord, Human Rhabdomyosarcoma, Smooth muscle, serum induced,re-exc,
Human Gall Bladder, Activated T-Cell (l2hs)/Thiouridine labelledEco, CD34
depleted Buffy Coat (Cord Blood), re-excision, Anergic T-cell, Activated T-
cell( 12h)/Thiouridine-re-excision, Soares placenta Nb2HP, Soares fetal liver
spleen
1 NFLS.
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 1601 of SEQ ID
N0:52, b
is an integer of 15 to 1615, 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
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.
gnlIPIDIe1291654 (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "rna
helicase", which
is thought to function by unwinding double-stranded RNA in a 3' to
5'direction. A
partial alignment demonstrating the observed homology is shown immediately
below.
>gnl~PID~e1291654 (AL023290) rna helicase [Schizosaccharomyces pombe]
Length = 1428
CA 02361277 2001-08-28
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47
Plus Strand HSPs:
Score = 435 (153.1 bits), Expect = 1.1e-37, P = 1.1e-37
Identiti=s = 122/429 (280), Positives = 229/429 (530), Frame = +2
S
Query: 2 GKMKEKRYDASKGMESLEDTFVSQANALQRKGRAGWASGVCFHLFTSHHYNHQLLKQQL
181
G +E RY++ + + L DTFVS+ANA QR GRAG V G+C+HLF+ ++ Q L Q
Sbjct: 936 GVHREMRYNSRRHLSRLTDTFVSKANAKQRSGRAGRVQEGICYHLFSKFKHDTQFLSYQT
l~ 1045
Query: 182 PEIQRVPLEQLCLRIKILEMFSAHNLQSVFSRLIEPPH-TDSLRASKIRLRDLGALTPDE
358
PEI R+ L+++ LR+K+ +M ++Q V + ++Pp T+ +RA + +L +GAL+ +E
IS Sbjct: 1046 PEILRLNLQEWLRVKMCQM---GDVQDVLGKALDPPSSTNIIRALE-KLHQVGALSENE
1101
Query: 359 RLTPLGYHLASLPVDVRIGKLMLFGSIFRCLDPALTIAASLAFKSPFVSPWDKKEEANQK
538
2O +LT LG L+ LPVD +GK+++ G ++C+D A +I A L SPF D + AN+
Sbjct: 1102 KLTKLGKFLSQLPVDANLGKILVLGCFYKCVDAASSIVAMLTIGSPFRKSVDNEFSANKA
1161
Query: 539 KLEFAFAN--SDYLALLQAYKGWQLSTKEGVRASYN-YCRQNFLSGRVLQEMASLKRQFT
2S 709
+L FA N SD + + AY W+ + + + ++ +L+ L SLK Q
Sbjct: 1162 RLSFAKENTRSDLVLMYYAYCAWREICLSPLGPDEDSFAKEKYLNLEALSMTESLKIQLL
1221
3O Query: 710 ELLSDIGF--AREGLRAREIEKRAQGGDGVLDATGEEANSNAENPKLISAMLCAALYPNV
883
L D+ A + + +++ V+ +E + N+ N +++ ++ A+LYPN+
Sbjct: 1222SELKDMKLLGASDVDTCKSLKRSICRRFAVIP---KEHDINSGNAEILCGVIAASLYPNI
1278
3S
Query: 884 VQVKSPEGKFQKTSTGAVRMQPKSAELKFVTKNDGYVHIHPSSVNYQVRHFDSPYLLYHE
1063
++ + ++ ST K + V+ N+ ++ + S ++ Y
Sbjct: 1279LRYDYEKRQWSTLSTN------KRVRILDVSVNN----------RSELPNMPSKFVAYTN
4~ 1322
Query: 1064 KIKTSRV--FIRDCSMVSVYPLVLFGGGQVNVQLQRGEFWSLDDGG7IRF--VAASHQVA
1231
+ ++R ++ + +MV++ L++ G +V ++ G+ LD+ + F V S ++
4S Sbjct: 1323 MMSSTRASEYVNETTMVTLRQLLMMCGLKVENRVSVGQ--AKLDNFTVYFENVWSASLS
1380
Query: 1232 ELVKELRCELDQLLQDKIK 1288
L + + L++ + K
S~ Sbjct: 1381 ILRRFIETSLNEFFAEPDK 1399
The segment of gnIIPIDIe1291654 that is shown as "Sbjct" above is set out as
SS sequences) SEQ ID NO. 143. Based on the structural similarity these
homologous
polypeptides are expected to share at least some biological activities. Such
activities
are known in the art and described elsewhere herein. Assays for determining
such
activities are also known in the art some of which have been described
elsewhere
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
48
herein. Preferred polypeptides of the invention comprise polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 144 which correspond to the Query
sequence in the alignment shown above (gaps introduced in the sequence by the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Synovial hypoxia-RSF subtracted and to a lesser extent in Soares
infant brain
INIB, HL-60, PMA 4H, re-excision. Nine Week Old Early Stage Human, Human
Lung Cancer, Supt cells, cyclohexamide treated, subtracted, Human Adult
Retina,
Synovial hypoxia, Stratagene fetal spleen (#937205), Stratagene lung
(#937210),
Brain frontal cortex, Adipocytes, Human Microvascular Endothelial Cells,
fract. A,
Monocyte activated, Human Bone Marrow, treated, Human Testes, Stratagene
endothelial cell 937223.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
102 as residues: Pro-29 to Lys-40, Glu-66 to Phe-79, Arg-112 to Gly-121, Thr-
128 to
Lys-140, Lys-158 to Ser-168.
The expression of this gene product in synovium suggests a role in the
detection and treatment of disorders and conditions affecting the skeletal
system, in
particular osteoporosis as well as disorders afflicting connective tissues
(e.g. arthritis,
trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis
or
treatment of various autoimmune disorders such as rheumatoid arthritis, lupus,
scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and
specific joint abnormalities as well as chondrodysplasias (ie.
spondyloepiphyseal
dysplasia congenita, familial arthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type Schmid). 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 N0:53 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
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
ao
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 1947 of SEQ ID
N0:53, b
is an integer of 15 to 1961, 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 the following
tissues: Soares NhHMPu_S 1 and to a lesser extent in Human Brain, Human Adult
Liver, subtracted, Human Adult Spleen, Human Heart, Human Gall Bladder, Human
Adult Pulmonary;re-excision, Human Osteoclastoma.
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 1 to 1343 of SEQ ID
N0:54, b
is an integer of 15 to 1357, 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
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 45
It has been discovered that this gene is expressed primarily in the following
tissues: Soares retina N2b4HR, Soares_fetal heart NbHHI9W, Soares infant brain
1NIB and to a lesser extent in Human Adult Retina, NCI CGAP_LuS, Human
Stomach;re-excision, Human Infant Brain, Soares adult brain N2b5HB55Y, Human
Amygdala, Human Cerebellum.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:55 and may have been publicly available prior to
conception of
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
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
5 general formula of a-b, where a is any integer between 1 to 771 of SEQ ID
N0:55, b
is an integer of 15 to 785. where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:55, and where b is greater than or
equal to a
+ 14.
10 FEATURES OF PROTEIN ENCODED BY GENE NO: 46
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.
gi13115348 (all information available through the recited accession number is
15 incorporated herein by reference) which is described therein as
"mitochondria)
processing peptidase beat subunit precursor", which is thought to function as
a
peptidase which cleaves the leader sequence from mitochondria) proteins
following
translocation. A partial alignment demonstrating the observed homology is
shown
immediately below.
>gi~3115348 (AC004668) mitochondria) processing peptidase beta subunit
precursor; similar to Q03346 (PID:g1171010) [Homo Sapiens]
Length = 489
Plus Strand HSPs:
Score = 2357 (829.7 bits), Expect = 5.3e-244, P = 5.3e-244
Identities = 456/462 (980), Positives = 457/462 (980), Frame = +2
Query: 65 WIRGA--RRLWGFSESLLIRGAAGRSLYFGENRLRSTQAATQWLNVPETRVTCLESGL
238
W+ A RRLWGFSESLLIRGAAGRSLYFGENRLRSTQAATQWLNVPETRVTCLESGL
Sbjct: 8 WLSSAARRRLWGFSESLLIRGAAGRSLYFGENRLRSTQAATQWLNVPETRVTCLESGL
67
Query: 239 RVASEDSGLSTCTVGLWIDAGSRYENEKNNGTAHFLEHMAFKGTKKRSQLDLELEIENMG
418
RVASEDSGLSTCTVGLWIDAGSRYENEKNNGTAHFLEHMAFKGTKKRSQLDLELEIENMG
Sbjct: 68 RVASEDSGLSTCTVGLWIDAGSRYENEKNNGTAHFLEHMAFKGTKKRSQLDLELEIENMG
4
0 127
Query: 419 AHLNAYTSREQTWYAKAFSKDLPRAVEILADIIQNSTLGEAEIERERGVILREMQEVET
598
AHLNAYTSREQTWYAKAFSKDLPRAVEILADIIQNSTLGEAEIERERGVILREMQEVET
WO-00/55177 CA 02361277 2001-08-28
PCT/US00/06058
>1
Sbjct: 122 AHLNAYTSREQTVYYAKAFSKDLPRAVEILADIIQNSTLGEAEIERERGVILREMQEVET
187
Query: 199 NLQEWFDYLHATAYQNTALGRTILGPTENIKSISRKDLVDYITTHYKGPRIVLAAAGGV
778
NLQEWFDYLHATAYQNTALGRTILGPTENIKSISRKDLVDYITTHYKGPRIVLAAAGGV
Sbjct: i88 NLQEWFDYLHATAYQNTALGRTILGPTENIKSISRKDLVDYITTHYKGPRIVLAAAGGV
247
lO Query: 779 SHDELLDLAKFHFGDSLCTHKGEIPALPPCKFTGSEIRVRDDKMPLAHLAIAVEAVGWAH
958
SHDELLDLAKFHFGDSLCTHKGEIPALPPCKFTGSEIRVRDDKMPLAHLAIAVEAVGWAH
Sbjct: 248 SHDELLDLAKFHFGDSLCTHKGEIPALPPCKFTGSEIRVRDDKMPLAHLAIAVEAVGWAH
307
Query: 959 PDTICLMVANTLIGNWDRSFGGGMNLSSKLAQLTCHGNLCHSFQSFNTSYTDTGLWGLYM
1138
PDTICLMVANTLIGNWDRSFGGGMNLSSKLAQLTCHGNLCHSFQSFNTSYTDTGLWGLYM
Sbjct: 308 PDTICLMVANTLIGNWDRSFGGGMNLSSKLAQLTCHGNLCHSFQSFNTSYTDTGLWGLYM
ZO 367
Query: 1139 VCESSTVADMLHWQKEGTMRLCTSVTESEVARARNLLKTNMLLQLDGSTPICEDIGRQML
1318
VCESSTVADMLHWQKEWMRLCTSVTESEVARARNLLKTNMLLQLDGSTPICEDIGRQML
ZS Sbjct: 368 VCESSTVADMLHWQKEWMRLCTSVTESEVARARNLLKTNMLLQLDGSTPICEDIGRQML
427
Query: 1319 CYNRRIPIPELEARIDAVNAEXIREVCTKYIYNRSPAIAAVG 1444
CYNRRIPIPELEARIDAVNAE IREVCTKYIYNRSPAIAAVG
3O Sbjct: 428 CYNRRIPIPELEARIDAVNAETIREVCTKYIYNRSPAIAAVG 469
The segment of gi13115348 that is shown as "Sbjct" above is set out as
sequences) SEQ ID NO. 145. Based on the structural similarity these homologous
polypeptides are expected to share at least some biological activities. Such
activities
35 are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 146 which correspond to the Query
sequence in the alignment shown above (gaps introduced in the sequence by the
40 computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Primary Dendritic Cells, lib 1 and to a lesser extent in Human
Adrenal Gland
Tumor, Soares_pregnant uterus_NbHPU, Human Adult Small Intestine, Human T-
Cell Lymphoma, Human Testes Tumor, Keratinocyte, Nine Week Old Early Stage
45 Human, Namalwa Cells, Liver Normal MetSNo, Human Prostate, subtracted,
Human
Colon, subtraction, Human Thyroid, Human Soleus, Human adult small
intestine,re-
excision, Human Amygdala;re-excision, B Cell lymphoma, Human Synovium,
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
52
Stratagene NT2 neuronal precursor 937230, Human Adipose Tissue. re-excision,
Human Osteosarcoma, Human Osteoclastoma, re-excision, Human Manic Depression
Tissue, KMH2, Human Dermal Endothelial Cells,untreated, L428, Human Fetal Dura
Mater, T-Cell PHA 24 hrs, Merkel Cells, Strata~ene liver (#937224).
Soares_fetal lung_NbHLI9W, Soares multiple_sclerosis_2NbHMSP,
Soares_senescent fibroblasts_NbHSF, Resting T-Cell Library,II, 12 Week Old
Early
Stage Human, Smooth muscle, serum treated, Normal colon, Endothelial cells-
control, Human Osteoclastoma, Human Amygdala, Human Bone Marrow, treated,
Human Testes, Soares NhHMPu_S 1, T cell helper II, Human Endometrial Tumor,
Hodgkin's Lymphoma II, Soares infant brain 1NIB.
The homology to mitochondria) processing peptidase beta subunit protein
suggests that the translation product of this gene is useful for the detection
and/or
treatment of disorders involving the post-translational processing of
proteins, and/or
disorders involving aberrant transport of mitochondria) proteins. Furthermore,
the
tissue distribution in primary dendritic cells suggests that the protein
product of this
clone is useful for the diagnosis and treatment of a variety of immune system
disorders. Expression of this gene product in tonsils suggests a role in the
regulation
of the proliferation; survival; differentiation; and/or activation of
potentially all
hematopoietic cell lineages, including blood stem cells. This gene product may
be
involved in the regulation of cytokine production, antigen presentation, or
other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the gene or protein, as well as, antibodies directed against the protein may
show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
Therefore it may be also used as an agent for immunological disorders
including
arthritis, asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid
arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In
addition, this
gene product may have commercial utility in the expansion of stem cells and
committed progenitors of various blood lineages, and in the differentiation
and/or
proliferation of various cell types. 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.
WO-00/55177 CA 02361277 2001-08-28
PCT/US00/06058
53
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 1 to 2426 of SEQ ID
N0:56, b
is an integer of 15 to 2440, 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: Human Adult Spleen, Human placenta cDNA (TFujiwara), Resting T-Cell;
re-excision, Macrophage-oxLDL.
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
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 796 of SEQ ID
N0:57, b
is an integer of 15 to 810, 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
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.
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
5-1
S
gnIIPIDIe206S44 (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "pval
[Plasmodium
vivax]." A partial alignment demonstrating the observed homology is shown
immediately below.
>gnl~PID~e206544 peal [Plasmodium vivax] >sp~Q26195~Q26195 PVA1 GENE.
Length = 200
Minus Strand HSPs:
Score = 139 (48.9 bits), Expect = 6.8e-09, P = 6.8e-09
Identities = 28/53 (52%), Positives = 31/53 (58%), Frame = -1
Query: 484 FFFFFFRDGVLICCPGWS*TPSS----------KRSSCFGIPKCWDYRHEPPH 356
IS FF FFFRD VL+C PGWS S K+SSC G+P WDYR PPH
Sbjct: 107 FFIFFFRDRVLLCHPGWSAWQSLFTVASTFLVKQSSCLGLPSSWDYRRIPPH 159
The segment of gnlIPIDIe206S44 that is shown as "Sbjct" above is set out as
sequenee(s) SEQ ID NO. 147. Based on the structural similarity these
homologous
polypeptides are expected to share at least some biological activities. Such
activities
are known in the art and 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 polypeptides having
the
amino acid sequences) set out as SEQ ID NO. 148 which correspond to the Query
2S sequence in the alignment shown above (gaps introduced in the sequence by
the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues: Human Amygdala.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
107 as residues: Ser-33 to Leu-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 NO:S8 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
3S 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 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
JJ
general formula of a-b, where a is any integer between I to 470 of SEQ ID
N0:58, b
is an integer of IS to 484, 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 GENE NO: 49
It has been discovered that this gene is expressed primarily in the following
tissues: Activated Monocytes and to a lesser extent in Human Normal
Cartilage,Fraction I, Human Normal Cartilage Fraction II, Human Brain,
Striatum,
Stratagene colon (#937204), Monocyte activated; re-excision, Human
Hippocampus,
Hepatocellular Tumor; re-excision, Colon Tumor, Human Substantia Nigra, Colon
Normal II, NCI CLAP Co3, NCI_CGAP_Kid3, NCI CLAP PrlO, Colon Tumor II,
Stratagene colon (#937204), Soares fetal liver spleen 1NFLS.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
108 as residues: Ala-17 to Gln-23.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:59 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 417 of SEQ ID
N0:59, b
is an integer of 15 to 431, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:59, and where b is greater than or
equal to a
+ 14.
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
56
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WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
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CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
58
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WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
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;,... ~ ~n o ~n a,
z
L -o
~, -- o ~ a
~ -- ~, a\
n
a~
U C/~ M f~ N oo d. d'
c
h
C
4-. M
~'
O ~Y
O ~
U v~
~
. z v~ M t~ N oo
E
zapx
x
x x x x
~ 0., a, a~ a. Q
0
v Q Q Q Q d o
j N N N N N
U ~ N ~ ~ ~ ~ ~ ~ ~
~ ~ ~ a ov a,
o C~ ov o~ o~ ~ ov o,
~ av ~ ov ~ ov
E-~ 00" O M M M M M M
,~ O\O 0\O O\O O\O 0\O O\O
z N O N O O O
~ ~h '~ ~ '
~ N ~t ~f
~
Q x a z ~ ~ a
z
n
o
_ x
x ~ x x
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61
Table 1 summarizes the information corresponding to each "Gene No." described
above. The nucleotide sequence identified as "NT SEQ ID NO:X" was assembled
from partially homologous ("overlapping") sequences obtained from the "cDNA
clone ID" identified in Table 1 and, in some cases, from additional related
DNA
clones. The overlapping sequences were assembled into a single contiguous
sequence
of high redundancy (usually three to five overlapping sequences at each
nucleotide
position), resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding
deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits
contain multiple different clones corresponding to the same gene. "Vector"
refers to
the type of vector contained in the cDNA Clone ID.
"Total NT Seq." refers to the total number of nucleotides in the contig
identified by "Gene No." The deposited clone may contain all or most of these
sequences, reflected by the nucleotide position indicated as "5' NT of Clone
Seq."
and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ
ID
NO:X of the putative start codon (methionine) is identified as "5' NT of Start
Codon."
Similarly , the nucleotide position of SEQ ID NO:X of the predicted signal
sequence
is identified as "5' NT of First AA of Signal Pep."
The translated amino acid sequence, beginning with the methionine, is
identified as "AA SEQ ID NO:Y," although other reading frames can also be
easily
translated using known molecular biology techniques. The polypeptides produced
by
these alternative open reading frames are specifically contemplated by the
present
invention.
The first and last amino acid position of SEQ 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
any of the polypeptide sequences disclosed in the sequence listing) are
sufficiently
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accurate and otherwise suitable for a variety of uses well known in the art
and
described further below. For instance. SEQ ID NO:X is useful for designing
nucleic
acid hybridization probes that will detect nucleic acid sequences contained in
SEQ ID
NO:X or the cDNA contained in the deposited clone. These probes will also
hybridize to nucleic acid molecules in biological samples, thereby enabling a
variety
of forensic and diagnostic methods of the invention. Similarly, polypeptides
identified from SEQ ID NO:Y may be used, for example, to generate antibodies
which bind specifically to proteins containing the polypeptides and the
secreted
proteins encoded by the cDNA clones identified in Table 1.
Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino
acid sequence. In these cases, the predicted amino acid sequence diverges from
the
actual amino acid sequence, even though the generated DNA sequence may be
greater
than 99.9% identical to the actual DNA sequence (for example, one base
insertion or
deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ ID NO:X and the predicted
translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of
plasmid DNA containing a human cDNA of the invention deposited with the ATCC,
as set forth in Table 1. The nucleotide sequence of each deposited clone can
readily
be determined by sequencing the deposited clone in accordance with known
methods.
The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can
also be directly determined by peptide sequencing or by expressing the protein
in a
suitable host cell containing the deposited human cDNA, collecting the
protein, and
determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can be
isolated in accordance with known methods using the sequence information
disclosed
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63
herein. Such methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from appropriate
sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or
species homologs. Procedures known in the art can be used to obtain full-
length
genes, allelic variants, splice variants, full-length coding portions,
orthologs, and/or
species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a
deposited clone, using information from the sequences disclosed herein or the
clones
deposited with the ATCC. For example, allelic variants and/or species homologs
may
be isolated and identified by making suitable probes or primers from the
sequences
provided herein and screening a suitable nucleic acid source for allelic
variants and/or
the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner.
Such polypeptides include isolated naturally occurring polypeptides,
recombinantly
produced polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means for preparing such
polypeptides
are well understood in the art.
The polypeptides may be in the form of the secreted protein, including the
mature form, or may be a part of a larger protein, such as a fusion protein
(see below).
It is often advantageous to include an additional amino acid sequence which
contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification ,
such as multiple histidine residues, or an additional sequence for stability
during
recombinant production.
The polypeptides of the present invention are preferably provided in an
isolated form, and preferably are substantially purified. A recombinantly
produced
version of a polypeptide, including the secreted polypeptide, can be
substantially
purified using techniques described herein or otherwise known in the art, such
as, for
example, by the one-step method described in Smith and Johnson, Gene 67:31-40
(1988). Polypeptides of the invention also can be purified from natural,
synthetic or
recombinant sources using techniques described herein or otherwise known in
the art,
such as, for example, antibodies of the invention raised against the secreted
protein.
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The present invention provides a polynucleotide comprising. or alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA
contained
in ATCC deposit Z. The present invention also provides a polypeptide
comprising, or
alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a
polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides
encoding a polypeptide comprising. or alternatively consisting of the
polypeptide
sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA
contained in ATCC deposit Z are also encompassed by the invention.
SignalSeauences
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)
protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses
the
information from the residues surrounding the cleavage site, typically
residues -13 to
+2, where +1 indicates the amino terminus of the secreted protein. The
accuracy of
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predicting the cleavage points of known mammalian secretory proteins for each
of
these methods is in the range of 75-80%. (von Heinje, supra.) However, the two
methods do not always produce the same predicted cleavage points) for a given
protein.
In the present case, the deduced amino acid sequence of the secreted
polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen
et
al., Protein Engineering 10:1-6 ( 1997)), which predicts the cellular location
of a
protein based on the amino acid sequence. As part of this computational
prediction of
localization, the methods of McGeoch and von Heinje are incorporated. The
analysis
10 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
15 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
20 invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally occurring signal sequence may be further upstream from the predicted
signal
sequence. However, it is likely that the predicted signal sequence will be
capable of
25 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 polynucleotide sequence contained in the cDNA of a deposited
clone, in a mammalian cell (e.g., COS cells, as desribed below). These
polypeptides,
and the polynucleotides encoding such polypeptides, are contemplated by the
present
30 invention.
Polynucleotide and Poly~eptide Variants
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66
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:Y and/or encoded by a deposited clone.
"Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential
properties thereof. Generally, variants are overall closely similar, and, in
many
regions, identical to the polynucleotide or polypeptide of the present
invention.
The present invention is also directed to nucleic acid molecules which
comprise, or alternatively consist of, a nucleotide sequence which is at least
80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide
coding sequence in SEQ ID NO:X or the complementary strand thereto, the
nucleotide coding sequence contained in a deposited cDNA clone or the
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% 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).
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
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67
other words, to obtain a nucleic acid having a nucleotide sequence at least
95%
identical to a reference nucleotide sequence. up to 5% of the nucleotides in
the
reference sequence may be deleted or substituted with another nucleotide, or a
number of nucleotides up to 5% of the total nucleotides in the reference
sequence may
be inserted into the reference sequence. The query sequence may be an entire
sequence shown inTable 1, the ORF (open reading frame), or any fragment
specified
as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
a
nucleotide sequence of the presence invention can be determined conventionally
using known computer programs. A preferred method for determining the best
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 both DNA sequences. An RNA sequence can be compared by
converting U's to T's. The result of said global sequence alignment is in
percent
identity. Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap
Penalty=5, Gap Size Penalty 0.05. Window Size=500 or the lenght of the subject
nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the FASTDB program does not account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
sequences truncated at the 5' or 3' ends, relative to the query sequence, the
percent
identity is corrected by calculating the number of bases of the query sequence
that are
5' and 3' of the subject sequence, which are not matched/aligned, as a percent
of the
total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
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6~
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent identity
score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
subject sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the 5' or 3' of
the subject
sequence which are not matched/aligned with the query. In this case the
percent
identity calculated by FASTDB is not manually corrected. Once again, only
bases 5'
and 3' of the subject sequence which are not matched/aligned with the query
sequence
are manually corrected for. No other manual corrections are to made for the
purposes
of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other
words, to obtain a polypeptide having an amino acid sequence at least 95%
identical
to a query amino acid sequence, up to 5% of the amino acid residues in the
subject
sequence may be inserted, deleted, (indels) or substituted with another amino
acid.
These alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those
terminal positions, interspersed either individually among residues in the
reference
sequence or m one or more contiguous groups within the reference sequence.
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69
As a practical matter, whether any particular polypeptide is at least 80%,
85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid
sequences shown in Table 1 (SEQ ID NO:Y) or to the amino acid sequence encoded
by cDNA contained in a deposited clone can be determined conventionally using
known computer programs. A preferred method for determing the best overall
match
between a query sequence (a sequence of the present invention) and a subject
sequence, also referred to as a global sequence alignment, can be determined
using
the FASTDB computer program based on the algorithm of Brutlag et al. (Comp.
App.
Biosci. 6:237-245(1990)). In a sequence alignment the query and subject
sequences
are either both nucleotide sequences or both amino acid sequences. The result
of said
global sequence alignment is in percent identity. Preferred parameters used in
a
FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l,
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/aligned is
determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from
the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score
is what is used for the purposes of the present invention. Only residues to
the N- and
C-termini of the subject sequence, which are not matched/aligned with the
query
sequence, are considered for the purposes of manually adjusting the percent
identity
score. That is, only query residue positions outside the farthest N- and C-
terminal
residues of the subject sequence.
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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
5 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
10 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
15 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
20 the properties or activities of the encoded polypeptide. Nucleotide
variants produced
by silent substitutions due to the degeneracy of the genetic code are
preferred.
Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted,
deleted, or
added in any combination are also preferred. Polynucleotide variants can be
produced
for a variety of reasons, e.g., to optimize codon expression for a particular
host
25 (change codons in the human mRNA to those preferred by a bacterial host
such as E.
coli).
Naturally occurring variants are called "allelic variants," and refer to one
of
several alternate forms of a gene occupying a given locus on a chromosome of
an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).)
These
30 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.
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71
Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics
of the
polypeptides of the present invention. For instance, one or more amino acids
can be
deleted from the N-terminus or C-terminus of the secreted protein without
substantial
loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988
(1993), reported variant KGF proteins having heparin binding activity even
after
deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma
exhibited up to ten times higher activity after deleting 8-10 amino acid
residues from
the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-
216
( 1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity similar to that of the naturally occurring protein. For example,
Gayle and
coworkers (J. Biol. Chem 268:22105-22111 ( 1993)) conducted extensive
mutational
analysis of human cytokine IL-la. They used random mutagenesis to generate
over
3,500 individual IL-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible amino acid position. The investigators found that "[m]ost of the
molecule
could be altered with little effect on either [binding or biological
activity]." (See,
Abstract.) In fact, only 23 unique amino acid sequences, out of more than
3,500
nucleotide sequences examined, produced a protein that significantly differed
in
activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or
C-terminus of a polypeptide results in modification or loss of one or more
biological
functions, other biological activities may still be retained. For example, the
ability of
a deletion variant to induce and/or to bind antibodies which recognize the
secreted
form will likely be retained when less than the majority of the residues of
the secreted
form are removed from the N-terminus or C-terminus. Whether a particular
polypeptide lacking N- or C-terminal residues of a protein retains such
immunogenic
activities can readily be determined by routine methods described herein and
otherwise known in the art.
Thus, the invention further includes polypeptide variants which show
substantial biological activity. Such variants include deletions, insertions,
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72
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 al.,
Science
247:1306-1310 (1990), wherein the authors indicate that there are two main
strategies
for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
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 244:1081-1085 (1989).) The resulting mutant
molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary~structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and
Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the
acidic
residues Asp and Glu; replacement of the amide residues Asn and Gln,
replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic residues
Phe, Tyr,
and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,
and Gly.
Besides conservative amino acid substitution, variants of the present
invention
include (i) substitutions with one or more of the non-conserved amino acid
residues,
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73
where the substituted amino acid residues may or may not be one encoded by the
genetic code, or (ii) substitution with one or more of amino acid residues
having a
substituent group, or (iii) fusion of the mature polypeptide with another
compound,
such as a compound to increase the stability and/or solubility of the
polypeptide (for
example, polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or leader or
secretory
sequence, or a sequence facilitating purification. 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); Robbins 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
amino acid substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and
still even more preferably, not more than 20 amino acid substitutions. Of
course, in
order of ever-increasing preference, it is highly preferable for a peptide or
polypeptide
to have an amino acid sequence which comprises the amino acid sequence of the
present invention, which contains at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3,
2 or 1 amino acid substitutions. In specific embodiments, the number of
additions,
substitutions, and/or deletions in the amino acid sequence of the present
invention or
fragments thereof (e.g., the mature form and/or other fragments described
herein), is
1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions
are
preferable.
Polynucleotide and Polypentide Fragments
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The present invention is also directed to polynucleotide fragments of the
polynucleotides of the invention.
In the present invention, a "polynucleotide fragment" refers to a short
polynucleotide having a nucleic acid sequence which: is a portion of that
contained in
a deposited clone, or encoding the polypeptide encoded by the cDNA in a
deposited
clone; is a portion of that shown in SEQ ID NO:X or the complementary strand
thereto, or is a portion of a polynucleotide sequence encoding the polypeptide
of SEQ
ID NO:Y. The nucleotide fragments of the invention are preferably at least
about 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
example, is intended to include 20 or more contiguous bases from the cDNA
sequence contained in a deposited clone or the nucleotide sequence shown in
SEQ ID
NO:X. In this context "about" includes the particularly recited value, a value
larger
or smaller by several (5, 4, 3, 2, or 1 ) nucleotides, at either terminus or
at both
termini. These nucleotide fragments have uses that include, but are not
limited to, as
diagnostic probes and primers as discussed herein. Of course, larger fragments
(e.g.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the
invention, include, for example, fragments comprising, or alternatively
consisting of,
a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-
250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-
750,
751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,
1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,
1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,
1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the
complementary strand thereto, or the cDNA contained in a deposited clone. In
this
context "about" includes the particularly recited ranges, and ranges larger or
smaller
by several (5, 4, 3, 2, or 1 ) nucleotides, at either terminus or at both
termini.
Preferably, these fragments encode a polypeptide which has biological
activity. More
preferably, these polynucleotides can be used as probes or primers as
discussed
herein. Polynucleotides which hybridize to these nucleic acid molecules under
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stringent hybridization conditions or lower stringency conditions are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
In the present invention, a "polypeptide fragment" refers to an amino acid
sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the
5 cDNA contained in a deposited clone. Protein (polypeptide) fragments may be
"free-
standing," or comprised within a larger polypeptide of which the fragment
forms a
part or region, most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, include, for example,
fragments
comprising, or alternatively consisting of, from about amino acid number 1-20,
21-40,
10 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 301 to the end of the
coding
region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70,
80, 90,
100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about"
includes the particularly recited ranges or values, and ranges or values
larger or
smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at
both extremes.
15 Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Preferred polypeptide fragments include the secreted protein as well as the
mature form. Further preferred polypeptide fragments include the secreted
protein or
the mature form having a continuous series of deleted residues from the amino
or the
carboxy terminus, or both. For example, any number of amino acids, ranging
from 1-
20 60, can be deleted from the amino terminus of either the secreted
polypeptide or the
mature form. Similarly, any number of amino acids, ranging from 1-30, can be
deleted from the carboxy terminus of the secreted protein or mature form.
Furthermore, any combination of the above amino and carboxy terminus deletions
are
preferred. Similarly, polynucleotides encoding these polypeptide fragments are
also
25 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
30 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
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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 [ability to bind (or compete
with a
polypeptide of the invention for binding) to an antibody to the polypeptide of
the
invention], immunogenicity (ability to generate antibody which binds to a
polypeptide
of the invention), ability to form multimers with polypeptides of the
invention, and
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
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77
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. 59:94-123. In anther
embodiment, physiological correlates of binding of a polypeptide of the
invention to
its substrates (signal transduction) can be assayed.
In addition, assays described herein (see Examples) and otherwise known in
the art may routinely be applied to measure the ability of polypeptides of the
invention and fragments, variants derivatives and analogs thereof to elicit
related
biological activity related to that of the polypeptide of the invention
(either in vitro or
in vivo). Other methods will be known to the skilled artisan and are within
the scope
of the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ ID
NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:X or contained in
ATCC deposit No. Z under stringent hybridization conditions or lower
stringency
hybridization conditions as defined supra. The present invention further
encompasses
polynucleotide sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID NO:X),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence
encoding an epitope of the invention, and polynucleotide sequences which
hybridize
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78
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
81:3998- 4002 ( 1983)). The term "antigenic epitope," as used herein, is
defined as a
portion of a protein to which an antibody can immunospecifically bind its
antigen as
determined by any method well known in the art, for example, by the
immunoassays
described herein. Immunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985),
further described in U.S. Patent No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 20,
at least 25, at
least 30, at least 40, at least 50, and, most preferably, between about 15 to
about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Additional non-exclusive preferred antigenic
epitopes
include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. Preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as any combination of two, three, four,
five or more
of these antigenic epitopes. Antigenic epitopes can be used as the target
molecules in
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79
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
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 (KL,H) or
tetanus
toxoid. For instance, peptides containing cysteine residues may be coupled to
a
carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester
(MBS),
while other peptides may be coupled to carriers using a more general linking
agent
such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized
with
either free or carrier- coupled peptides, for instance, by intraperitoneal
and/or
intradermal injection of emulsions containing about 100 pg of peptide or
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
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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.
5 As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention comprising an immunogenic or antigenic
epitope can be fused to other polypeptide sequences. For example, the
polypeptides
of the present invention may be fused with the constant domain of
immunoglobulins
(IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
10 thereof and portions thereof) resulting in chimeric polypeptides. Such
fusion proteins
may facilitate purification and may increase half-life in vivo. This has been
shown
for chimeric proteins consisting of the first two domains of the human CD4-
polypeptide and various domains of the constant regions of the heavy or light
chains
of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al.,
Nature,
15 331:84-86 ( 1988). Enhanced delivery of an antigen across the epithelial
barrier to the
immune system has been demonstrated for antigens (e.g., insulin) conjugated to
an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications
WO
96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG portion desulfide bonds have also been found
to be
20 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
25 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
30 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+
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81
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, andlor 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 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-
33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
( 1998) (each of these patents and publications are hereby incorporated by
reference in
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.
Antibodies
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,
polyclonal, monoclonal, multispecific, human, humanized or chimeric
antibodies,
single chain antibodies, Fab fragments, F(ab') fragments, fragments produced
by a
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82
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., IgGI, IgG2, IgG3, IgG4, IgAl 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
fragments comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the variable
regions)
alone or in combination with the entirety or a portion of the following: hinge
region,
CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CH1, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human immunoglobulin and include antibodies isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
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83
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,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least
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%, 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, or more of the
specific
antigenic and/or immunogenic polypeptides disclosed herein. Further included
in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides
which hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the present
invention
may also be described or specified in terms of their binding affinity to a
polypeptide
of the invention. Preferred binding affinities include those with a
dissociation
constant or Kd less than S X 10-Z M, 10-2 M, 5 X 10-~ M, 10-~ M, 5 X 10-'' M,
104 M, 5
X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 10' M, 5 X 10-8 M, 10-g M, 5
X 10-~
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84
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-" M, 5 X 10-'4 M, 10-'4 M, 5 X 10-'S M, or 10-'5 M.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art
for determining competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively inhibits binding
to the
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%.
Antibodies of the present invention may act as agonists or antagonists of the
polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. Preferrably, antibodies of the
present~invention
bind an antigenic epitope disclosed herein, or a portion thereof. The
invention
features both receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor-specific antibodies which do not prevent
ligand
binding but prevent receptor activation. Receptor activation (i.e., signaling)
may be
determined by techniques described herein or otherwise known in the art. For
example, receptor activation can be determined by detecting the
phosphorylation
(e.g., tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example, as
described
supra). In specific embodiments, antibodies are provided that inhibit ligand
activity
or receptor activity by at least 95%, at least 90%, at least 85%, at least
80%, at least
75%, at least 70%, at least 60%, or at least 50% of the activity in absence of
the
antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
well as antibodies which bind the ligand, thereby preventing receptor
activation, but
do not prevent the ligand from binding the receptor. Further included in the
invention
are antibodies which activate the receptor. These antibodies may act as
receptor
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
agonists, i.e., potentiate or activate either all or a subset of the
biological activities of
the ligand-mediated receptor activation, for example, by inducing dimerization
of the
receptor. The antibodies may be specified as agonists, antagonists or inverse
agonists
for biological activities comprising the specific biological activities of the
peptides of
5 the invention disclosed herein. The above antibody agonists can be made
using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu
et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-
10 3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et
al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et
al.,
Neuron 14(4):755-762 ( 1995); Muller et al., Structure 6(9):1153-1167 ( 1998);
Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference
15 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
20 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
25 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,
30 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.
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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
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
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"monoclonal antibody" refers to an antibody that is derived from a single
clone,
including any eukaryotic, prokaryotic, or phage clone, and not the method by
which it
is produced.
Methods for producing and screening for specific antibodies using hybridoma
technology are routine and well known in the art and are discussed in detail
in the
Examples (e.g., Example 16). In a non-limiting example, mice can be immunized
with a polypeptide of the invention or a cell expressing such peptide. Once an
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
CH 1 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
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phage can be utilized to display antigen binding domains expressed from a
repertoire
or combinatorial antibody library (e.g., human or murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or
identified
with antigen, e.g., using labeled antigen or antigen bound or captured to a
solid
surface or bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab, Fv or
disulfide stabilized Fv antibody domains recombinantly fused to either the
phage
gene III or gene VIII protein. Examples of phage display methods that can be
used to
make the antibodies of the present invention include those disclosed in
Brinkman et
al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 ( 1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (
1994); Persic
et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;
5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI
34:26-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston
et al., Methods in Enzymology 203:46-88 ( 1991 ); Shu et al., PNAS 90:7995-
7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including
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in vivo use of antibodies in humans and in vitro detection assays, it may be
preferable
to use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule
in which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entirety. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known in the art, e.g., by modeling of the interactions of the CDR and
framework
residues to identify framework residues important for antigen binding and
sequence
comparison to identify unusual framework residues at particular positions.
(See, e.g.,
Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (
1988),
which are incorporated herein by reference in their entireties.) Antibodies
can be
humanized using a variety of techniques known in the art including, for
example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al.,
Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973
(1994)),
and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
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98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
5 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.
10 The mouse heavy and light chain immunoglobulin genes may be rendered non-
functional separately or simultaneously with the introduction of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of the JH region prevents endogenous antibody production. The
modified
embryonic stem cells are expanded and microinjected into blastocysts to
produce
15 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
20 immunoglobulin transgenes harbored by the transgenic mice rearrange during
B cell
differentiation, and subsequently undergo class switching and somatic
mutation.
Thus, using such a technique, it is possible to produce therapeutically useful
IgG, IgA,
IgM and IgE antibodies. For an overview of this technology for producing human
antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 ( 1995). For a
25 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
30 incorporated by reference herein in their entirety. In addition, companies
such as
Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to
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provide human antibodies directed against a selected antigen using technology
similar
to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Biotechnology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized
to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using
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.
Polvnucleotides 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 polypeptide 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
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92
in Kutmeier et al., BioTechniques 17:242 ( 1994)), which, briefly, involves
the
synthesis of overlapping oligonucleotides containing portions of the sequence
encoding the antibody, annealing and ligating of those oligonucleotides, and
then
amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from
nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a
particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoglobulin may be chemically
synthesized
or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA
library
generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any
tissue
or cells expressing the antibody, such as hybridoma cells selected to express
an
antibody of the invention) by PCR amplification using synthetic primers
hybridizable
to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide
probe
specific for the particular gene sequence to identify, e.g., a cDNA clone from
a
cDNA library that encodes the antibody. Amplified nucleic acids generated by
PCR
may then be cloned into replicable cloning vectors using any method well known
in
the art.
Once the nucleotide sequence and corresponding amino acid sequence of the
antibody is determined, the nucleotide sequence of the antibody may be
manipulated
using methods well known in the art for the manipulation of nucleotide
sequences,
e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see,
for
example, the techniques described in Sambrook et al., 1990, Molecular Cloning,
A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John
Wiley & Sons, NY, which are both incorporated by reference herein in their
entireties ), to generate antibodies having a different amino acid sequence,
for
example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light
chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the
art, e.g., by comparison to known amino acid sequences of other heavy and
light
chain variable regions to determine the regions of sequence hypervariability.
Using
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93
routine recombinant DNA techniques, one or more of the CDRs may be inserted
within framework regions, e.g., into human framework regions to humanize a non-
human antibody. as described supra. The framework regions may be naturally
occurring or consensus framework regions, and preferably human framework
regions
(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of
human
framework regions). Preferably, the polynucleotide generated by the
combination of
the framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the framework regions, and, preferably, the
amino
acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
alterations to the polynucleotide are encompassed by the present invention and
within
the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 ( 1984); Neuberger et al.,
Nature
312:604-608 ( 1984); Takeda et al., Nature 314:452-454 ( 1985)) by splicing
genes
from a mouse antibody molecule of appropriate antigen specificity together
with
genes from a human antibody molecule of appropriate biological activity can be
used.
As described supra, a chimeric antibody is a molecule in which different
portions are
derived from different animal species, such as those having a variable region
derived
from a murine mAb and a human immunoglobulin constant region, e.g., humanized
antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et
al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature
334:544-54 (1989)) can be adapted to produce single chain antibodies. Single
chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al.,
Science 242:1038- 1041 ( 1988)).
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9-1
Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the
art for the synthesis of antibodies, in particular, by chemical synthesis or
preferably,
by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, (e.g., a heavy or light chain of an antibody of
the
invention or a single chain antibody of the invention), requires construction
of an
expression vector containing a polynucleotide that encodes the antibody. Once
a
polynucleotide encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or light chain
variable
domain), of the invention has been obtained, the vector for the production of
the
antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding nucleotide
sequence are
described herein. Methods which are well known to those skilled in the art can
be
used to construct expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals. These methods
include,
for example, in vitro recombinant DNA techniques, synthetic techniques, and in
vivo
genetic recombination. The invention, thus, provides replicable vectors
comprising a
nucleotide sequence encoding an antibody molecule of the invention, or a heavy
or
light chain thereof, or a heavy or light chain variable domain, operably
linked to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant
region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of
the antibody may be cloned into such a vector for expression of the entire
heavy or
light chain.
The expression vector is transferred to a host cell by conventional techniques
and the transfected cells are then cultured by conventional techniques to
produce an
antibody of the invention. Thus, the invention includes host cells containing
a
polynucleotide encoding an antibody of the invention, or a heavy or light
chain
thereof, or a single chain antibody of the invention, operably linked to a
heterologous
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JS
promoter. In preferred embodiments for the expression of double-chained
antibodies,
vectors encoding both the heavy and light chains may be co-expressed in the
host cell
for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced and
subsequently
purified, but also represent cells which may, when transformed or transfected
with
the appropriate nucleotide coding sequences, express an antibody molecule of
the
invention in situ. These include but are not limited to microorganisms such as
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)).
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
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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 fratgiperda 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
transcriptionltranslation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region
El or E3) will result in a recombinant virus that is viable and capable of
expressing
the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad.
Sci. USA 81:355-359 ( 1984)). Specific initiation signals may also be required
for
efficient translation of inserted antibody coding sequences. These signals
include the
ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding sequence to
ensure
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translation of the entire insert. These exogenous translational control
signals and
initiation codons can be of a variety of origins, both natural and synthetic.
The
efficiency of expression may be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc. (see Bittner
et al.,
Methods in Enzymol. 153:51-544 ( 1987)).
In addition, a host cell strain may be chosen which modulates the expression
of the inserted sequences, or modifies and processes the gene product in the
specific
fashion desired. Such modifications (e.g., glycosylation) and processing
(e.g.,
cleavage) of protein products may be important for the function of the
protein.
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 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
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Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the
herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc.
Natl.
Acad. Sci. USA 48:202 ( 1992)), and adenine phosphoribosyltransferase (Lowy et
al.,
Cell 22:817 ( 1980)) genes can be employed in tk-, hgprt- or aprt- cells,
respectively.
Also, antimetabolite resistance can be used as the basis of selection for the
following
genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl.
Acad. Sci.
USA 77:357 ( 1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 ( 1981
)); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad.
Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside
6-
418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 ( 1993); Mulligan,
Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, 1993, TIB TECH 11(Sj:155-215); and hygro, which confers
resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in
the art of recombinant DNA technology may be routinely applied to select the
desired
recombinant clone, and such methods are described, for example, in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expression. A Laboratory Manual, Stockton Press,
NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.
Biol. 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
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
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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
acids of the polypeptide) of the present invention. For example, antibodies
may be
used to target the polypeptides of the present invention to particular cell
types, either
in vitro or in vivo, by fusing or conjugating the polypeptides of the present
invention
to antibodies specific for particular cell surface receptors. Antibodies fused
or
conjugated to the polypeptides of the present invention may also be used in in
vitro
immunoassays and purification methods using methods known in the art. See
e.g.,
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Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095: Naramura et
al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al.,
PNAS
89:1428-1432 ( 1992); Fell et al., J. Immunol. 146:2446-2452( 1991 ), which
are
incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example. the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any
combination of whole domains or portions thereof. The polypeptides may also be
fused or conjugated to the above antibody portions to form multimers. For
example,
Fc portions fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher multimeric forms can
be
made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing
or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad. Sci.
USA 89:11337- 11341 ( 1992) (said references incorporated by reference in
their
entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated
to
the above antibody portions to increase the in vivo half life of the
polypeptides or for
use in immunoassays using methods known in the art. Further, the polypeptides
corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody
portions to facilitate purification. One reported example describes chimeric
proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The
polypeptides of the present invention fused or conjugated to an antibody
having
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disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964
(1995)). In
many cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and
thus can result in, for example, improved pharmacokinetic properties. (EP A
232,262). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition
8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can be
fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311), among others, many of which are commercially available. As described
in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for convenient purification of the fusion protein. Other
peptide tags
useful for purification include, but are not limited to, the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson
et al., Cell 37:767 ( 1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given
treatment regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include various
enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, radioactive materials, positron emitting metals using various
positron
emission tomographies, and nonradioactive paramagnetic metal ions. The
detectable
substance may be coupled or conjugated either directly to the antibody (or
fragment
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thereof) or indirectly, through an intermediate (such as, for example, a
linker known
in the art) using techniques known in the art. See, for example, U.S. Patent
No.
4,741,900 for metal ions which can be conjugated to antibodies for use as
diagnostics
according to the present invention. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin
and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material
includes luminol; examples of bioluminescent materials include luciferase,
luciferin,
and aequorin; and examples of suitable radioactive material include 125I,
131I, 11 lIn
or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or
a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells. Examples
include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to
classical chemical therapeutic agents. For example, the drug moiety may be a
protein
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or polypeptide possessing a desired biological activity. Such proteins may
include,
for example, a toxin such as abrin, ricin A, pseudomonas exotoxin. or
diphtheria
toxin; a protein such as tumor necrosis factor, a-interferon, 13-interferon,
nerve growth
factor, platelet derived growth factor, tissue plasminogen activator, an
apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899), AIM II (See, International Publication No. WO 97/34911 ), Fas
Ligand
(Takahashi etal., IfZt. Irmnunol., 6:1567-1574 (1994)), VEGI (See,
International
Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent,
e.g.,
angiostatin or endostatin; or, biological response modifiers such as, for
example,
lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6
("IL-6"),
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
In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 ( 1985); "Analysis,
Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev. 62:119-58 ( 1982).
Alternatively, an antibody can be conjugated to a second antibody to form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980,
which
is incorporated herein by reference in its entirety.
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An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Immunophenotyping
The antibodies of the invention may be utilized for immunophenotyping of
cell lines and biological samples. The translation product of the gene of the
present
invention may be useful as a cell specific marker, or more specifically as a
cellular
marker that is differentially expressed at various stages of differentiation
and/or
maturation of particular cell types. Monoclonal antibodies directed against a
specific
epitope, or combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be utilized using
monoclonal antibodies to screen for cellular populations expressing the
marker(s), and
include magnetic separation using antibody-coated magnetic beads, "panning"
with
antibody attached to a solid matrix (i.e., plate), and flow cytometry (See,
e.g., U.S.
Patent 5,985,660; and Morrison et al., Cell, 96:737-49 ( 1999)).
These techniques allow for the screening of particular populations of cells,
such as might be found with hematological malignancies (i.e. minimal residual
disease (MRD) in acute leukemic patients) and "non-self" cells in
transplantations to
prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for
the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood.
Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding
by any method known in the art. The immunoassays which can be used include but
are not limited to competitive and non-competitive assay systems using
techniques
such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions,
gel diffusion precipitin reactions, immunodiffusion assays, agglutination
assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
protein A immunoassays, to name but a few. Such assays are routine and well
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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 ( 1 % NP-40 or Triton X- 100, 1 % sodium
deoxycholate, 0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1 %
Trasylol) supplemented with protein phosphatase and/or protease inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C,
adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an
hour or
more at 4° C, washing the beads in lysis buffer and resuspending the
beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a
particular antigen can be assessed by, e.g., western blot analysis. One of
skill in the
art would be knowledgeable as to the parameters that can be modified to
increase the
binding of the antibody to an antigen and decrease the background (e.g., pre-
clearing
the cell lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-
fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking
the membrane with primary antibody (the antibody of interest) diluted in
blocking
buffer, washing the membrane in washing buffer, blocking the membrane with a
secondary antibody (which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase
or
alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in
blocking
buffer, washing the membrane in wash buffer, and detecting the presence of the
antigen. One of skill in the art would be knowledgeable as to the parameters
that can
WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
106
be modified to increase the signal detected and to reduce the background
noise. For
further discussion regarding western blot protocols see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New
York
at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate with the antigen, adding the antibody of interest conjugated to a
detectable
compound such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline
phosphatase) to the well and incubating for a period of time, and detecting
the
presence of the antigen. In ELISAs the antibody of interest does not have to
be
conjugated to a detectable compound: instead, a second antibody (which
recognizes
the antibody of interest) conjugated to a detectable compound may be added to
the
well. Further, instead of coating the well with the antigen, the antibody may
be
coated to the well. In this case, a second antibody conjugated to a detectable
compound may be added following the addition of the antigen of interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that
can be modified to increase the signal detected as well as other variations of
ELISAs
known in the art. For further discussion regarding ELISAs see, e.g., Ausubel
et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc.,
New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody bound to the labeled antigen. The affinity of the antibody of
interest for a
particular antigen and the binding off-rates can be determined from the data
by
scatchard plot analysis. Competition with a second antibody can also be
determined
using radioimmunoassays. In this case, the antigen is incubated with antibody
of
interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence
of
increasing amounts of an unlabeled second antibody.
Therapeutic Uses
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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.
A summary of the ways in which the antibodies of the present invention may
be used therapeutically includes binding polynucleotides or polypeptides of
the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed with the
teachings provided herein, one of ordinary skill in the art will know how to
use the
antibodies of the present invention for diagnostic, monitoring or therapeutic
purposes
without undue experimentation.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and 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
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lOh
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-' M, 10-' M, 5 X 10-~ M, 10-' M,
5 X 10~~
M, 10-'' M, 5 X 105 M, 10-5 M, 5 X 10-6 M, 10-6 M, 5 X 10-' M, 10~' M, 5 X 10-
g M,
10-8 M, 5 X 10-9 M, 10-~ M, 5 X 10-'° M, 10-'° M, 5 X 10-" M, 10-
" M, 5 X 10-'' M, 10-
''- M, 5 X 10-' ' M, 10'' ' M, 5 X 10-' ~' M, 10~' ~' M, 5 X 10-' S M, and 10-
'' M.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art
of recombinant DNA technology which can be used are described in Ausubel et
al.
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(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY ( 1993);
and
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
( 1990).
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors
that express the antibody or fragments or chimeric proteins or heavy or light
chains
thereof in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or
constitutive, and, optionally, tissue- specific. In another particular
embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other
desired sequences are flanked by regions that promote homologous recombination
at a
desired site in the genome, thus providing for intrachromosomal expression of
the
antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci.
USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific
embodiments, the expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy
and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case
the patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or
indirect, in which case, cells are first transformed with the nucleic acids in
vitro, then
transplanted into the patient. These two approaches are known, respectively,
as in
mvo 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
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administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic
acid-ligand complexes can be formed in which the ligand comprises a fusogenic
viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO
93/20221 ). Alternatively, the nucleic acid can be introduced intracellularly
and
incorporated within host cell DNA for expression, by homologous recombination
(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al.,
Nature 342:435-438 ( 1989)).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can
be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These
retroviral
vectors contain the components necessary for the correct packaging of the
viral
genome and integration into the host cell DNA. The nucleic acid sequences
encoding
the antibody to be used in gene therapy are cloned into one or more vectors,
which
facilitates delivery of the gene into a patient. More detail about retroviral
vectors can
be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem cells in
order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-
651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,
Human
Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in
Genetics
and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of
being capable of infecting non-dividing cells. Kozarsky and Wilson, Current
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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).
Another approach to gene therapy involves transferring a gene to cells in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes the
transfer of a selectable marker to the cells. The cells are then placed under
selection
to isolate those cells that have taken up and are expressing the transferred
gene.
Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method known in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
containing the nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes into
cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,
Meth.
Enzymol. 217:618-644 ( 1993); Cline, Pharmac. Ther. 29:69-92m ( 1985) and may
be
used in accordance with the present invention, provided that the necessary
developmental and physiological functions of the recipient cells are not
disrupted.
The technique should provide for the stable transfer of the nucleic acid to
the cell, so
that the nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell progeny.
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The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of
the present invention (see e.g. PCT Publication WO 94/08598; Stemple and
Anderson, Cell 71:973-985 ( 1992); Rheinwald, Meth. Cell Bio. 21 A: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
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113
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.
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 invention, preferably an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired side-effects). The
subject is
preferably an animal, including but not limited to animals such as cows, pigs,
horses,
chickens, cats, dogs, etc., and is preferably a mammal, and most preferably
human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor-
mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432
(1987)),
construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of
introduction include but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes. The
compounds or
compositions may be administered by any convenient route, for example by
infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered
together
with other biologically active agents. Administration can be systemic or
local. In
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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 rnay be desirable to administer the
pharmaceutical
compounds or compositions of the invention locally to the area in need of
treatment;
this may be achieved by, for example, and not by way of limitation, local
infusion
during surgery, topical application, e.g., in conjunction with a wound
dressing after
surgery, by injection, by means of a catheter, by means of a suppository, or
by means
of an implant, said implant being of a porous, non-porous, or gelatinous
material,
including membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention, care must be
taken to
use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 ( 1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in
a controlled release system. In one embodiment, a pump may be used (see
Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 ( 1987); Buchwald et al.,
Surgery
88:507 ( 1980); Saudek et al., N. Engl. J. Med. 321:574 ( 1989)). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974);
Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen
and
Ball (eds.), Wiley, New York ( 1984); Ranger and Peppas, J., Macromol. Sci.
Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During
et al., Ann. Neurol. 25:351 ( 1989); Howard et al., J.Neurosurg. 71:105 (
1989)). In yet
another embodiment, a controlled release system can be placed in proximity of
the
therapeutic target, i.e., the brain, thus requiring only a fraction of the
systemic dose
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llj
(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 Lancer
(Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic
acid expression vector and administering it so that it becomes intracellular,
e.g., by
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 diluent, adjuvant, excipient, or vehicle with which the
therapeutic
is administered. Such pharmaceutical carriers can be sterile liquids, such as
water
and oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
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agents. These COITIpOSItIOIls Can take the fOI'ITl Of sOlutI0IlS,
suspenS1011s, emUlslOn,
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 trialycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington'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 for proper administration to the patient. The
formulation
should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an
infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition
is administered by injection, an ampoule of sterile water for injection or
saline can be
provided so that the ingredients may be mixed prior to administration.
2$ The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with canons such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
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expression and/or activity of a polypeptide of the invention can be determined
by
standard clinical techniques. In addition, in vitro assays may optionally be
employed
to help identify optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and the
seriousness of
the disease or disorder, and should be decided according to the judgment of
the
practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a
patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
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 and/or disorders 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
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1 1 ~i
increase or decrease in the assayed polypeptide gene expression level compared
to
the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder,
comprising (a) assaying the expression of the polypeptide of interest in cells
or body
fluid of an individual using one or more antibodies specific to the
polypeptide interest
and (b) comparing the level of gene expression with a standard gene expression
level,
whereby an increase or decrease in the assayed polypeptide gene expression
level
compared to the standard expression level is indicative of a particular
disorder. With
respect to cancer, the presence of a relatively high amount of transcript in
biopsied
tissue from an individual may indicate a predisposition for the development of
the
disease, or may provide a means for detecting the disease prior to the
appearance of
actual clinical symptoms. A more definitive diagnosis of this type may allow
health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et
al., J. Cell .
Biol. 105:3087-3096 ( 1987)). Other antibody-based methods useful for
detecting
protein gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as iodine (125I,.121I), carbon (14C), sulfur (35S),
tritium (3H),
indium ( 1 l2In), 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
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unbound labeled molecule to be cleared to background level); c) determining
background level; and d) detecting the labeled molecule in the subject, such
that
detection of labeled molecule above the background level indicates that the
subject
has a particular disease or disorder associated with aberrant expression of
the
polypeptide of interest. Background level can be determined by various methods
including, comparing the amount of labeled molecule detected to a standard
value
previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode
of administration, the time interval following the administration for
permitting the
labeled molecule to preferentially concentrate at sites in the subject and for
unbound
labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24
hours or
6 to 12 hours. In another embodiment the time interval following
administration is 5
to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is 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 (CT), whole
body
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scan such as position emission tomography (PET), magnetic resonance imaging
(MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et
al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is
labeled with
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
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kit may include a recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to a solid
support.
In a more specific embodiment the detecting means of the above-described kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may
also include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can be detected
by
binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The detecting means of the kit may include a second,
labeled
monoclonal antibody. Alternatively, or in addition, the detecting means may
include
a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
bound anti-antigen antibody on the solid support. The reagent is again washed
to
remove unbound labeled antibody, and the amount of reporter associated with
the
reagent is determined. Typically, the reporter is an enzyme which is detected
by
incubating the solid phase in the presence of a suitable fluorometric,
luminescent or
colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques
for attaching protein material to solid support material, such as polymeric
beads, dip
sticks, 96-well plate or filter material. These attachment methods generally
include
non-specific adsorption of the protein to the support or covalent attachment
of the
protein, typically through a free amine group, to a chemically reactive group
on the
solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
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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
polypeptide to facilitate purification. Such regions may be removed prior to
final
preparation of the polypeptide. The addition of peptide moieties to facilitate
handling
of polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and
specifically epitopes, can be combined with parts of the constant domain of
immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and
any
combination thereof, including both entire domains and portions thereof),
resulting in
chimeric polypeptides. These fusion proteins facilitate purification and show
an
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
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increased half-life in vivo. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 ( 1988).)
Fusion proteins having disulfide-linked dimeric structures (due to the IgG)
can also be
more efficient in binding and neutralizing other molecules, than the monomeric
secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 (1995).)
Similarly, EP-A-O 464 533 (Canadian counterpart 2045869 j discloses fusion
proteins comprising various portions of constant region of immunoglobulin
molecules
together with another human protein or part thereof. In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for
example, human proteins, such as hIL-5, have been fused with Fc portions for
the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
(See,
D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et
al., J. Biol.
Chem.270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide.
In preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 91311 ), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
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Vectors, Host Cells. and Protein Production
The present invention also relates to vectors containing the polynucleotide of
the present invention, host cells, and the production of polypeptides by
recombinant
techniques. The vector may be, for example, a phage, plasmid, viral, or
retroviral
vector. Retroviral vectors may be replication competent or replication
defective. In
the latter case, viral propagation generally will occur only in complementing
host
cells.
The polynucleotides may be joined to a vector containing a selectable marker
for propagation in a host. Generally, a plasmid vector is introduced in a
precipitate,
such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the
vector is a virus, it may be packaged in vitro using an appropriate packaging
cell line
and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
resistance for eukaryotic cell culture and tetracycline, kanamycin or
ampicillin
resistance genes for culturing in E. coli and other bacteria. Representative
examples
of appropriate hosts include, but are not limited to, bacterial cells, such as
E. coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells;
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.
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Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,
pNHBA,
pNH 16a, pNH 18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and
ptrc99a, pKK223-3, pKK233-3, pDR~40, pRITS available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. 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, DEAF-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection, or other methods. Such
methods
are described in many standard laboratory manuals, such as Davis et al., Basic
Methods In Molecular Biology (1986). It is specifically contemplated that the
polypeptides of the present invention may in fact be expressed by a host cell
lacking a
recombinant vector.
A polypeptide of this invention can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction. anion or canon exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification.
Polypeptides of the present invention, and preferably the secreted form, can
also be recovered from: products purified from natural sources, including
bodily
fluids, tissues and cells, whether directly isolated or cultured; products of
chemical
synthetic procedures; and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial, yeast,
higher plant,
insect, and mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may be
glycosylated
or may be non-glycosylated. In addition, polypeptides of the invention may
also
~0 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
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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 addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous Genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide
sequences) that is operably associated with the polynucleotides of the
invention, and
which activates, alters, and/or amplifies endogenous polynucleotides. For
example,
techniques known in the art may be used to operably associate heterologous
control
regions (e.g., promoter and/or enhancer) and endogenous polynucleotide
sequences
via homologous recombination, resulting in the formation of a new
transcription unit
(see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; U.S. Patent No.
5,733,761, issued March 31, 1998; International Publication No. WO 96/2941 l,
published September 26, 1996; International Publication No. WO 94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-
8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of
each of
which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and
Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al.,
Nature,
310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of
a
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-
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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 Qeneral. 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 protecting/blocking groups, proteolytic
cleavage,
linkage to an antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques, including but
not
limited, to specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin,
papain, V8 protease. NaBH~; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic
host cell expression. The polypeptides may also be modified with a detectable
label,
such as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
Also provided by the invention are chemically modified derivatives of the
polypeptides of the invention which may provide additional advantages such as
increased solubility, stability and circulating time of the polypeptide, or
decreased
immunogenicity (see U.S. Patent NO: 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,
dextran, polyvinyl alcohol and the like. The polypeptides may be modified at
random
positions within the molecule, or at predetermined positions within the
molecule and
may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations of
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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 0 401 384. herein incorporated by reference
(coupling PEG
to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
be covalently bound through amino acid residues via a reactive group, such as,
a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene glycol molecule may be bound. The amino acid residues having a
free
amino group may include lysine residues and the N-terminal amino acid
residues;
those having a free carboxyl group may include aspartic acid residues glutamic
acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used
as a reactive group for attaching the polyethylene glycol molecules. Preferred
for
therapeutic purposes is attachment at an amino group, such as attachment at
the
N-terminus or lysine group.
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
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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
tetramers. In additional embodiments, the multimers of the invention are at
least
dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer, refers to a multimer containing only
polypeptides
corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA
contained in a deposited clone (including fragments, variants, splice
variants, and
fusion proteins, corresponding to these polypeptides as described herein).
These
homomers may contain polypeptides having identical or different amino acid
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only polypeptides having an identical amino acid sequence. In
another
specific embodiment, a homomer of the invention is a multimer containing
polypeptides having different amino acid sequences. In specific embodiments,
the
multimer of the invention is a homodimer (e.g., containing polypeptides having
identical or different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid sequences). In
additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at
least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or
more heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to
the polypeptides of the invention. In a specific embodiment, the multimer of
the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional
embodiments, the heteromeric multimer of the invention is at least a
heterodimer, at
least a heterotrimer, or at least a heterotetramer.
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Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked, by for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as,
for example, homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of
the invention, such as, for example, heterotrimers or heterotetramers, are
formed
when polypeptides of the invention contact antibodies to the polypeptides of
the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, multimers of the
invention are formed by covalent associations with and/or between the
polypeptides
of the invention. Such covalent associations may involve one or more amino
acid
residues contained in the polypeptide sequence ( e.g., that recited in the
sequence
listing, or contained in the polypeptide encoded by a deposited clone). In one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring) polypeptide. In another instance, the covalent associations are the
consequence of chemical or recombinant manipulation. Alternatively, such
covalent
associations may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence
contained in a fusion protein of the invention (see, e.g., US Patent Number
5,478,925). In a specific example, the covalent associations are between the
heterologous sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent associations of
fusion
proteins of the invention are between heterologous polypeptide sequence from
another protein that is capable of forming covalently associated multimers,
such as for
example, oseteoprotegerin (see, e.g., International Publication NO: WO
98/49305, the
contents of which are herein incorporated by reference in its entirety). In
another
embodiment, two or more polypeptides of the invention are joined through
peptide
linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627
(hereby incorporated by reference). Proteins comprising multiple polypeptides
of the
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invention separated by peptide linkers may be produced using conventional
recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves
use of polypeptides of the invention fused to a leucine zipper or isoleucine
zipper
polypeptide sequence. Leucine zipper and isoleucine zipper domains are
polypeptides
that promote multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins (Landschulz
et al.,
Science 240:1759, ( 1988)), and have since been found in a variety of
different
proteins. Among the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize. Examples of leucine zipper
domains
suitable for producing soluble multimeric proteins of the invention are those
described
in PCT application WO 94/10308, hereby incorporated by reference. Recombinant
fusion proteins comprising a polypeptide of the invention fused to a
polypeptide
sequence that dimerizes or trimerizes in solution are expressed in suitable
host cells,
and the resulting soluble multimeric fusion protein is recovered from the
culture
supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are
those that preferentially form trimers. One example is a leucine zipper
derived from
lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters
344:191,
( 1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by
reference. Other peptides derived from naturally 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 Flag~ polypeptide seuqence. In a further embodiment, associations
proteins of the invention are associated by interactions between heterologous
polypeptide sequence contained in Flag~ fusion proteins of the invention and
anti-
Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in the art. For example, polypeptides desired to be contained in the
multimers
of the invention may be chemically cross-linked using linker molecules and
linker
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molecule length optimization techniques known in the art (see, e.g., US Patent
Number 5,478.925, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques
known in
the art to form one or more inter-molecule cross-links between the cysteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). Further, polypeptides of the invention may be
routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the
polypeptide components desired to be contained in the multimer of the
invention (see,
e.g., US Patent Number 5,478,925, which is herein incorporated by reference in
its
entirety).
Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In a
specific embodiment, polynucleotides coding for a homodimer of the invention
are
generated by ligating a polynucleotide sequence encoding a polypeptide of the
invention to a sequence encoding a linker polypeptide and then further to a
synthetic
polynucleotide encoding the translated product of the polypeptide in the
reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). In another embodiment, recombinant techniques
described
herein or otherwise known in the art are applied to generate recombinant
polypeptides
of the invention which contain a transmembrane domain (or hyrophobic or signal
peptide) and which can be incorporated by membrane reconstitution techniques
into
liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety).
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Uses of the Polvnucleotides
Each of the polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes
known techniques.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are presently available. Each polynucleotide of the present
invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be
selected using computer analysis so that primers do not span more than one
predicted
exon in the genomic DNA. These primers are then used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only those
hybrids
containing the human gene corresponding to the SEQ ID NO:X will yield an
amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal 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 Techniques," Pergamon
Press, New York ( 1988).
For chromosome mapping, the polynucleotides can be used individually (to
mark a single chromosome or a single site on that chromosome) or in panels
(for
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marking multiple sites and/or multiple chromosomes). Preferred polynucleotides
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.
Once a polynucleotide has been mapped to a precise chromosomal location,
the physical position of the polynucleotide can be used in linkage analysis.
Linkage
analysis establishes coinheritance between a chromosomal location and
presentation
of a particular disease. (Disease mapping data are found, for example, in V.
McKusick, Mendelian Inheritance in Man (available on line through Johns
Hopkins
University Welch Medical Library) .) Assuming 1 megabase mapping resolution
and
one gene per 20 kb, a cDNA precisely localized to a chromosomal region
associated
with the disease could be one of 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 polymorphism. 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
WO 00/55177 cA 02361277 2001-08-28 pCT/US00/06058
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level, whereby an increase or decrease in the gene expression level compared
to the
standard is indicative of a disorder.
In still another embodiment, the invention includes a kit for analyzing
samples
for the presence of proliferative and/or cancerous polynucleotides derived
from a test
subject. In a general embodiment, the kit includes at least one polynucleotide
probe
containing a nucleotide sequence that will specifically hybridize with a
polynucleotide of the present invention and a suitable container. In a
specific
embodiment, the kit includes two polynucleotide probes defining an internal
region of
the polynucleotide of the present invention, where each probe has one strand
containing a 31'mer-end internal to the region. In a further embodiment, the
probes
may be useful as primers for polymerise 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 polypeptide level or mRNA level, the
standard
being taken from a second biological sample obtained from an individual not
having
the disorder or being determined by averaging levels from a population of
individuals
not having a disorder. As will be appreciated in the art, once a standard
polypeptide
level or mRNA level is known, it can be used repeatedly as a standard for
comparison.
By "biological sample" is intended any biological sample obtained from an
individual, body fluid, cell line, tissue culture, or other source which
contains the
polypeptide of the present invention or mRNA. As indicated, biological samples
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include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and
spinal fluid) which contain the polypeptide of the present invention. and
other tissue
sources found to express the polypeptide of the present invention. Methods for
obtaining tissue biopsies and body fluids from mammals are well known in the
art.
Where the biological sample is to include mRNA, a tissue biopsy is the
preferred
source.
The methods) provided above may preferrably be applied in a diagnostic
method and/or kits in which polynucleotides 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
WO 00/55177 CA 02361277 2001-08-28
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backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider
range of stringency conditions than DNA/DNA duplexes, making it easier to
perform
multiplex hybridization. Smaller probes can be used than with DNA due to the
strong
binding. In addition, it is more likely that single base mismatches can be
determined
with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (Tm) by 8°-20° C, vs. 4°-
16° C for the DNA/DNA 15-
mer duplex. Also, the absence of charge groups in PNA means that hybridization
can
be done at low ionic strengths and reduce possible interference by salt during
the
analysis.
The present invention is useful for detecting cancer in mammals. In particular
the invention is useful during diagnosis of pathological cell proliferative
neoplasias
which include, but are not limited to: acute myelogenous leukemias including
acute
monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,
acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic
leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous
leukemias including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs,
horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative disorders 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)
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For example, c-myc expression is highly amplified in the non-lymphocytic
leukemia
cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation, the
level of c-myc is found to be downregulated. (International Publication Number
WO
91/15580) However, it has been shown that exposure of HL-60 cells to a DNA
construct that is complementary to the 5' end of c-myc or c-myb blocks
translation of
the corresponding mRNAs which downreQulates 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 disorders of hematopoietic cells and
tissues, in
light of the numerous cells and cell types of varying origins which are known
to
exhibit proliferative phenotypes.
In addition to the foregoing, a polynucleotide can be used to control gene
expression through triple helix formation or antisense DNA or RNA. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRCPress,
Boca
Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et
al.,
Nucleic Acids Research 6: 3073 ( 1979); Cooney et al., Science 241: 456 (
1988); and
Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques, preferred
polynucleotides are usually oligonucleotides 20 to 40 bases in length and
complementary to either the region of the gene involved in transcription
(triple helix -
see Lee et al., Nucl. Acids Res. 6:3073 ( 1979); Cooney et al., Science
241:456
( 1988); and Dervan et al., Science 251:1360 ( 1991 ) ) or to the mRNA itself
(antisense
- Okano, J. Neurochem. 56:560 ( 1991 ); Oligodeoxy-nucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988).) Triple
helix
formation optimally results in a shut-off of RNA transcription from DNA, while
antisense RNA hybridization blocks translation of an mRNA molecule into
polypeptide. Both techniques are effective in model systems, and the
information
disclosed herein can be used to design antisense or triple helix
polynucleotides in an
effort to treat disease.
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Polynucleotides of the present invention are also useful in gene therapy. One
goal of gene therapy is to insert a normal gene into an organism having a
defective
gene, in an effort to correct the genetic defect. The polynucleotides
disclosed in the
present invention offer a means of targeting such genetic defects in a highly
accurate
manner. Another goal is to insert a new gene that was not present in the host
genome,
thereby producing a new trait in the host cell.
The polynucleotides are also useful for identifying individuals from minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel.
In this technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of
"Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
The polynucleotides of the present invention can also be used as an
alternative
to RFLP, by determining the actual base-by-base DNA sequence of selected
portions
of an individual's genome. These sequences can be used to prepare PCR primers
for
amplifying and isolating such selected DNA, which can then be sequenced. Using
this technique, individuals can be identified because each individual will
have a
unique set of DNA sequences. Once an unique ID database is established for an
individual, positive identification of that individual, living or dead, can be
made from
extremely small tissue samples.
Forensic biology also benefits from using DNA-based identification
techniques as disclosed herein. DNA sequences taken from very small biological
samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, semen,
synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or
surfactant,urine,fecal matter, etc., can be amplified using PCR. In one prior
art
technique, gene sequences amplified from polymorphic loci, such as DQa class
II
HLA gene, are used in forensic biology to identify individuals. (Erlich, H.,
PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are
amplified, they are digested with one or more restriction enzymes, yielding an
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1-t0
identifying set of bands on a Southern blot probed with DNA corresponding to
the
DQa class II HLA gene. Similarly, polynucleotides of the present invention can
be
used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a
particular tissue. Such need arises, for example, in forensics when presented
with
tissue of unknown origin. Appropriate reagents can comprise, for example, DNA
probes or primers specific to particular tissue prepared from the sequences of
the
present invention. Panels of such reagents can identify tissue by species
and/or by
organ type. In a similar fashion, these reagents can be used to screen tissue
cultures
for contamination.
In the very least, the polynucleotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences
in the process of discovering novel polynucleotides, for selecting and making
oligomers for attachment to a "gene chip" or other support, to raise anti-DNA
antibodies using DNA immunization techniques, and as an antigen to elicit an
immune response.
Uses of the Pol~~eptides
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 immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium
(112In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
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In addition to assaying secreted protein levels in a biological sample,
proteins
can also be detected in vivo by imaging. Antibody labels or markers for in
vivo
imaging of protein include those detectable by X-radiography, NMR or ESR. For
X-
radiography, suitable labels include radioisotopes such as barium or cesium,
which
emit detectable radiation but are not overtly harmful to the subject. Suitable
markers
for NMR and ESR include those with a detectable characteristic spin, such as
deuterium, which may be incorporated into the antibody by labeling of
nutrients for
the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
131I,
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 99mTc. The labeled antibody or antibody fragment will
then
preferentially accumulate at the location of cells which contain the specific
protein.
In vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics
of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. ( 1982).)
Thus, the invention provides a diagnostic method of a disorder, which
involves (a) assaying the expression of a polypeptide of the present invention
in cells
or body fluid of an individual; (b) comparing the level of gene expression
with a
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard expression level is
indicative of a disorder. With respect to cancer, the presence of a relatively
high
amount of transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical symptoms. A
more
definitive diagnosis of this type may allow health professionals to employ
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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 disease.
For example, patients can be administered a polypeptide of the present
invention in an
effort to replace absent or decreased levels of the polypeptide (e.g.,
insulin), to
supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S
for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity
of a
polypeptide (e.g., an oncogene or tumor supressor), to activate the activity
of a
polypeptide (e.g., by binding to a receptor), to reduce the activity of a
membrane
bound receptor by competing with it for free ligand (e.g., soluble TNF
receptors used
in reducing inflammation), or to bring about a desired response (e.g., blood
vessel
growth inhibition, enhancement of the immune response to proliferative cells
or
tissues).
Similarly, antibodies directed to a polypeptide of the present invention can
also be used to treat disease. For example, administration of an antibody
directed to a
polypeptide of the present invention can bind and reduce overproduction of the
polypeptide. Similarly, administration of an antibody can activate the
polypeptide,
such as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art. Polypeptides
can also
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 Therapy Methods
Another aspect of the present invention is to gene therapy methods for
treating
disorders, diseases and conditions. The gene therapy methods relate to the
introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences
into
an animal to achieve expression of a polypeptide of the present invention.
This
method requires a polynucleotide which codes for a polypeptide of the
invention that
operatively linked to a promoter and any other Qenetic elements necessary for
the
expression of the polypeptide by the target tissue. Such gene therapy and
delivery
techniques are known in the art, see, for example, W090/11092, which is herein
incorporated by reference.
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) comprising a promoter operably linked to a
polynucleotide of the invention ex vivo, with the engineered cells then being
provided
to a patient to be treated with the polypeptide. Such methods are well-known
in the
art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993);
Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J.
Immunology 153: 4604-4615 ( 1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229
( 1995); Ogura et al., Cancer Research 50: 5102-5106 ( 1990); Santodonato, et
al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-
1255
( 1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 ( 1996)), which are
herein
incorporated by reference. In one embodiment, the cells which are engineered
are
arterial cells. The arterial cells may be reintroduced into the patient
through direct
injection to the artery, the tissues surrounding the artery, or through
catheter injection.
As discussed in more detail below, the polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells of an
animal,
such as, injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically
acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
that are free from any delivery vehicle that acts to assist, promote or
facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations,
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lipofectin or precipitating agents and the like. However, the polynucleotides
of the
invention can also be delivered in liposome formulations and lipofectin
formulations
and the like can be prepared by methods well known to those skilled in the
art. Such
methods are described, for example, in U.S. Patent Nos. 5,593,972, 5.589,466,
and
5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs of the invention used in the gene
therapy method are preferably constructs that will not integrate into the host
genome
nor will they contain sequences that allow for replication. Appropriate
vectors
include pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene;
pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI/V5,
pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will
be
readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving
the expression of polynucleotide sequence of the invention. Suitable promoters
include adenoviral promoters, such as the adenoviral major late promoter; or
heterologous promoters, such as the cytomegalovirus (CMV) promoter; the
respiratory syncytial virus (RSV) promoter; inducible promoters, such as the
MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter;
the ApoAI promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex 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,
gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye,
gland, and connective tissue. Interstitial space of the tissues comprises the
intercellular,
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14i
fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues,
elastic
fibers in the walls of vessels or chambers. collagen fibers of fibrous
tissues, or that
same matrix within connective tissue ensheathina muscle cells or in the
lacunae of
bone. It is similarly the space occupied by the plasma of the circulation and
the lymph
fluid of the lymphatic channels. Delivery to the interstitial space of muscle
tissue is
preferred for the reasons discussed below. They may be conveniently delivered
by
injection into the tissues comprising these cells. They are preferably
delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression may be achieved in non-differentiated or less completely
differentiated
cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells
are particularly competent in their ability to take up and express
polynucleotides.
For the nakednucleic acid sequence injection, an effective dosage amount of
DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about
50
mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to
about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 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.
The constructs may also be delivered with delivery vehicles such as viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents, etc.
Such methods of delivery are known in the art.
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In certain embodiments, the polynucleotide constructs of the invention are
complexed in a liposome preparation. Liposomal preparations for use in the
instant
invention include cationic (positively charged), anionic (negatively charged)
and
neutral preparations. However, cationic liposomes are particularly preferred
because a
tight charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci.
USA ,
84:7413-7416 ( 1987), which is herein incorporated by reference); mRNA (Malone
et
al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 ( 1989), which is herein
incorporated
by reference): and purified transcription factors (Debs et al., J. Biol.
Chem.,
265:10189-10192 (1990), which is herein incorporated by reference), in
functional
form.
Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly useful and are available under the trademark Lipofectin, from
GIBCO
BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA
,
84:7413-7416 ( 1987), which is herein incorporated by reference). Other
commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
Other cationic liposomes can be prepared from readily available materials
using techniques well known in the art. See, e.g. PCT Publication NO: WO
90/11092
(which is herein incorporated by reference) for a description of the synthesis
of
DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation
of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al.,
Proc.
Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference.
Similar methods can be used to prepare liposomes from other cationic lipid
materials.
Similarly, anionic and neutral liposomes are readily available, such as from
Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using
readily
available materials. Such materials include phosphatidyl, choline,
cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE),
among others. These materials can also be mixed with the DOTMA and DOTAP
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starting materials in appropriate ratios. Methods for making liposomes using
these
materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can be used in various combinations to make conventional liposomes,
with or
without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be
prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas
into a sonication vial. The sample is placed under a vacuum pump overnight and
is
hydrated the following day with deionized water. The sample is then sonicated
for 2
hours in a capped vial, using a Heat Systems model 350 sonicator equipped with
an
inverted cup (bath type) probe at the maximum setting while the bath is
circulated at
15EC. Alternatively, negatively charged vesicles can be prepared without
sonication
to produce multilamellar vesicles or by extrusion through nucleopore membranes
to
produce unilamellar vesicles of discrete size. Other methods are known and
available
to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (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
then the preformed liposomes are mixed directly with the DNA. The liposome and
DNA form a very stable complex due to binding of the positively charged
liposomes
to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are
prepared by a number of methods, well known in the art. Commonly used methods
include Ca'+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta,
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394:483 ( 1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer
et al.,
Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.
Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348
(1979));
detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci. USA , 76:145 (
1979)); and
reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431
(1980);
Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 ( 1978); Schaefer-Ridder et
al.,
Science, 215:166 ( 1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about
1:10. Preferably, the ration will be from about 5:1 to about 1:5. More
preferably, the
ration will be about 3:1 to about 1:3. Still more preferably, the ratio will
be about 1:1.
U.S. Patent NO: 5,676,954 (which is herein incorporated by reference) reports
on the injection of genetic material, complexed with cationic liposomes
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 94/9469 (which are
herein incorporated by reference) provide methods for delivering DNA-cationic
lipid
complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a
retroviral particle containing RNA which comprises a sequence encoding
polypeptides of the invention. Retroviruses from which the retroviral plasmid
vectors
may be derived include, but are not limited to, Moloney Murine Leukemia Virus,
spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian
leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form producer cell lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317, R-2, R-AM, PA 12, T 19-14X,
VT-
19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described
in Miller, Human Gene Therapy , 1:5-14 (1990), which is incorporated herein by
reference in its entirety. The vector may transduce the packaging cells
through any
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means known in the art. Such means include, but are not limited to,
electroporation,
the use of liposomes, and CaPO~ 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 Qenerates infectious retroviral vector particles which
include polynucleotide encoding polypeptides of the invention. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or in
vivo. The transduced eukaryotic cells will express polypeptides of the
invention.
In certain other embodiments, cells are engineered, ex vivo or i~z vivo, with
polynucleotides of the invention contained in an adenovirus vector. Adenovirus
can
be manipulated such that it encodes and expresses polypeptides of the
invention, and
at the same time is inactivated in terms of its ability to replicate in a
normal lytic viral
life cycle. Adenovirus expression is achieved without integration of the viral
DNA
into the host cell chromosome, thereby alleviating concerns about insertional
mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines
for
many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir.
Dis.,
109:233-238 ( 1974)). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and
CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991);
Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to
attempt
to establish adenovirus as a causative agent in human cancer were uniformly
negative
(Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 ( 1979)).
Suitable adenoviral vectors useful in the present invention are described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 ( 1993);
Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther.,
4:759-769 ( 1993); Yang et al., Nature Genet., 7:362-369 ( 1994); Wilson et
al.,
Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2 is useful
and can
be grown in human 293 cells. These cells contain the E 1 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
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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: E 1
a, E 1 b,
E3, E4, E2a, or L 1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
Curr.
Topics in Microbiol. Immunol., 158:97 ( 1992)). It is also one of the few
viruses that
may integrate its DNA into non-dividing cells. Vectors containing as little as
300 base
pairs of AAV can be packaged and can integrate, but space for exogenous DNA is
limited to about 4.5 kb. Methods for producing and using such AAVs are known
in
the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will
include all the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The polynucleotide construct containing polynucleotides of the
invention
is inserted into the AAV vector using standard cloning methods, such as those
found
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989). The recombinant AAV vector is then transfected into packaging
cells
which are infected with a helper virus, using any standard technique,
including
lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper
viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses.
Once the packaging cells are transfected and infected, they will produce
infectious
AAV viral particles which contain the polynucleotide construct of the
invention.
These viral particles are then used to transduce eukaryotic cells, either ex
vivo or in
vivo. The transduced cells will contain the polynucleotide construct
integrated into its
genome, and will express the desired gene product.
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Another method of gene therapy involves operably associating heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding the
polypeptide sequence of interest) via homologous recombination (see, e.g.,
U.S.
Patent NO: 5,641,670, issued June 24, 1997; International Publication NO: WO
96/29411, published September 26, 1996; International Publication NO: WO
94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 ( 1989); and Zijlstra et al., Nature, 342:435-438 ( 1989). This
method
involves the activation of a gene which is present in the target cells, but
which is not
normally expressed in the cells, or is expressed at a lower level than
desired.
Polynucleotide constructs are made, using standard techniques known in the
art, which contain the promoter with targeting sequences flanking the
promoter.
Suitable promoters are described herein. The targeting sequence is
sufficiently
complementary to an endogenous sequence to permit homologous recombination of
the promoter-targeting sequence with the endogenous sequence. The targeting
sequence will be sufficiently near the 5' end of the desired endogenous
polynucleotide sequence so the promoter will be operably linked to the
endogenous
sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5'
and 3' ends. Preferably, the 3' end of the first targeting sequence contains
the same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the
second targeting sequence contains the same restriction site as the 3' end of
the
amplified promoter. The amplified promoter and targeting sequences are
digested
and ligated together.
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.
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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-Cj, VEGF-3 (VEGF-B), epidermal growth factor
alpha and beta, platelet-derived endothelial cell growth factor, platelet-
derived growth
factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like
growth
factor, colony stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
Preferably, the polynucleotide encoding a polypeptide of the invention
contains a secretory signal sequence that facilitates secretion of the
protein.
Typically, the signal sequence is positioned in the coding region of the
polynucleotide
to be expressed towards or at the 5' end of the coding region. The signal
sequence
may be homologous or heterologous to the polynucleotide of interest and may be
homologous or heterologous to the cells to be transfected. Additionally, the
signal
sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect. This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available
depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial
solid
(tablet or pill) pharmaceutical formulations, and decanting or topical
applications
during surgery. For example, direct injection of naked calcium
phosphate-precipitated plasmid into rat liver and rat spleen or a protein-
coated
plasmid into the portal vein has resulted in gene expression of the foreign
gene in the
rat livers. (Kaneda et al., Science, 243:375 ( 1989)).
A preferred method of local administration is by direct injection. Preferably,
a
recombinant molecule of the present invention complexed with a delivery
vehicle is
WO 00/55177 cA o23si2~~ 2ooi-oa-2a pCT/US00/06058
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administered by direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries refers to
injecting
the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide
construct of the present invention in or around a surgical wound. For example,
a
patient can undergo surgery and the polynucleotide construct can be coated on
the
surface of tissue inside the wound or the construct can be injected into areas
of tissue
inside the wound.
Therapeutic compositions useful in systemic administration, include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
administration comprise liposomes comprising ligands for targeting the vehicle
to a
particular site.
Preferred methods of systemic administration, include intravenous injection,
aerosol, oral and percutaneous (topical) delivery. Intravenous injections can
be
performed using methods standard in the art. Aerosol delivery can also be
performed
using methods standard in the art (see, for example, Stribling et al., Proc.
Natl. Acad.
Sci. USA , 189:11277-11281 (1992), which is incorporated herein by reference).
Oral
delivery can be performed by complexing a polynucleotide construct of the
present
invention to a carrier capable of withstanding degradation by digestive
enzymes in the
gut of an animal. Examples of such carriers, include plastic capsules or
tablets, such
as those known in the art. Topical delivery can be performed by mixing a
polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
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
polynucleotide constructs administered per dose, as well as the health and
history of
the subject. The precise amount, number of doses, and timing of doses will be
determined by the attending physician or veterinarian. Therapeutic
compositions of
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the present invention can be administered to any animal, preferably to mammals
and
birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle,
horses and pigs, with humans being particularly
Biological Activities
The polynucleotides or polypeptides, or agonists or antagonists of the present
invention can be used in assays to test for one or more biological activities.
If these
polynucleotides and polypeptides do exhibit activity in a particular assay, it
is likely
that these molecules may be involved in the diseases associated with the
biological
activity. Thus, the polynucleotides or polypeptides, or agonists or
antagonists could
be used to treat the associated disease.
Immune Activity
The polynucleotides or polypeptides, or agonists or antagonists of the present
invention may be useful in treating deficiencies or disorders 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 deficiencies or disorders may be genetic,
somatic, such
as cancer or some autoimmune disorders, 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 or detecting deficiencies or disorders of
hematopoietic cells. A polynucleotides or polypeptides, or agonists or
antagonists of
the present invention could be used to increase differentiation and
proliferation of
hematopoietic cells, including the pluripotent stem cells, in an effort to
treat those
disorders associated with a decrease in certain (or many) types hematopoietic
cells.
Examples of immunologic deficiency syndromes include, but are not limited to:
blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),
ataxia
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telangiectasia, common variable immunodeficiency, DiQeor~e Syndrome, HIV
infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia,
or hemoglobinuria.
Moreover, 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 blood coagulation
disorders
(e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (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 of
heart attacks (infarction), strokes, or scarring.
A polynucleotides or polypeptides, or agonists or antagonists of the present
invention may also be useful in treating or detecting autoimmune disorders.
Many
autoimmune disorders 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
disorders.
Examples of autoimmune disorders that can be treated 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, Ophthalmia, Bullous
Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man
Syndrome,
Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary
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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 by a
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 and/or prevent 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 response
is
also involved in GVHD, but, in this case, the foreign transplanted immune
cells
destroy the host tissues. 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 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 polynucleotide or
agonists
or antagonist may inhibit the proliferation and differentiation of cells
involved in an
inflammatory response. These molecules can be used to treat inflammatory
conditions, both chronic and acute conditions, including inflammation
associated with
infection (e.g., septic shock, sepsis, or systemic inflammatory response
syndrome
(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-
mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung
injury,
inflammatory bowel disease, Crohn's disease, or resulting from over production
of
cytokines (e.g., TNF or IL-1
H~perproliferative Disorders
A polynucleotides or polypeptides, or agonists or antagonists of the invention
can be used to treat or detect hyperproliferative disorders, including
neoplasms. A
polynucleotides or polypeptides, or agonists or antagonists of the present
invention
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may inhibit the proliferation of the disorder through direct or indirect
interactions.
Alternatively, a polynucleotides or polypeptides, or agonists or antagonists
of the
present invention may proliferate other cells which can inhibit the
hyperproliferative
disorder.
For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing T-cells, hyperproliferative disorders can be
treated.
This immune response may be increased by either enhancing an existing immune
response, or by initiating a new immune response. Alternatively, decreasing an
immune response may also be a method of treating hyperproliferative disorders,
such
as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by a
polynucleotides or polypeptides, or agonists or antagonists of the present
invention
include, but are not limited to neoplasms located in the: 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 disorders can also be treated or detected
by
a polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Examples of such hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,
purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's
Disease, histiocytosis, and any other hyperproliferative disease, besides
neoplasia,
located in an organ system listed above.
One preferred embodiment utilizes polynucleotides of the present invention to
inhibit aberrant cellular division, by gene therapy using the present
invention, and/or
protein fusions or fragments thereof.
Thus, the present invention provides a method for treating cell proliferative
disorders by inserting into an abnormally proliferating cell a polynucleotide
of the
present W vention, wherein said polynucleotide represses said expression.
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Another embodiment of the present invention provides a method of treating
cell-proliferative disorders 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 construct encoding the poynucleotides of the
present
invention is inserted into cells to be treated utilizing a retrovirus, or more
preferrably
an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is
hereby incorporated by reference). In a most preferred embodiment, the viral
vector
is defective and will not transform non-proliferating cells, only
proliferating cells.
Moreover, in a preferred embodiment, the polynucleotides of the present
invention
inserted into proliferating cells either alone, or in combination with or
fused to other
polynucleotides, can then be modulated via an external stimulus (i.e.
magnetic,
specific small molecule, chemical, or drug administration, etc.), which acts
upon the
promoter upstream of said polynucleotides to induce expression of the encoded
protein product. As such the beneficial therapeutic affect of the present
invention
may be expressly modulated (i.e. to increase, decrease, or inhibit expression
of the
present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing
expression of oncogenic genes or antigens. By "repressing expression of the
oncogenic genes " is intended the suppression of the transcription of the
gene, the
degradation of the gene transcript (pre-message RNA), the inhibition of
splicing, the
destruction of the messenger RNA, the prevention of the post-translational
modifications of the protein, the destruction of the protein, or the
inhibition of the
normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of
the present invention may be administered by any method known to those of
skill in
the art including, but not limited to transfection, electroporation,
microinjection of
cells, or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any
other method described throughout the specification. The polynucleotide of the
present invention may be delivered by known gene delivery systems such as, but
not
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limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature
320:275 ( 1986): Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014),
vaccinia virus
system (Chakrabarty et al., Mol. Cell Biol. 5:3403 ( 1985) or other efficient
DNA
delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled
in the
art. These references are exemplary only and are hereby incorporated by
reference.
In order to specifically deliver or transfect cells which are abnormally
proliferating
and spare non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as
described in the art and elsewhere herein) delivery system known to those of
skill in
the art. Since host DNA replication is required for retroviral DNA to
integrate and
the retrovirus will be unable to self replicate due to the lack of the
retrovirus genes
needed for its life cycle. Utilizing such a retroviral delivery system for
polynucleotides of the present invention will target said gene and constructs
to
abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell
proliferative disorder/disease sites in internal organs, body cavities and the
like by use
of imaging devices used to guide an injecting needle directly to the disease
site. The
polynucleotides of the present invention may also be administered to disease
sites at
the time of surgical intervention.
By "cell proliferative disease" is meant any human or animal disease or
disorder, affecting any one or any combination of organs, cavities, or body
parts,
which is characterized by single or multiple local abnormal proliferations of
cells,
groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be
administered as long as it has a biologically inhibiting effect on the
proliferation of
the treated cells. Moreover, it is possible to administer more than one of the
polynucleotide of the present invention simultaneously to the same site. By
"biologically inhibiting" is meant partial or total growth inhibition as well
as
decreases in the rate of proliferation or growth of the cells. The
biologically
inhibitory dose may be determined by assessing the effects of the
polynucleotides of
the present invention on target malignant or abnormally proliferating cell
growth in
tissue culture, tumor growth in animals and cell cultures, or any other method
known
to one of ordinary skill in the art.
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The present invention is further directed to antibody-based therapies which
involve administering of anti-polypeptides and anti-polynucleotide antibodies
to a
mammalian, preferably human, patient for treating one or more of the described
disorders. Methods for producing anti-polypeptides and anti-polynucleotide
antibodies polyclonal and monoclonal antibodies are described in detail
elsewhere
herein. Such antibodies may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may
be used therapeutically includes binding polynucleotides or polypeptides of
the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without
undue experimentation.
In particular, the antibodies, fragments and derivatives of the present
invention
are useful for treating a subject having or developing cell proliferative
and/or
differentiation disorders as described herein. Such treatment comprises
administering
a single or multiple doses of the antibody, or a fragment, derivative, or a
conjugate
thereof.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors, for example, which serve to increase the number
or
activity of effector cells which interact with the antibodies.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy of disorders 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 SX 10-6M, 10-6M, SX 10-'M, 10-'M, SX 10-~M, 10-~M, SX 10-''M, 10-9M,
SX 10-
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'°M, 10-'°M, SX 10-"M, 10-"M, SX 10-'-M, 10-''M, SX 10-''M, 10-
''M, SX 10-'~M, 10~
'~'M, SX10~''M, and 10-''M.
Moreover, polypeptides of the present invention are useful in inhibiting the
angiogenesis of proliferative cells or tissues, either alone, as a protein
fusion, or in
combination with other polypeptides directly or indirectly, as described
elsewhere
herein. In a most preferred embodiment, said anti-angiogenesis effect may be
achieved indirectly, for example, through the inhibition of hematopoietic,
tumor-
specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J
Natl
Cancer Inst, 90(21 ):1648-53 ( 1998), which is hereby incorporated by
reference).
Antibodies directed to polypeptides or polynucleotides of the present
invention may
also result in inhibition of angiogenesis directly, or indirectly (See Witte
L, et al.,
Cancer Metastasis Rev. 17(2):155-61 ( 1998), which is hereby incorporated by
reference)).
Polypeptides, including protein fusions, of the present invention, or
fragments
thereof may be useful in inhibiting proliferative cells or tissues through the
induction
of apoptosis. Said polypeptides may act either directly, or indirectly to
induce
apoptosis of proliferative cells and tissues, for example in the activation of
a death-
domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-
1),
TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K,
et.al.,
Eur J Biochem 254(3):439-59 ( 1998), which is hereby incorporated by
reference).
Moreover, in another preferred embodiment of the present invention, said
polypeptides may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or through
stimulating the
expression of said proteins, either alone or in combination with small
molecule drugs
or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory
proteins
(See for example, Mutat Res 400( 1-2):447-55 ( 1998), Med Hypotheses.50(5):423-
33
(1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-
12
( 1998), Int J Tissue React;20( 1 ):3-15 ( 1998), which are all hereby
incorporated by
reference).
Polypeptides, including protein fusions to, or fragments thereof, of the
present
invention are useful in inhibiting the metastasis of proliferative cells or
tissues.
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Inhibition may occur as a direct result of administering polypeptides, or
antibodies
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 Microbiol Immunol 1998;231:125-41, which is
hereby
incorporated by reference). Such thereapeutic affects of the present invention
may be
achieved either alone, or in combination with small molecule drugs or
adjuvants.
In another embodiment, the invention provides a method of delivering
compositions containing the polypeptides of the invention (e.g., compositions
containing polypeptides or polypeptide antibodes associated with heterologous
polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted
cells
expressing the polypeptide of the present invention. Polypeptides or
polypeptide
antibodes of the invention may be associated with with heterologous
polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic,
ionic
and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present
invention are
useful in enhancing the immunogenicity and/or antigenicity of proliferating
cells or
tissues, either directly, such as would occur if the polypeptides of the
present
invention 'vaccinated' the immune response to respond to proliferative
antigens and
immunogens, or indirectly, such as in activating the expression of proteins
known to
enhance the immune response (e.g. chemokines), to said antigens and
immunogens.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the invention
may be used to treat cardiovascular disorders, including peripheral artery
disease,
such as limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-
arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital
heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects
include aortic coarctation, cor triatriatum, coronary vessel anomalies,
crisscross heart,
dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger
complex,
hypoplastic left heart syndrome, levocardia, tetralogy of fallot,
transposition of great
vessels, double outlet right ventricle, tricuspid atresia, persistent truncus
arteriosus,
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and heart septal defects, such as aortopulmonary septal defect, endocardial
cushion
defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal
defects.
Cardiovascular disorders also include heart disease, such as arrhythmias,
carcinoid heart disease, high cardiac output, low cardiac output, cardiac
tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive
heart
failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right
ventricular hypertrophy, post-infarction heart rupture, ventricular septal
rupture, heart
valve diseases, myocardial diseases, myocardial ischemia, pericardial
effusion,
pericarditis (including constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease,
ventricular dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-
type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias include
paroxysmal tachycardia, supraventricular tachycardia, accelerated
idioventricular
rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial
tachycardia, ectopic
functional tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia,
Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear murmurs, aortic valve prolapse, mural valve prolapse, tricuspid valve
prolapse,
mural valve insufficiency, mural 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,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
reperfusion injury, and myocarditis.
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Myocardial ischemias include coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis. coronary
vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema,
aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive
diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular
disorders, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia,
hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension,
ischemia,
peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease,
Raynaud's
disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena
cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and
venous
insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication,
carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion,
Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis
obliterans.
Cerebrovascular disorders 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 (including transient),
subclavian
steal syndrome, periventricular leukomalacia, vascular headache, cluster
headache,
~0 migraine, and vertebrobasilar insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
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thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion
injuries, and peripheral limb ischemia. Vasculitis includes aortitis,
arteritis, Behcet's
Syndrome. Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch
purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
Polynucleotides or polypeptides, or agonists or antagonists of the invention,
are especially effective for the treatment of critical limb ischemia and
coronary
disease.
Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators. gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides of the invention may be administered as part of a
Therapeutic,
described in more detail below. Methods of delivering polynucleotides of the
invention are described in more detail herein.
Anti-Angio~enesis Activitx
The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell 56:345-355 ( 1989). In those rare instances in which
neovascularization occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited. Under conditions of pathological angiogenesis such as that
characterizing
solid tumor growth, these regulatory controls fail. Unregulated angiogenesis
becomes
pathologic and sustains progression of many neoplastic and non-neoplastic
diseases.
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A number of serious diseases are dominated by abnormal neovascularization
including solid tumor growth and metastases, arthritis, some types of eye
disorders,
and psoriasis. See, e. g., reviews by Moses et al., Biotech. 9:630-634 ( 1991
); Folkman
et al., N. Efigl. J. Mecl., 333:1757-1763 ( 1995); Auerbach et al., J.
Microvcrsc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and
Weinhouse, Academic Press, New York. pp. 175-203 (1985); Patz, Arn. J.
OpthalnZOl. 94:715-743 ( 1982); and Folkman et al., Science 221:719-725
(1983). In a
number of pathological conditions, the process of angiogenesis contributes to
the
disease state. For example, significant data have accumulated which suggest
that the
growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun,
Science 235:442-447 ( 1987).
The present invention provides for treatment of diseases or disorders
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 an angiogenesis-related disease and/or
disorder, comprising administering to an individual in need thereof a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
of the
invention. For example, polynucleotides, polypeptides, antagonists and/or
agonists
may be utilized in a variety of additional methods in order to therapeutically
treat a
cancer or tumor. Cancers which may be treated with polynucleotides,
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; glioblastoma;
Kaposi's
sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer;
advanced
malignancies; and blood born tumors such as leukemias. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be delivered
topically,
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in order to treat 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 other disorders, besides cancers. which involve angiogenesis. These
disorders include, but are not limited to: benign tumors, for example
hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example, diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed
wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial
angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous
malformations;
ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia;
wound
granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided
for treating 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 polynucleotides,
polypeptides, antagonists and/or agonists are directly injected into a
hypertrophic scar
or keloid, in order to prevent the progression of these lesions. This therapy
is of
particular value in the prophylactic treatment of conditions which are known
to result
in the development of hypertrophic scars and keloids (e.g., burns), and is
preferably
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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 neovascular
diseases
of the eye, including for example, corneal neovascularization, neovascular
glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and macular
degeneration.
Moreover, Ocular disorders associated with neovascularization which can be
treated 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 ccl., Am. J.
Ophtl2al. 85:704-
710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for
treating neovascular diseases of the eye such as corneal neovascularization
(including
corneal graft neovascularization), comprising the step of administering to a
patient a
therapeutically effective amount of a compound (as described above) to the
cornea,
such that the formation of blood vessels is inhibited. Briefly, the cornea is
a tissue
which normally lacks blood vessels. In certain pathological conditions
however,
capillaries may extend into the cornea from the pericorneal vascular plexus of
the
limbus. When the cornea becomes vascularized, it also becomes clouded,
resulting in
a decline in the patient's visual acuity. Visual loss may become complete if
the
cornea completely opacitates. A wide variety of disorders can result in
corneal
neovascularization, including for example, corneal infections (e.g., trachoma,
herpes
simplex keratitis, leishmaniasis and onchocerciasis), immunological processes
(e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma,
inflammation
(of any cause), toxic and nutritional deficiency states, and as a complication
of
wearing contact lenses.
Within particularly preferred embodiments of the invention, may be prepared
for topical administration in saline (combined with any of the preservatives
and
antimicrobial agents commonly used in ocular preparations), and administered
in
eyedrop form. The solution or suspension may be prepared in its pure form and
WO 00/55177 CA 02361277 2001-08-28 pCT/US00/06058
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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-angioaenic composition is prepared with a muco-
adhesive polymer which binds to cornea. Within further embodiments, the anti-
s angiogenic factors or anti-angiogenic compositions may be utilized as an
adjunct to
conventional steroid therapy. Topical therapy may also be useful
prophylactically in
corneal lesions which are known to have a high probability of inducing an
angiogenic
response (such as chemical burns). In these instances the treatment, likely in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
Within other embodiments, the compounds described above may be injected
directly into the corneal stroma by an ophthalmologist under microscopic
guidance.
The preferred site of injection may vary with the morphology of the individual
lesion,
but the goal of the administration would be to place the composition at the
advancing
front of the vasculature (i.e., interspersed between the blood vessels and the
normal
cornea). In most cases this would involve perilimbic corneal injection to
"protect" the
cornea from the advancing blood vessels. This method may also be utilized
shortly
after a corneal insult in order to prophylactically prevent corneal
neovascularization.
In this situation the material could be injected in the perilimbic cornea
interspersed
between the corneal lesion and its undesired potential limbic blood supply.
Such
methods may also be utilized in a similar fashion to prevent capillary
invasion of
transplanted corneas. In a sustained-release form injections might only be
required 2-
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 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
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
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compound is continuously released into the aqueous humor. Within another
aspect of
the present invention, methods are provided for treating proliferative
diabetic
retinopathy, comprising the step of administering to a patient a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
to the
eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in order to increase the local concentration of the polynucleotide,
polypeptide, antagonist and/or agonist in the retina. Preferably, this
treatment should
be initiated prior to the acquisition of severe disease requiring
photocoagulation.
Within another aspect of the present invention, methods are provided for
treatW g retrolental fibroplasia, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous injection and/or via
intraocular implants.
Additionally, disorders which can be treated with the polynucleotides,
polypeptides, agonists and/or agonists include, but are not limited to,
hemangioma,
arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound
healing,
granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-
Weber
syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated with be treated 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
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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.
Polynucleotides, polypeptides, agonists and/or agonists of the present
invention may be incorporated into surgical sutures in order to prevent stitch
granulomas.
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a
wide variety of surgical procedures. For example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
to coat or spray an area prior to removal of a tumor, in order to isolate
normal
surrounding tissues from malignant tissue, and/or to prevent the spread of
disease to
surrounding tissues. Within other aspects of the present invention,
compositions (e.g.,
in the form of a spray) may be delivered via endoscopic procedures in order to
coat
tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects
of the
present invention, surgical meshes which have been coated with anti-
angiogenic
compositions of the present invention may be utilized in any procedure wherein
a
surgical mesh might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition may be
utilized
during abdominal cancer resection surgery (e.g., subsequent to colon
resection) in
order to provide support to the structure, and to release an amount of the
anti
angiogenic factor.
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Within further aspects of the present invention, methods are provided for
treating tumor excision sites, comprising administering a polynucleotide,
polypeptide,
agonist and/or agonist to the resection margins of a tumor subsequent to
excision,
such that the local recurrence of cancer and the formation of new blood
vessels at the
site is inhibited. Within one embodiment of the invention, the anti-angiogenic
compound is administered directly to the tumor excision site (e.g., applied by
swabbing, brushing or otherwise coating the resection margins of the tumor
with the
anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be
incorporated into known surgical pastes prior to administration. Within
particularly
preferred embodiments of the invention, the anti-angiogenic compounds are
applied
after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, polynucleotides, polypeptides,
agonists and/or agonists may be administered to the resection margin of a wide
variety of tumors, including for example, breast, colon, brain and hepatic
tumors. For
example, within one embodiment of the invention, anti-angiogenic compounds may
be administered to the site of a neurological tumor subsequent to excision,
such that
the formation of new blood vessels at the site are inhibited.
The polynucleotides, polypeptides, agonists and/or agonists of the present
invention may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-l, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-l, 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
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complexes include. for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
Representative examples of tungsten and molybdenum complexes also include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide complexes. Suitable tungstate complexes include ammonium tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
A wide variety of other anti-angiogenic factors may also be utilized within
the
context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells),
(Murata et al., Cancer Res. 51:22-26, 1991 ); Sulphated Polysaccharide
Peptidoglycan
Complex (SP- PG) (the function of this compound may be enhanced by the
presence
of steroids such as estrogen, and tamoxifen citrate); Staurosporine;
modulators of
matrix metabolism, including for example, proline analogs, cishydroxyproline,
d,L-
3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile
fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.
267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480,
1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin
(Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-
serum;
alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987);
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
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316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole;
and metalloproteinase inhibitors such as BB94
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of
apoptosis
that could be treated or detected by the polynucleotides or polypcptides
and/or
antagonists or agonists of the invention, include cancers (such as follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors, pancreatic cancer,
melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast
cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune
disorders
(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
and
viral infections (such as herpes viruses, pox viruses and adenoviruses),
inflammation,
graft v. host disease, acute graft rejection, and chronic graft rejection. In
preferred
embodiments, the polynucleotides or polypeptides, and/or agonists or
antagonists of
the invention are used to inhibit growth, progression, and/or metasis of
cancers, in
particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be treated or detected by the polynucleotides or 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., Hodgkin's disease
and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
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osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphanaioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated or detected
by the polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, include AIDS; neurodegenerative disorders (such as Alzheimer's
disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa,
Cerebellar
degeneration and brain tumor or prior associated disease); autoimmune
disorders
(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
myelodysplastic syndromes (such as aplastic anemia), graft v. host disease,
ischemic
injury (such as that caused by myocardial infarction, stroke and reperfusion
injury),
liver injury (e.g., hepatitis related liver injury, ischemialreperfusion
injury, cholestosis
(bile duct injury) and liver cancer); toxin-induced liver disease (such as
that caused by
alcohol), septic shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing the polynucleotides or polypeptides, and/or
agonists
or antagonists of the invention, for therapeutic purposes, for example, to
stimulate
epithelial cell proliferation and basal keratinocytes for the purpose of wound
healing,
and to stimulate hair follicle production and healing of dermal wounds.
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Polynucleotides or polypeptides, as well as agonists or antagonists of the
invention,
may be clinically useful in stimulating wound healing including surgical
wounds,
excisional wounds, deep wounds involving damage of the dermis and epidermis,
eye
tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers,
dermal
ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting
from heat
exposure or chemicals, and other abnormal wound healing conditions such as
uremia,
malnutrition, vitamin deficiencies and complications associted with systemic
treatment with steroids, radiation therapy and antineoplastic drugs and
antimetabolites. Polynucleotides or polypeptides, and/or agonists or
antagonists of
the invention, could be used to promote dermal reestablishment subsequent to
dermal
loss
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could be used to increase the adherence of skin grafts to a wound
bed and
to stimulate re-epithelialization from the wound bed. The following are a non-
exhaustive list of grafts that polynucleotides or polypeptides, agonists or
antagonists
of the invention, could be used to increase adherence to a wound bed:
autografts,
artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular
grafts, Blair-
Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft,
dermic graft,
epidermis graft, fascia graft, full thickness graft, heterologous graft,
xenograft,
homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal
graft, Ollier-
Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft,
split skin
graft, thick split graft. The polynucleotides or polypeptides, and/or agonists
or
antagonists of the invention, can be used to promote skin strength and to
improve the
appearance of aged skin.
It is believed that the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, will also produce changes in hepatocyte
proliferation,
and epithelial cell proliferation in the lung, breast, pancreas, stomach,
small intesting,
and large intestine. The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could promote proliferation of epithelial cells
such as
sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing
goblet
cells, and other epithelial cells and their progenitors contained within the
skin, lung,
liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or
agonists
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or antagonists of the invention, may promote proliferation of endothelial
cells,
keratinocytes, and basal keratinocytes.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could also be used to reduce the side effects of gut toxicity that
result from
radiation, chemotherapy treatments or viral infections. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may have a
cytoprotective effect on the small intestine mucosa. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may also
stimulate
healing of mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could further be used in full regeneration of skin in full and
partial
thickness skin defects, including burns, (i.e., repopulation of hair
follicles, sweat
glands, and sebaceous glands), treatment of other skin defects such as
psoriasis. The
polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could
be used to treat epidermolysis bullosa, a defect in adherence of the epidermis
to the
underlying dermis which results in frequent, open and painful blisters by
accelerating
reepithelialization of these lesions. The polynucleotides or polypeptides,
and/or
agonists or antagonists of the invention, could also be used to treat gastric
and
doudenal ulcers and help heal by scar formation of the mucosal lining and
regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis,
are
diseases which result in destruction of the mucosal surface of the small or
large
intestine, respectively. Thus, the polynucleotides or polypeptides, and/or
agonists or
antagonists of the invention, could be used to promote the resurfacing of the
mucosal
surface to aid more rapid healing and to prevent progression of inflammatory
bowel
disease. Treatment with the polynucleotides or polypeptides, and/or agonists
or
antagonists of the invention, is expected to have a significant effect on the
production
of mucus throughout the gastrointestinal tract and could be used to protect
the
intestinal mucosa from injurious substances that are ingested or following
surgery.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention,
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could be used to treat diseases associate with the under expression of the
polynucleotides of the invention.
Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists
of
the invention, could be used to prevent and heal damage to the lungs due to
various
pathological states. A growth factor such as the polynucleotides or
polypeptides,
and/or agonists or antagonists of the invention, which could stimulate
proliferation
and differentiation and promote the repair of alveoli and brochiolar
epithelium to
prevent or treat acute or chronic lung damage. For example, emphysema, which
results in the progressive loss of aveoli, and inhalation injuries, i.e.,
resulting from
smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium
and
alveoli could be effectively treated using the polynucleotides or
polypeptides, and/or
agonists or antagonists of the invention. Also, the polynucleotides or
polypeptides,
and/or agonists or antagonists of the invention, could be used to stimulate
the
proliferation of and differentiation of type II pneumocytes, which may help
treat or
prevent disease such as hyaline membrane diseases, such as infant respiratory
distress
syndrome and bronchopulmonary displasia, in premature infants.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could stimulate the proliferation and differentiation of
hepatocytes and,
thus, could be used to alleviate or treat liver diseases and pathologies such
as
fulminant liver failure caused by cirrhosis, liver damage caused by viral
hepatitis and
toxic substances (i.e., acetaminophen, carbon tetraholoride and other
hepatotoxins
known in the art).
In addition, the polynucleotides or polypeptides, and/or agonists or
antagonists
of the invention, could be used treat or prevent the onset of diabetes
mellitus. In
patients with newly diagnosed Types I and II diabetes, where some islet cell
function
remains, the polynucleotides or polypeptides, and/or agonists or antagonists
of the
invention, could be used to maintain the islet function so as to alleviate,
delay or
prevent permanent manifestation of the disease. Also, the polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, could be used
as an
auxiliary in islet cell transplantation to improve or promote islet cell
function.
Neurological Diseases
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Nervous system disorders, which can be treated with the compositions of the
invention (e.g., polypeptides, polynucleotides, and/or agonists or
antagonists),
include, but are not limited to, nervous system injuries, and diseases or
disorders
which result in either a disconnection of axons, a diminution or degeneration
of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient
(including human and non-human mammalian patients) according to the invention,
include but are not limited to, the following lesions of either the central
(including
spinal cord, brain) or peripheral nervous systems: ( 1 ) 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 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 or disorders, in which a portion of the nervous
system is
destroyed or injured by a nutritional disorder or disorder of metabolism
including but
not limited to, vitamin B 12 deficiency, folic acid deficiency, Wernicke
disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration
of
the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological
lesions
associated with systemic diseases including, but not limited to, diabetes
(diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or
sarcoidosis;
(8) lesions caused by toxic substances including alcohol, lead, or particular
neurotoxins; and (9) demyelinated lesions in which a portion of the nervous
system
is destroyed or injured by a demyelinating disease including, but not limited
to,
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multiple sclerosis, human immunodeficiency virus-associated myelopathy,
transverse
myelopathy or various etiologies, progressive multifocal leukoencephalopathy,
and
central pontine myelinolysis.
In a preferred embodiment, the polypeptides, polynucleotides. or agonists or
antagonists of the invention are used to protect neural cells from the
damaging effects
of cerebral hypoxia. According to this embodiment, the compositions of the
invention are used to treat or prevent 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 or prevent 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 or prevent 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 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 or prevent neural cell
injury associated
with a heart attack.
The compositions of the invention which are useful for treating or preventing
a nervous system disorder may be selected by testing for biological activity
in
promoting the survival or differentiation of neurons. For example, and not by
way of
limitation, compositions of the invention which elicit any of the following
effects may
be useful according to the invention: ( 1 ) increased survival time of neurons
in culture;
(2) increased sprouting of neurons in culture or in vivo; (3) increased
production of a
neuron-associated molecule in culture or in vivo, e.g., choline
acetyltransferase or
acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms
of
neuron dysfunction in vivo. Such effects may be measured by any method known
in
the art. In preferred, non-limiting embodiments, increased survival of neurons
may
routinely be measured using a method set forth herein or otherwise known in
the art,
such as, for example, the method set forth in Arakawa et al. (J. Neurosci.
10:3507-3515 ( 1990)); increased sprouting of neurons may be detected by
methods
known in the art, such as, for example, the methods set forth in Pestronk et
al. (Exp.
Neurol. 70:65-82 ( 1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 ( 1981
));
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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 disorders that may be treated
according to the invention include, but are not limited to, disorders such as
infarction,
infection, exposure to toxin, trauma, surgical damage, degenerative disease or
malignancy that may affect motor neurons as well as other components of the
nervous
system, as well as disorders that selectively affect neurons such as
amyotrophic lateral
sclerosis, and including, but not limited to, progressive spinal muscular
atrophy,
progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile
muscular
atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary Motorsensory
Neuropathy
(Charcot-Marie-Tooth Disease).
Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present
invention can be used to treat or detect infectious agents. For example, by
increasing
the immune response, particularly increasing the proliferation and
differentiation of B
and/or T cells, infectious diseases may be treated. 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 or detected by a polynucleotide or polypeptide
and/or
agonist or antagonist of the present invention. Examples of viruses, include,
but are
not limited to Examples of viruses, include, but are not limited to the
following DNA
and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae,
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Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavinis (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 or detect any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat: 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 AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can be treated or detected by a polynucleotide or polypeptide and/or
agonist or
antagonist of the present invention include, but not limited to, include, but
not limited
to, the following Gram-Negative and Gram-positive bacteria and bacterial
families
and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium),
Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia
burgdorferi),
Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic
E.
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coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and
Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter,
Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae,
Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Meniaococcal),
Meisseria meningitides, Pasteurellacea Infections (e.g., Actinobacillus,
Heamophilus
(e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas,
Rickettsiaceae,
Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal,
Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the following
diseases or
symptoms, including, but not limited to: bacteremia, endocarditis, eye
infections
(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections
(e.g.; AIDS
related infections), paronychia, prosthesis-related infections, Reiter's
Disease,
respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme
Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,
Typhoid, pneumonia, Gonorrhea, 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 or detect any of these
symptoms or
diseases. In specific embodiments, Ppolynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat: tetanus, Diptheria, botulism,
and/or
meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated or
detected by a polynucleotide or polypeptide and/or agonist or antagonist of
the
present invention include, but not limited to, the following families or
class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,
Dourine,
Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,
Toxoplasmosis,
Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,
Plasmodium falciparium, Plasmodium malariae and Plasmodium ovate). These
parasites can cause a variety of diseases or symptoms, including, but not
limited to:
Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,
giardiasis),
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liver disease, lung disease, opportunistic infections (e.g., AIDS related),
malaria,
pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides,
or
agonists or antagonists of the invention. can be used to treat or detect any
of these
symptoms or diseases. In specific embodiments, polynucleotides, polypeptides,
or
agonists or antagonists of the invention are used to treat malaria.
Preferably, treatment using a polypeptide or polynucleotide and/or agonist or
antagonist of the present invention could either be by administering an
effective
amount of a polypeptide to the patient, or by removing cells from the patient,
supplying the cells with a polynucleotide of the present invention, and
returning the
engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide
or
polynucleotide of the present invention can be used as an antigen in a vaccine
to raise
an immune response against infectious disease.
Regeneration
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention can be used to differentiate, proliferate, and attract cells,
leading to the
regeneration of tissues. (See, Science 276:59-87 ( 1997).) The regeneration of
tissues
could be used to repair, replace, or protect tissue damaged by congenital
defects,
trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery, including
cosmetic plastic
surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
or cardiac), vasculature (including vascular and lymphatics), nervous,
hematopoietic,
and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration
occurs without or decreased scarring. Regeneration also may include
angiogenesis.
Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention may increase regeneration of tissues difficult to heal. For
example,
increased tendon/ligament regeneration would quicken recovery time after
damage.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention
could also be used prophylactically in an effort to avoid damage. Specific
diseases
that could be treated include of tendinitis, carpal tunnel syndrome, and other
tendon or
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ligament defects. A further example of tissue regeneration of non-healing
wounds
includes pressure ulcers, ulcers associated with vascular insufficiency,
surgical, and
traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide or polypeptide and/or agonist or antagonist of the present
invention to
proliferate and differentiate nerve cells. Diseases that could be treated
using this
method include central and peripheral nervous system diseases, neuropathies,
or
mechanical and traumatic disorders (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 using the
polynucleotide or
polypeptide and/or agonist or antagonist of the present invention.
Chemotaxis
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes
cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils,
epithelial and/or endothelial cells) to a particular site in the body, such as
inflammation, infection, or site of hyperproliferation. The mobilized cells
can then
fight off and/or heal the particular trauma or abnormality.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may increase chemotaxic activity of particular cells. These
chemotactic
molecules can then be used to treat inflammation, infection,
hyperproliferative
disorders, 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 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 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
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could also be used to treat disorders. Thus, a polynucleotide or polypeptide
and/or
agonist or antagonist of the present invention could be used as an inhibitor
of
chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules
that bind to the polypeptide or for molecules to which the polypeptide binds.
The
binding of the polypeptide and the molecule may activate (agonist), increase,
inhibit
(antagonist). or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleotides, proteins (e.g.,
receptors),or
small molecules.
Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand,
a structural
or functional mimetic. (See, Coligan et al., Current Protocols in Immunology
1 (2):Chapter 5 ( 1991 ).) Similarly, the molecule can be closely related to
the natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide, either as a secreted protein or on the
cell
membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli.
Cells expressing the polypeptide (or cell membrane containing the expressed
polypeptide) are then preferably contacted with a test compound potentially
containing the molecule to observe binding, stimulation, or inhibition of
activity of
either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the
polypeptide, wherein binding is detected by a label, or in an assay involving
competition with a labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
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mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The
antibody can measure polypeptide level or activity by either binding, directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
Additionally, the receptor to which a polypeptide of the invention binds can
be
identified by numerous methods known to those of skill in the art, for
example, ligand
panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1 (2),
Chapter 5, ( 1991 )). For example, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the polypeptides, for
example, NIH3T3 cells which are known to contain multiple receptors for the
FGF
family proteins, and SC-3 cells, and a cDNA library created from this RNA is
divided
into pools and used to transfect COS cells or other cells that are not
responsive to the
polypeptides. Transfected cells which are grown on glass slides are exposed to
the
polypeptide of the present invention, after they have been labelled. The
polypeptides
can be labeled by a variety of means including iodination or inclusion of a
recognition
site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-
radiographic analysis. Positive pools are identified and sub-pools are
prepared and re-
transfected using an iterative sub-pooling and re-screening process,
eventually
yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffinity linked with cell membrane or extract preparations that
express
the receptor molecule. Cross-linked material is resolved by PAGE analysis and
exposed to X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and subjected to
protein
microsequencing. The amino acid sequence obtained from microsequencing would
be used to design a set of degenerate oligonucleotide probes to screen a cDNA
library
to identify the genes encoding the putative receptors.
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Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA shuffling") may be
employed to modulate the activities of polypeptides of the invention thereby
effectively generating agonists and antagonists of polypeptides of the
invention. See
generally, U.S. Patent Nos. 5,605,793. 5,811,238, 5,830,721, 5,834,252, and
5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (
1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 ( 1998); Hansson, L. O., et al.,
J. Mol.
Biol. 287:265-76 ( 1999); and Lorenzo, M. M. and Blasco, R. Biotechniques
24(2):308-13 (1998) (each of these patents and publications are hereby
incorporated
by reference). In one embodiment, alteration of polynucleotides and
corresponding
polypeptides of the invention may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments into a desired
polynucleotide
sequence of the invention molecule by homologous, or site-specific,
recombination.
In another embodiment, polynucleotides and corresponding polypeptides of the
invention may be alterred by being subjected to random mutagenesis by error-
prone
PCR, random nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections, parts, domains,
fragments, etc., of the polypeptides of the invention may be recombined with
one or
more components, motifs, sections, parts, domains, fragments, etc. of one or
more
heterologous molecules. In preferred embodiments, the heterologous molecules
are
family members. In further preferred embodiments, the heterologous molecule is
a
growth factor such as, for example, platelet-derived growth factor (PDGF),
insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha,
epidermal
growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic
protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B,
decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors
(GDFs),
nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-betas, and
glial-
derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the
polypeptides of the invention. Biologically active fragments are those
exhibiting
activity similar, but not necessarily identical, to an activity of the
polypeptide. The
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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.
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 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
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(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.
Additional preferred embodiments of the invention are directed to polypeptides
which
comprise, or alternatively consist of, the amino acid sequence of each of the
beta
pleated sheet regions in one of the polypeptide sequences of the invention.
Additional
embodiments of the invention are directed to polypeptides which comprise, or
alternatively consist of, any combination or all of the beta pleated sheet
regions in one
of the polypeptide sequences of the invention.
Targeted Delivery
In another embodiment, the invention provides a method of delivering
compositions to targeted cells expressing a receptor for a polypeptide of the
invention,
or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be
associated with heterologous polypeptides, heterologous nucleic acids, toxins,
or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In
one
embodiment, the invention provides a method for the specific delivery of
compositions of the invention to cells by administering polypeptides of the
invention
(including antibodies) that are associated with heterologous polypeptides or
nucleic
acids. In one example, the invention provides a method for delivering a
therapeutic
protein into the targeted cell. In another example, the invention provides a
method for
delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or
double
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stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or
replicate
episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention (e.g., polypeptides of the invention or antibodies of the
invention) in
association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic
effector systems, radioisotopes, holotoxins, modified toxins, catalytic
subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell
that under defined conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not limited to,
radioisotopes
known in the art, compounds such as, for example, antibodies (or complement
fixing
containing portions thereof) that bind an inherent or induced endogenous
cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin,
abrin,
Pseudomorzcis exotoxin A, diphtheria toxin, saporin, momordin, gelonin,
pokeweed
antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is
meant a
non-toxic compound that is converted by an enzyme, normally present in the
cell, into
a cytotoxic compound. Cytotoxic prodrugs that may be used according to the
methods of the invention include, but are not limited to, glutamyl derivatives
of
benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or
mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives
of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention,
or
the polynucleotides encoding these polypeptides, to screen for molecules which
modify the activities of the polypeptides of the present invention. Such a
method
would include contacting the polypeptide of the present invention with a
selected
compounds) suspected of having antagonist or agonist activity, and assaying
the
activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by
using the polypeptides of the present invention, or binding fragments thereof,
in any
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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 anent being tested and a polypeptide of the present
invention.
Thus, the present invention provides methods of screening for drugs or any
other agents which affect activities mediated by the polypeptides of the
present
invention. These methods comprise contacting such an agent with a polypeptide
of the
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 13, 1984, which is incorporated herein by reference
herein.
Briefly stated, large numbers of different small peptide test compounds are
synthesized on a solid substrate, such as plastic pins or some other surface.
The
peptide test compounds are reacted with polypeptides of the present invention
and
washed. Bound polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in the
aforementioned
drug screening techniques. In addition, non-neutralizing antibodies may be
used to
capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing antibodies capable of binding polypeptides of the
present
invention specifically compete with a test compound for binding to the
polypeptides
or fragments thereof. In this manner, the antibodies are used to detect the
presence of
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any peptide which shares one or more antigenic epitopes with a polypeptide of
the
invention.
Antisense And Ribozvme (Antagonists)
In specific embodiments, antagonists according to the present invention are
nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the
complementary strand thereof, and/or to nucleotide sequences contained a
deposited
clone. In one embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is separately
administered
(see, for example, O'Connor, Neurochem., 56:60 (1991). Oligodeoxynucleotides
as
Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988).
Antisense technology can be used to control gene expression through antisense
DNA
or RNA, or through triple-helix formation. Antisense techniques are discussed
for
example, in Okano, Neurochem., 56:560 ( 1991 ); Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix
formation is discussed in, for instance, Lee et al., Nucleic Acids Research,
6:3073
(1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science,
251:1300
(1991). The methods are based on binding of a polynucleotide to a
complementary
DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit
the growth of the non-lymphocytic leukemia cell line HL-60 and other cell
lines was
previously described. (Wickstram et al. ( 1988); Anfossi et al. ( 1989)).
These
experiments were performed in vitro by incubating cells with the
oligoribonucleotide.
A similar procedure for in vivo use is described in WO 91/15580. Briefly, a
pair of
oligonucleotides for a given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is flanked by an
EcoRl
site on the 5 end and a HindIII site on the 3 end. Next, the pair of
oligonucleotides is
heated at 90°C for one minute and then annealed in 2X ligation buffer
(20mM TRIS
HCl pH 7.5, IOmM MgCl2, IOMM dithiothreitol (DTT) and 0.2 mM ATP) and then
ligated to the EcoRl/Hind III site of the retroviral vector PMV7 (WO
91/15580).
For example, the 5' coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an antisense RNA
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oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oli~onucleotide
is designed to be complementary to a region of the gene involved in
transcription
thereby preventing transcription and the production of the receptor. The
antisense
RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of
the
mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced
intracellularly by transcription from an exogenous sequence. For example, a
vector or
a portion thereof. is transcribed, producing an antisense nucleic acid (RNA)
of the
invention. Such a vector would contain a sequence encoding the antisense
nucleic
acid of the invention. Such a vector can remain episomal or become
chromosomally
integrated, as long as it can be transcribed to produce the desired antisense
RNA.
Such vectors can be constructed by recombinant DNA technology methods standard
in the art. Vectors can be plasmid, viral, or others known in the art, used
for
replication and expression in vertebrate cells. Expression of the sequence
encoding a
polypeptide of the invention, or fragments thereof, can be by any promoter
known in
the art to act in vertebrate, preferably human cells. Such promoters can be
inducible
or constitutive. Such promoters include. but are not limited to, the SV40
early
promoter region (Bernoist and Chambon, Nature, 29:304-310 ( 1981 ), the
promoter
contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et
al.,
Cell, 22:787-797 ( 1980), the herpes thymidine promoter (Wagner et al., Proc.
Natl.
Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a gene of
interest.
However, absolute complementarity, although preferred, is not required. A
sequence
"complementary to at least a portion of an RNA," referred to herein, means a
sequence having sufficient complementarity to be able to hybridize with the
RNA,
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
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invention it may contain and still form a stable duplex (or triplex as the
case may be).
One skilled in the art can ascertain a tolerable degree of mismatch by use of
standard
procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the message, e.g.,
the 5' untranslated sequence up to and including the AUG initiation codon,
should
work most efficiently at inhibiting translation. However, sequences
complementary
to the 3' untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See Generally, Wagner, R., Nature,
372:333-335 ( 1994). Thus, oligonucleotides complementary to either the 5' -
or 3' -
non- translated, non-coding regions of a polynucleotide sequence of the
invention
could be used in an antisense approach to inhibit translation of endogenous
mRNA.
Oligonucleotides complementary to the 5' untranslated region of the mRNA
should
include the complement of the AUG start codon. Antisense oligonucleotides
complementary to mRNA coding regions are less efficient inhibitors of
translation but
could be used in accordance with the invention. Whether designed to hybridize
to the
5' -, 3' - or coding region of mRNA, antisense nucleic acids should be at
least six
nucleotides in length, and are preferably oligonucleotides ranging from 6 to
about 50
nucleotides in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety, or
phosphate backbone, for example, to improve stability of the molecule,
hybridization,
etc. The oligonucleotide may include other appended groups such as peptides
(e.g.,
for targeting host cell receptors in vivo), or agents facilitating transport
across the cell
membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-
6556
( 1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 ( 1987); PCT
Publication
NO: W088/09810, published December 15, 1988) or the blood-brain barrier (see,
e.g., PCT Publication NO: W089/10134, published April 25, 1988), hybridization-
triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976
(1988))
or intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549 ( 1988)). To
this end,
the oligonucleotide may be conjugated to another molecule, e.g., a peptide,
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hybridization tri;~ered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including, but not limited to. 5-
fluorouracil.
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine,
5-(carboxyhydroxylmethyl) uracil. ~-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methyl guanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, ~-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid
(v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar
moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment. the antisense oligonucleotide comprises at least
one modified phosphate backbone selected from the group including, but not
limited
to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual b-units, the
strands
run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (
1987)).
The oligonucleotide is a 2-0-methylribonucleotide (moue et al., Nucl. Acids
Res.,
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (moue et al., FEBS Lett.
215:327-330 (1987)).
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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 oliaonucleotides may be synthesized by the method of Stein et
al.
(Nucl. Acids Res., 16:3209 ( 1988)), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports (Sarin et al., Proc.
Natl.
Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.
While antisense nucleotides complementary to the coding region sequence of
the invention could be used, those complementary to the transcribed
untranslated
region are most preferred.
Potential antagonists according to the invention also include catalytic RNA,
or
a ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes
that
cleave mRNA at site specific recognition sequences can be used to destroy
mRNAs
corresponding to the polynucleotides of the invention, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated
by flanking regions that form complementary base pairs with the target mRNA.
The
sole requirement is that the target mRNA have the following sequence of two
bases:
5' -UG-3' . The construction and production of hammerhead ribozymes is well
known in the art and is described more fully in Haseloff and Gerlach, Nature,
334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage
sites within each nucleotide sequence disclosed in the sequence listing.
Preferably,
the ribozyme is engineered so that the cleavage recognition site is located
near the 5'
end of the mRNA corresponding to the polynucleotides of the invention; i.e.,
to
increase efficiency and minimize the intracellular accumulation of non-
functional
mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be composed
of modified oligonucleotides (e.g. for improved stability, targeting, etc.)
and should
be delivered to cells which express the polynucleotides of the invention in
vivo.
DNA constructs encoding the ribozyme may be introduced into the cell in the
same
manner as described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct "encoding" the
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ribozyme under the control of a strong constitutive promoter, such as, for
example,
pol III or pol II promoter, so that transfected cells will produce sufficient
quantities of
the ribozyme to destroy endogenous messages and inhibit translation. Since
ribozymes unlike antisense molecules. are catalytic, a lower intracellular
concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth
and proliferation effects of the polypeptides of the present invention on
neoplastic
cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore,
retard or
prevent abnormal cellular growth and proliferation, for example, in tumor
formation
or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular
diseases, and prevent the proliferation of epithelial lens cells after
extracapsular
cataract surgery. Prevention of the mitogenic activity of the polypeptides of
the
present invention may also be desirous in cases such as restenosis after
balloon
angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar
tissue during wound healing.
The antagonist/agonist may also be employed to treat the diseases described
herein.
Thus, the invention provides a method of treating disorders or diseases,
including but
not limited to the disorders or diseases 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
Other Activities
The polypeptide of the present invention, as a result of the ability to
stimulate
vascular endothelial cell growth, may be employed in treatment for stimulating
re-
vascularization of ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions. These
polypeptide
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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 fibroblast cells and skeletal muscle cells, and
therefore,
facilitate the repair or replacement of damaged or diseased tissue.
The polypeptide of the present invention may also be employed stimulate
neuronal growth and to treat and prevent neuronal damage which occurs in
certain
neuronal disorders or neuro-degenerative conditions such as Alzheimer's
disease,
Parkinson's disease, and AIDS-related complex. The polypeptide of the
invention
may have the ability to stimulate chondrocyte growth, therefore, they may be
employed to enhance bone and periodontal regeneration and aid in tissue
transplants
or bone grafts.
The polypeptide of the present invention may be also be employed to prevent
skin aging due to sunburn by stimulating keratinocyte growth.
The polypeptide of the invention may also be employed for preventing hair
loss, since FGF family members activate hair-forming cells and promotes
melanocyte
growth. Along the same lines, the polypeptides of the present invention may be
employed to stimulate growth and differentiation of hematopoietic cells and
bone
marrow cells when used in combination with other cytokines.
The polypeptide of the invention may also be employed to maintain organs
before transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing
tissue of mesodermal origin to differentiate in early embryos.
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 andlor agonist or antagonists of the
present invention may also be used to modulate mammalian characteristics, such
as
body height, weight, hair color, eye color, skin, percentage of adipose
tissue,
pigmentation, size, and shape (e.g., cosmetic surgery). Similarly,
polypeptides or
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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 aaonist 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
disorders), tendency for violence, tolerance for pain, reproductive
capabilities
(preferably by Activin or Inhibin-like activity), hormonal or endocrine
levels,
appetite, libido, memory, stress, or other cognitive qualities.
Polypeptide or polynucleotides and/or agonist or antagonists of the present
invention may also be used as a food additive or preservative, such as to
increase or
decrease storage capabilities, fat content, lipid, protein, carbohydrate,
vitamins,
minerals, cofactors or other nutritional components.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical to a sequence of at least about 50 contiguous nucleotides in the
nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5 ~
Nucleotide of
the Clone Sequence and ending with the nucleotide at about the position of the
3
Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of
the Start Codon and ending with the nucleotide at about the position of the 3'
Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
Similarly preferred is a nucleic acid molecule wherein said sequence of
contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X
in the
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range of positions beginning with the nucleotide at about the position of the
5'
Nucleotide of the First Amino Acid of the Signal Peptide and ending with the
nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as
defined for SEQ ID NO:X in Table 1.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 150
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 500
contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide sequence which is at least 95% identical to the nucleotide sequence
of SEQ
ID NO:X beginning with the nucleotide at about the position of the 5'
Nucleotide of
the First Amino Acid of the Signal Peptide and ending with the nucleotide at
about
the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID
NO:X
in Table 1.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence of SEQ ID NO:X.
Also preferred is an isolated nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule, wherein said
nucleic
acid molecule which hybridizes does not hybridize under stringent
hybridization
conditions to a nucleic acid molecule having a nucleotide sequence consisting
of only
A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which
comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1,
which DNA molecule is contained in the material deposited with the American
Type
Culture Collection and given the ATCC Deposit Number shown in Table 1 for said
cDNA Clone Identifier.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in the nucleotide sequence of a human cDNA clone identified by a
cDNA
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Clone Identifier in Table l, which DNA molecule is contained in the deposit
given the
ATCC Deposit Number shown in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of
at least 50 contiguous nucleotides is included in the nucleotide sequence of
the
complete open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to sequence of at least 150
contiguous
nucleotides in the nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to sequence
of at
least 500 contiguous nucleotides in the nucleotide sequence encoded by said
human
cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is a method for detecting in a biological
sample a nucleic acid molecule comprising a nucleotide sequence which is at
least
95% identical to a sequence of at least 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; which method comprises a step of comparing a nucleotide sequence of at
least one
nucleic acid molecule in said sample with a sequence selected from said group
and
determining whether the sequence of said nucleic acid molecule in said sample
is at
least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a nucleic acid molecule comprising said sequence
selected from said group. Similarly, also preferred is the above method
wherein said
step of comparing sequences is performed by comparing the nucleotide sequence
determined from a nucleic acid molecule in said sample with said sequence
selected
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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 continuous nucleotides in a
sequence
selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table 1; and a nucleotide sequence
encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
The method for identifying the species, tissue or cell type of a biological
sample can comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide sequences, wherein
at least
one sequence in said panel is at least 95% identical to a sequence of at least
50
contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein identified in Table 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 l;
and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA
Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at
least two nucleotide sequences, wherein at least one sequence in said panel is
at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from said group.
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Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise
a panel of at least two nucleotide sequences, wherein at least one sequence in
said
panel is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a
sequence selected from the group consisting of: a nucleotide sequence of SEQ
ID
NO:X wherein X is any integer as defined in Table l; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about 10 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1.
Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in the amino acid sequence of SEQ ID NO:Y in the range of
positions beginning with the residue at about the position of the First Amino
Acid of
the Secreted Portion and ending with the residue at about the Last Amino Acid
of the
Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to a sequence of at least about 100 contiguous
amino
acids in the amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to the complete amino acid sequence of SEQ ID
NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 90% identical to a sequence of at least about 10 contiguous
amino
acids in the complete amino acid sequence of a secreted protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
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Also preferred is a polypeptide wherein said sequence of contiguous amino
acids is included in the amino acid sequence of a secreted portion of the
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in
the amino acid sequence of the secreted portion of the protein encoded by a
human
cDNA clone identified by a eDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to 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
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table l; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
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
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defined in Table 1; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1; which method comprises a step of comparing an amino acid sequence of at
least
one polypeptide molecule in said sample with a sequence selected from said
group
and determining whether the sequence of said polypeptide molecule in said
sample is
at least 90% identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino
acid sequence of at least one polypeptide molecule in said sample with a
sequence
selected from said group comprises determining the extent of specific binding
of
polypeptides in said sample to an antibody which binds specifically to a
polypeptide
comprising an amino acid sequence that is at least 90% identical to a sequence
of at
least 10 contiguous amino acids in a sequence selected from the group
consisting of:
an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
IS Table 1; and a complete amino acid sequence of a protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is the above method wherein said step of comparing sequences
is performed by comparing the amino acid sequence determined from a
polypeptide
molecule in said sample with said sequence selected from said group.
Also preferred is a method for identifying the species, tissue or cell type of
a
biological sample which method comprises a step of detecting polypeptide
molecules
in said sample, if any, comprising an amino acid sequence that is at least 90%
identical to a sequence of at least 10 contiguous amino acids in a sequence
selected
from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y
is
any integer as defined in Table l; and a complete amino acid sequence of a
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is the above method for identifying the species, tissue or cell
type of a biological sample, which method comprises a step of detecting
polypeptide
molecules comprising an amino acid sequence in a panel of at least two amino
acid
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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 l, which method comprises a step of detecting in a
biological sample obtained from said subject polypeptide molecules comprising
an
amino acid sequence in a panel of at least two amino acid sequences, wherein
at least
one sequence in said panel is at least 90% identical to a sequence of at least
10
contiguous amino acids in a sequence selected from the group consisting of: an
amino
acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
In any of these methods, the step of detecting said polypeptide molecules
includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence encoding a
polypeptide wherein said polypeptide comprises an amino acid sequence that is
at
least 90% identical to a sequence of at least 10 contiguous amino acids in a
sequence
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 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 l;
and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
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identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Further preferred is a method of making a recombinant vector comprising
inserting any of the above isolated nucleic acid molecule into a vector. Also
preferred
is the recombinant vector produced by this method. Also preferred is a method
of
making a recombinant host cell comprising introducing the vector into a host
cell, as
well as the recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising
culturing this recombinant host cell under conditions such that said
polypeptide is
expressed and recovering said polypeptide. Also preferred is this method of
making
an isolated polypeptide, wherein said recombinant host cell is a eukaryotic
cell and
said polypeptide is a secreted portion of a human secreted protein comprising
an
amino acid sequence selected from the group consisting of: an amino acid
sequence of
SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid
of
the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table
1 and
said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y
is
defined in Table 1; and an amino acid sequence of a secreted portion of a
protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The isolated polypeptide produced by this method is also
preferred.
Also preferred is a method of treatment of an individual in need of an
increased level of a secreted protein activity, which method comprises
administering
to such an individual a pharmaceutical composition comprising an amount of an
isolated polypeptide, polynucleotide, or antibody of the claimed invention
effective to
increase the level of said protein activity in said individual.
The above-recited applications have uses in a wide variety of hosts. Such
hosts include, but are not limited to, human, murine, rabbit, goat, guinea
pig, camel,
horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog,
cat, non-
human primate, and human. In specific embodiments, the host is a mouse,
rabbit,
goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred
embodiments, the host is a mammal. In most preferred embodiments, the host is
a
human.
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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
2 HAGFF43 12 821316 N48187, N49746, N52675, AAl
15107
7 HKACA25 17 824087 T71107. 807491, 807544, 802367,
802473,
R I 2602, 874032, 874123, 879290,
881173,
881277, 886952, H49320, N54909,
AA196897
9 HAQBG57 19 837545 869327, AA507216, AA593594
10 HAQBY85 20 832384 T64857, H88471, W89023, W89024,
AA036826,
AA158032, AA253382, AA688389,
AA748991,
AA837024, AA888418, AA935978,
C21449
14 HAGEW83 24 61711 AA424534, AA424600
1
HAGEX49 25 834502 T89093. 860840, H16750, H51569,
H51939.
AI00561 1
16 HAGFM58 26 604536 H05533
HOEBI94 30 795312 T77192, 802068, 839545, H03581,
883018,
892004, H57629, H57730, N30564,
N63606,
N73133, N79282, N94803, W32888,
W35158,
AA053126, AA064861, AA065119,
AA081354,
AA088706, AA088774, AA088775,
AA088772,
AA101806, AA182658, AA196568,
AA196670,
AA233292, AA236666, AA256263,
AA256359,
AA425103. AA429324
22 HAIBT20 32 741086 AA461563
23 HAICE62 33 834523 N35973, N70484, AA236276, AA234286,
AA417589, AA482028, AA488888,
AA554535,
AA633532, AA715033, AA765790,
AA923504,
AA987966, AI027570, C00553
26 HAJAZ56 36 716168 869788, N62743, N79684, W52081,
AA084615
29 HAMGGO1 39 783864 855333, N24631, N35833. W63691,
AA070411,
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AA 147489. AA253000, AA252999,
AA255718.
AA262075
31 HAPBU86 41 789544 T77303, 820876, 838187. 842056.
W56173,
AA030021
32 HAPNL62 42 790340 T92717, T92796. 822380, N77136,
AA018552,
A.A037814, AA037815. AA 146872.
AA 146873.
AA 192996. AA I 93047, AA424312
33 HAPN050 43 834384 852145, N80724, W52200, W56688,
W56696,
W76539. AA588352, AA593340,
AA593332,
AA742572, AA865071, AA977531,
AA988767.
AI002202, AI081543
37 HAPQU71 47 752580 AA459527
42 HAQCF94 52 795993 823565, H52947
43 HARAT69 53 769389 T50337, T61914, T79997. 837580,
H29047,
H29149, AA 100250
45 HAGEQ79 55 828055 T72606, 855798, H46138, H91757.
W73069,
W92767, AA044775, AA975324
46 HAGHN57 56 773286 T60588, H16095, N58884, W01852,
W31025,
AA101864, AA134574, AA151047,
AA182513,
AA227675
47 HASAY07 57 834511 C18262
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Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Examples
Example l: Isolation of a Selected eDNA 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 1 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), pBluescript (pBS) (Short, J. M. et al., Nucleic
Acids Res.
16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res.
17:9494 (1989)) and pBK (Aping-Mees, M. A. et al., Strategies 5:58-61 (1992))
are
commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey
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Pines Road. La Jolla, CA, 92037. pBS contains an ampicillin resistance gene
and
pBK contains a neomycin resistance gene. Both can be transformed into E. coli
strain
XL-1 Blue, also available from Strataaene. pBS comes in 4 forms SK+, SK-, KS+
and KS. The S and K refers to the orientation of the polylinker to the T7 and
T3
primer sequences which flank the polylinker region ("S" is for SacI and "K" is
for
KpnI which are the first sites on each respective end of the linker). "+" or "-
" refer to
the orientation of the fl origin of replication ("ori"), such that in one
orientation,
single stranded rescue initiated from the f 1 on Generates sense strand DNA
and in the
other, antisense.
Vectors pSportl, pCMVSport 2.0 and pCMVSport 3.0, were obtained from
Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport
vectors
contain an ampicillin resistance gene and may be transformed into E. coli
strain
DH10B, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University,
NY) contains an ampicillin resistance gene and can be transformed into E. coli
strain
XL-1 Blue. Vector pCR"2.1, which is available from Invitrogen, 1600 Faraday
Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed into E. coli strain DH l OB, available from Life Technologies.
(See, for
instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 ( 1988) and Mead, D. et
al.,
Bio/Technology 9: ( 1991 ).) Preferably, a polynucleotide of the present
invention
does not comprise the phage vector sequences identified for the particular
clone in
Table 1, as well as the corresponding plasmid vector sequences designated
above.
The deposited material in the sample assigned the ATCC Deposit Number
cited in Table 1 for any given cDNA clone also may contain one or more
additional
plasmids, each comprising a cDNA clone different from that given clone. Thus,
deposits sharing the same ATCC Deposit Number contain at least a plasmid for
each
cDNA clone identified in Table 1. Typically, each ATCC deposit sample cited in
Table 1 comprises a mixture of approximately equal amounts (by weight) of
about 50
plasmid DNAs, each containing a different cDNA clone; but such a deposit
sample
may include plasmids for more or less than 50 cDNA clones, up to about 500
cDNA
clones.
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21;
Two approaches can be used to isolate a particular clone from the deposited
sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is
directly
isolated by screening the clones usiny> 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 ''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 Harbor Laboratory Press, pages 1.93 to
1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the
SEQ 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
1.5-5 mM MgCh, 0.01 % (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25
pmol of each primer and 0.25 Unit of Taq polymerise. Thirty five cycles of PCR
(denaturation at 94 degree C for 1 min; annealing at 55 degree C for 1 min;
elongation
at 72 degree C for 1 min) are performed with a Perkin-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and
the DNA band with expected molecular weight is excised and purified. The PCR
product is verified to be the selected sequence by subcloning and sequencing
the
DNA product.
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Several methods are available for the identification of the 5' or 3' non-
coding
portions of a gene which may not be present in the deposited clone. These
methods
include but are not limited to, filter probing, clone enrichment using
specific probes,
and protocols similar or identical to 5' and 3' "RACE" protocols which are
well
known in the art. For instance, a method similar to 5' RACE is available for
generating the missing 5' end of a desired full-length transcript. (Fromont-
Racine et
al., Nucleic Acids Res. 21 (7):1683-1684 ( 1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a
population of RNA presumably containing full-length gene RNA transcripts. A
primer set containing a primer specific to the ligated RNA oliQonucleotide and
a
primer specific to a known sequence of the gene of interest is used to PCR
amplify
the 5' portion of the desired full-length gene. This amplified product may
then be
sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source,
although poly-A+ RNA can be used. The RNA preparation can then be treated with
phosphatase if necessary to eliminate 5' phosphate groups on degraded or
damaged
RNA which may interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid pyrophosphatase in
order
to remove the cap structure present at the 5' ends of messenger RNAs. This
reaction
leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can
then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction is
used as a template for PCR amplification of the desired 5' end using a primer
specific
to the ligated RNA oligonucleotide and a primer specific to the known sequence
of
the gene of interest. The resultant product is then sequenced and analyzed to
confirm
that the 5' end sequence belongs to the desired gene.
Example 2: Isolation of Genomic Clones Corresponding to a Polvnucleotide
A human genomic Pl library (Genomic Systems, Inc.) is screened by PCR
using primers selected for the cDNA sequence corresponding to SEQ ID NO:X.,
according to the method described in Example 1. (See also, Sambrook.)
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Example 3: Tissue Distribution of PolXpeptide
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 P'' using the rediprimeTM DNA labeling
system (Amersham Life Science), according to manufacturer's instructions.
After
labeling, the probe is purified using CHROMA SPIN-100~'~M column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The
purified labeled probe is then used to examine various human tissues for mRNA
expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H)
or human immune system tissues (IM) (Clontech) are examined with the labeled
probe using ExpressHybTM hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization and washing,
the
blots are mounted and exposed to film at -70 degree C overnight, and the films
developed according to standard procedures.
Example 4: Chromosomal Ma~in~ of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5'
end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This
primer set is then used in a polymerise chain reaction under the following set
of
conditions : 30 seconds,95 degree C; 1 minute, 56 degree C; 1 minute, 70
degree C.
This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C.
Human, mouse, and hamster DNA is used as template in addition to a somatic
cell
hybrid panel containing individual chromosomes or chromosome fragments (Bios,
Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5 %
agarose
gels. Chromosome mapping is determined by the presence of an approximately 100
by PCR fragment in the particular somatic cell hybrid.
Example 5: Bacterial Expression of a Polypeptide
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216
A polynucleotide encoding a polypeptide of the present invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA
sequence, as outlined in Example l, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHI and XbaI, at the 5' end of the primers in order to clone the amplified
product
into the expression vector. For example, BamHI and XbaI correspond to the
restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc.,
Chatsworth, CA). This plasmid vector encodes antibiotic resistance (Ampr), a
bacterial origin of replication (ori), an IPTG-regulatable promoter/operator
(P/O), a
ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme
cloning
sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified
fragment is ligated into the pQE-9 vector maintaining the reading frame
initiated at
the bacterial RBS. The ligation mixture is then used to transform the E. coli
strain
M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4,
which
expresses the lacI repressor and also confers kanamycin resistance (Kanr).
Transformants are identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated
and
confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/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 1
mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading
to
increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCl by 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-NTA") affinity resin column
(available from
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QIAGEN, Inc., supr-n). 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
QIAexpressionist ( 1995) QIAGEN, Inc., supra).
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-HCI pH 6, and finally the polypeptide
is
eluted with 6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI.
Alternatively, the protein can be successfully refolded while immobilized on
the Ni-
NTA column. The recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCI, 20% glycerol, 20 mM Tris/HCI pH 7.4,
containing protease inhibitors. The renaturation should be performed over a
period of
1.5 hours or more. After renaturation the proteins are eluted by the addition
of 250
mM immidazole. Immidazole is removed by a final dialyzing step against PBS or
50
mM sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored
at
4 degree C or frozen at -80 degree C.
In addition to the above expression vector, the present invention further
includes an expression vector comprising phage operator and promoter elements
operatively linked to a polynucleotide of the present invention, called pHE4a.
(ATCC
Accession Number 209645, deposited on February 25, 1998.) This vector
contains:
1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli
origin of
replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences,
5) a
Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The
origin of replication (oriC) is derived from pUC 19 (LTI, Gaithersburg, MD).
The
promoter sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and
isolating
the larger fragment (the stuffer fragment should be about 310 base pairs). The
DNA
insert is generated according to the PCR protocol described in Example 1,
using PCR
primers having restriction sites for NdeI (5' primer) and XbaI, BamHI, XhoI,
or
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218
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 Pol~eptide 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, all of the following steps are conducted at 4-10 degree
C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is cooled to 4-10 degree C and the cells harvested by continuous
centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected
yield
of protein per unit weight of cell paste and the amount of purified protein
required, an
appropriate amount of cell paste, by weight, is suspended in a buffer solution
containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a
homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate
is then mixed with NaCI solution to a final concentration of 0.5 M NaCI,
followed by
centrifugation at 7000 xg for 15 min. The resultant pellet is washed again
using O.SM
NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 xg centrifugation for 15 min.,
the
pellet is discarded and the polypeptide containing supernatant is incubated at
4 degree
C overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 xg) to remove insoluble particles,
the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract
with
20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA
by vigorous stirring. The refolded diluted protein solution is kept at 4
degree C
without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential
filtration unit equipped with 0.16 um membrane filter with appropriate surface
area
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(e.g., Filtronj, 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,
Perseptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a
stepwise manner. The abcorbance at 280 nm of the effluent is continuously
monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of
tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak
anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are
equilibrated with 40 mM sodium acetate, pH 6Ø Both columns are washed with
40
mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using
a 10 column volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium
acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are
collected under constant A~BO monitoring of the effluent. Fractions containing
the
polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the
above refolding and purification steps. No major contaminant bands should be
observed from Commassie blue stained 16% SDS-PAGE gel when 5 ug of purified
protein is loaded. The purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml according
to LAL
assays.
Example 7: Cloning and Expression of a Polvpeptide in a Baculovirus
Expression S. sy 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 Autograpl2a califorfiica
nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as
BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40
("SV40")
is used for efficient polyadenylation. For easy selection of recombinant
virus, the
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plasmid contains the beta-aalactosidase gene from E. coli under control of a
weak
Drosophila promoter in the same orientation, followed by the polyadenylation
signal
of the polyhedrin gene. The inserted genes are flanked on both. sides by viral
sequences for cell-mediated homologous recombination with wild-type viral DNA
to
generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such
as pAc373, pVL941, and pAcIMI, as one skilled in the art would readily
appreciate,
as long as the construct provides appropriately located signals for
transcription,
translation, secretion and the like, including a signal peptide and an in-
frame AUG as
required. Such vectors are described, for instance, in Luckow et al., Virology
170:31-
39 ( 1989).
Specifically, the cDNA sequence contained in the deposited clone, including
the AUG initiation codon and the naturally associated leader sequence
identified in
Table 1, is amplified using the PCR protocol described in Example 1. If the
naturally
occurring signal sequence is used to produce the secreted protein, the pA2
vector does
not need a second signal peptide. Alternatively, the vector can be modified
(pA2 GP)
to include a baculovirus leader sequence, using the standard methods described
in
Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures," Texas Agricultural Experimental Station Bulletin No. 1555
( 1987).
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with appropriate restriction enzymes and again purified on a
1 %
agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. coli HB 101 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
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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 ("BaculoGoldTM
baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method
described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
One ug
of BaculoGoldTM virus DNA and 5 ug of the plasmid are mixed in a sterile well
of a
microtiter plate containing 50 ul of serum-free Grace'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
culture and baculovirology distributed by Life Technologies Inc.,
Gaithersburg, page
9-10.) After appropriate incubation, blue stained plaques are picked with the
tip of a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 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
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medium is removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Rockville, MD). After 42
hours, 5
uCi of ;~S-methionine and 5 uCi "S-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 of a Polvneptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian
cell. A typical mammalian expression vector contains a promoter element, which
mediates the initiation of transcription of mRNA, a protein coding sequence,
and
signals required for the termination of transcription and polyadenylation of
the
transcript. Additional elements include enhancers, Kozak sequences and
intervening
sequences flanked by donor and acceptor sites for RNA splicing. Highly
efficient
transcription is achieved with the early and late promoters from SV40, the
long
terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
promoter of the cytomegalovirus (CMV). However, cellular elements can also be
used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention
include,
for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBCI2MI (ATCC 67109),
pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos l,
Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
Alternatively, the polypeptide can be expressed in stable cell lines
containing
the polynucleotide integrated into a chromosome. The co-transfection with a
selectable marker such as dhfr, gpt, neomycin, hygromycin allows the
identification
and isolation of the transfected cells.
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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 (ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et
al., Molecular and Cellular Biology. 438-447 (March, 1985)) plus a fragment of
the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g.,
with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate
the
cloning of the gene of interest. The vectors also contain the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin gene. and the
mouse
DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate
restriction enzymes and then dephosphorylated using calf intestinal phosphates
by
procedures known in the art. The vector is then isolated from a 1 % aaarose
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
vector can be
modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
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a
then is digested with appropriate restriction enzymes and again purified on a
1 alo
agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a 19o 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 pg 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 iteo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including 6418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml 6418. After 2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in
alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1
mg/ml 6418. After about 10-14 days single clones are trypsinized and then
seeded in
6-well petri dishes or 10 ml flasks using different concentrations of
methotrexate (50
nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well plates
containing
even higher concentrations of methotrexate ( 1 uM, 2 uM, 5 uM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained which grow at a
concentration of 100 - 200 uM. Expression of the desired gene product is
analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9: Protein Fusions
The polypeptides of the present invention are preferably fused to other
proteins. These fusion proteins can be used for a variety of applications. For
example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG
domains, and maltose binding protein facilitates purification. (See Example 5;
see
also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly,
fusion to
IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear
localization
signals fused to the polypeptides of the present invention can target the
protein to a
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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. 209646) is used, the human Fc portion
can be ligated into the BamHI cloning site. Note that the 3' BamHI site should
be
destroyed. Next, the vector containing the human Fc portion is re-restricted
with
BamHI, linearizing the vector, and a polynucleotide of the present invention,
isolated
by the PCR protocol described in Example 1. is ligated into this BamHI site.
Note
that the polynucleotide is cloned without a stop codon, otherwise a fusion
protein will
not be produced.
If the naturally 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 IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGT
GGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
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CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1 )
Example 10: Production of an Antibody from a Polvneptide
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such methods,
cells
expressing a polypeptide of the present invention is administered to an animal
to
induce the production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the secreted protein is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonal antisera of greater specific
activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology. (Kohler et al., Nature
256:495 ( 1975); Kohler et al., Eur. J. Immunol. 6:511 ( 1976); Kohler et al.,
Eur. J.
Immunol. 6:292 ( 1976); Hammerling et al., in: Monoclonal Antibodies and T-
Cell
Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures
involve immunizing an animal (preferably a mouse) with polypeptide or, more
preferably, with a secreted polypeptide-expressing cell. Such cells may be
cultured in
any suitable tissue culture medium; however, it is preferable to culture cells
in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated
at
about 56 degrees C), and supplemented with about 10 g/1 of nonessential amino
acids,
about 1,000 U/ml of penicillin, and about 100 uglml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
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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-
specific antibody and can be used to immunize an animal to induce formation of
further protein-specific antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies of the present invention may be used according to the methods
disclosed
herein. Such fragments are typically produced by proteolytic cleavage, using
enzymes such as papain (to produce Fab fragments) or pepsin (to produce
F(ab')2
fragments). Alternatively, secreted protein-binding fragments can be produced
through the application of recombinant DNA technology or through synthetic
chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using genetic constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric antibodies are
known in
the art. (See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567;
Taniguchi et
al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;
Robinson
et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,
Nature
314:268 (1985).)
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Example 11: Production Of Secreted Protein For High-Throughput Screening
Assays
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/ml. 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
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
(12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx
Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 ul Lipofectamine
(18324-012 GibcoBRL) and Sml Optimem I (31985070 GibcoBRL)/96-well plate.
With a small volume mufti-channel pipetter, aliquot approximately tug of an
expression vector containing a polynucleotide insert, produced by the methods
described in Examples 8 or 9, into an appropriately labeled 96-well round
bottom
plate. With a mufti-channel pipetter, add SOuI of the Lipofectamine/Optimem I
mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45
minutes. After about 20 minutes, use a mufti-channel pipetter to add 150u1
Optimem
I to each well. As a control, one plate of vector DNA lacking an insert should
be
transfected with each set of transfections.
Preferably, the transfection should be performed by tag-teaming the following
tasks. By tag-teaming, hands on time is cut in half, and the cells do not
spend too
much time on PBS. First, person A aspirates off the media from four 24-well
plates
of cells, and then person B rinses each well with .5-lml PBS. Person A then
aspirates
off PBS rinse, and person B, using alt-channel pipetter with tips on every
other
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229
channel, adds the 200u1 of DNA/Lipofectamine/Optimem I complex to the odd
wells
first, then to the even wells, to each row on the 24-well plates. Incubate at
37 degrees
C for 6 hours.
While cells are incubating, prepare appropriate media, either 1 %BSA in
DMEM with 1 x penstrep, or CHO-5 media ( 116.6 mg/L of CaCl2 (anhyd); 0.00130
mg/L CuS04-SH,O; 0.050 mg/L of Fe(N03)~-9H20; 0.417 mg/L of FeS04-7H~0;
311.80 mg/L of Kcl; 28.64 mg/L of MgCI,; 48.84 mg/L of MgS04; 6995.50 mg/L of
NaCI; 2400.0 mg/L of NaHCO~; 62.50 mg/L of NaHZP04-H20; 71.02 mg/L of
Na2HP04; .4320 mg/L of ZnS04-7H~0; .002 mg/L of Arachidonic Acid ; 1.022 mg/L
of Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of
Linoleic
Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100
mg/L of
Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of
D-
Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50
mg/ml
of L-Asparagine-HBO; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-HZO; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-
H20; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-
Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0
mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-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/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L
of
Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319
mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and
O.b80 mg/L of Vitamin B,2; 25 mM of HEPES Buffer; 2.39 mg/L of Na
Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20uM of
Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-
Cyclodextrin
complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed
with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal)
with 2mm glutamine and lx penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in
1L
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230
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.Sml 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 be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any.of the
assays
described below using a supernatant, the activity originates from either the
polypeptide directly (e.g., as a secreted protein) or by the polypeptide
inducing
expression of other proteins, which are then secreted into the supernatant.
Thus, the
invention further provides a method of identifying the protein in the
supernatant
characterized by an activity in a particular assay.
Example 12: Construction of ~AS 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 cells by many cytokines.
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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 Darnell, Ann. Rev. Biochem. 64:621-
51 (1995).) A cytokine receptor family, capable of activating Jaks, is divided
into two
groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL,-6, IL-7, IL-
9, IL-1 l, IL-
12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)
Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a
conserved
cysteine motif (a set of four conserved cysteines and one tryptophan) and a
WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID N0:2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
activate STATs, which then translocate and bind to GAS elements. This entire
process is encompassed in the Jaks-STATs signal transduction pathway.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the GAS or the ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth factors and
cytokines
are known to activate the Jaks-STATs pathway. (See Table below.) Thus, by
using
GAS elements linked to reporter molecules, activators of the Jaks-STATs
pathway
can be identified.
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232
JAKs STATS GAS(elementsl or ISRE
Ligand t,~ akl ak2 Jak3
J
J
IFN family
IFN-a/B + + - - 1,2,3 ISRE
~N-g + + - 1 GAS (IRF 1 >Lys6>IFP)
Il-10 + ? ? - 1,3
gp 130 family
IL-6 (Pleiotrophic)+ + + ? 1,3 GAS (IRF1>Lys6>IFP)
Il-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 familX
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)- + - + 5 GAS
IL-13 (lymphocyte)- + ? ? 6 GAS
IL-15 ? + ? + 5 GAS
gp 140 family
IL-3 (myeloid) - - + - 5 GAS (IRF 1 >IFPLy6)
IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid)- - + - 5 GAS
Growth hormone
family
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRFl=IFPLy6)
Receptor Tyrosine
Kinases
EGF ? + + - 1,3 GAS (IRF 1 )
PDGF ? + + - 1,3
CSF-1 ? + + - 1,3 GAS (not IRF1)
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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-S V40 promoter sequence. The 5' primer contains
four
tandem copies of the GAS binding site found in the IRFI 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':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC
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)
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':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAA
TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCG
CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT
CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC
TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT
AGGCTTTTGCAAAAAGCTT: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
acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase,
green
fluorescent protein (GFP), or any protein detectable by an antibody.
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The above sequence confirmed synthetic GAS-SV40 promoter element is
subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII
and
XhoI, 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 NotI, and inserted into a backbone vector containing the neomycin
resistance
gene, such as pGFP-1 (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 NFK-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, IL-2, NFAT, or Osteocalcin promoters can
be
substituted, alone or in combination (e.g., GAS/NF-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), HUVAC (aortic), or Cardiomyocyte.
Example 13: High-Throughput Screening Assay for T-cell Activity
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.
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.
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Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate
stable cell lines, approximately 2 million Jurkat cells are transfected with
the GAS-
SEAP/neo vector using DMRIE-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/ml 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 wells
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 %Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM
containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 rains.
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 1 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 RPMI + 10%
serum, 1 mg/ml Genticin, and 1 % Pen-Strep. These cells are treated with
supernatants containing polypeptides of the invention and/or induced
polypeptides of
the invention as produced by the protocol described in Example 11.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh RPMI + 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.
Transfer the cells to a triangular reservoir boat, in order to dispense the
cells
into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette,
transfer
200 ul of cells into each well (therefore adding 100, 000 cells per well).
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After all the plates have been seeded, 50 ul of the supernatants are
transferred
directly from the 96 well plate containing the supernatants into each well
using a 12
channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0,
10 ng)
is added to wells H9, H 10, and H 11 to serve as additional positive controls
for the
assay.
The 96 well dishes containing Jurkat cells treated with supernatants are
placed
in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35
ul
samples from each well are then transferred to an opaque 96 well plate using a
12
channel pipette. The opaque plates should be covered (using sellophene covers)
and
stored at -20 degrees C until SEAP assays are performed according to Example
17.
The plates containing the remaining treated cells are placed at 4 degrees C
and serve
as a source of material for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is
known to activate Jurkat T cells. Over 30 fold induction is typically observed
in the
positive control wells.
The above protocol may be used in the generation of both transient, as well
as;
stable transfected cells, which would be apparent to those of skill in the
art.
Example 14: High-Throughput Screening Assay Identif~~ Myeloid Activity
The following protocol is used to assess myeloid activity by determining
whether polypeptides of the invention proliferates and/or differentiates
myeloid cells.
Myeloid 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. The myeloid cell used in this
assay is
U937, a pre-monocyte cell line, although TF-1, HL60, or KG 1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct
produced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell
Growth & Differentiation, 5:259-265) is used. First, harvest 2x 10e7 U937
cells and
wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing
10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml
penicillin and 100 mg/ml streptomycin.
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Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing
0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 rrllVl NaCI, 5 mM
KCI, 375 uM Na2HP04.7H20, 1 mM MgCl2, and 675 uM CaCl2. Incubate at 37
degrees C for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then
resuspend in 10 ml complete medium and incubate at 37 degrees C for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400
ug/ml 6418. The 6418-free medium is used for routine growth but every one to
two
months, the cells should be re-grown in 400 ug/ml 6418 for couple of passages.
These cells are tested by harvesting 1x108 cells (this is enough for ten 96-
well
plates assay) and wash with PBS. Suspend the cells in 200 ml above described
growth medium, with a final density of Sx 105 cells/ml. Plate 200 ul cells per
well in
the 96-well plate (or 1x105 cells/well).
Add 50 ul of the supernatant prepared by the protocol described in Example
11. Incubate at 37 degrees C for 48 to 72 hr. As a positive control, 100
Unit/ml
interferon gamma can be used which is known to activate U937 cells. Over 30
fold
induction is typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 17.
Examule 15: High-Throughout Screening_Assay Identif.~~ Neuronal Activity
When cells undergo differentiation and proliferation, a group of genes are
activated through many different signal transduction pathways. One of these
genes,
EGRI (early growth response gene 1), is induced in various tissues and cell
types
upon activation. The promoter of EGR1 is responsible for such induction. Using
the
EGRl promoter linked to reporter molecules, activation of cells can be
assessed.
Particularly, the following protocol is used to assess neuronal activity in PC
12
cell lines. PC 12 cells (rat phenochromocytoma cells) are known to proliferate
and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl
phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth
factor).
The EGRl gene expression is activated during this treatment. Thus, by stably
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transfecting PC 12 cells with a construct containing an EGR promoter linked to
SEAP
reporter, activation of PC 12 cells can be assessed.
The EGR/SEAP reporter construct can be assembled by the following
protocol. The EGR-1 promoter sequence (-633 to +1)(Sakamoto K et al., Oncogene
6:867-871 ( 1991 )) can be PCR amplified from human genomic DNA using the
following primers:
5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID N0:6)
5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID N0:7)
Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified
product can then be inserted into this vector. Linearize the GAS:SEAP/Neo
vector
using restriction enzymes XhoI/HindIII, removing the GAS/S V40 stuffer.
Restrict the
EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1
promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution (
1:30
dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol
(filter
sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well
plate, and
allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)
containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-
inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin
and
100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four
split is
done every three to four days. Cells are removed from the plates by scraping
and
resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine
protocol described in Example 11. EGR-SEAP/PC 12 stable cells are obtained by
growing the cells in 300 ug/ml 6418. The 6418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in 300 ug/ml
6418
for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80%
confluent is screened by removing the old medium. Wash the cells once with PBS
(Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-
1640
containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
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The next morning, remove the medium and wash the cells with PBS. Scrape
off the cells from the plate, suspend the cells well in 2 ml low serum medium.
Count
the cell number and add more low serum medium to reach final cell density as
Sx 105
cells/ml.
Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to
1x105 cells/well). Add 50 ul supernatant produced by Example 11, 37oC for 48
to 72
hr. As a positive control, a growth factor known to activate PC 12 cells
through EGR
can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold
induction of SEAP is typically seen in the positive control wells. SEAP assay
the
supernatant according to Example 17.
Example 16: High-Throughout Screening Assay for T-cell Activitx
NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide
variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and
CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin,
and by expression of certain viral gene products. As a transcription factor,
NF-KB
regulates the expression of genes involved in immune cell activation, control
of
apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell
development,
anti-viral and antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF- KB is retained in the cytoplasm with I-KB
(Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and
degraded,
causing NF- KB to shuttle to the nucleus, thereby activating transcription of
target
genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM-l and
class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter
constructs utilizing the NF-KB promoter element are used to screen the
supernatants
produced in Example 11. Activators or inhibitors of NF-KB would be useful in
treating diseases. For example, inhibitors of NF-KB could be used to treat
those
diseases related to the acute or chronic activation of NF-KB, such as
rheumatoid
arthritis.
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To construct a vector containing the NF-KB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-
KB binding site (GGGGACTTTCCC) (SEQ ID N0:8), 18 by of sequence
complementary to the 5' end of the S V40 early promoter sequence, and is
flanked
with an XhoI site:
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC
TTTCCATCCTGCCATCTCAATTAG:3' (SEQ ID N0:9)
The downstream primer is complementary to the 3' end of the S V40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID N0:4)
PCR amplification is performed using the SV40 promoter template present in
the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene)
Sequencing with the T7 and T3 primers confirms the insert contains the
following
sequence:
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC
ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC
ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA
CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTA
TTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA
GCTT:3' (SEQ ID NO:10)
Next, replace the SV40 minimal promoter element present in the pSEAP2-
promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and
HindIII. However, this vector does not contain a neomycin resistance gene, and
therefore, is not preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP
cassette is removed from the above NF-KB/SEAP vector using restriction enzymes
SaII and NotI, and inserted into a vector containing neomycin resistance.
Particularly,
the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing
the
GFP gene, after restricting pGFP-1 with SaII and NotI.
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Once NF-KB/S V40/SEAP/Neo vector is created, stable Jurkat T-cells are
created and maintained according to the protocol described in Example 13.
Similarly,
the method for assaying supernatants with these stable Jurkat T-cells is also
described
in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is
added to
wells H9, H10, and Hl 1, with a 5-10 fold activation typically observed.
Example 17: Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP
activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according
to the
following general procedure. The Tropix Phospho-light Kit supplies the
Dilution,
Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul of 2.5x
dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the
plates with
a plastic sealer and incubate at 65 degree C for 30 min. Separate the
Optiplates to
avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser
and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room
temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see
the
table below). Add 50 ul Reaction Buffer and incubate at room temperature for
20
minutes. Since the intensity of the chemiluminescent signal is time dependent,
and it
takes about 10 minutes to read 5 plates on luminometer, one should treat 5
plates at
each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print
the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:
_ # of Rxn buffer diluent CSPD (ml)
plates (ml)
10 60 3
11 65 3.25
12 70 3.5
13 75 3.75
14 80 4
15 85 4.25
16 90 4.5
17 95 4.75
18 100 5
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19 105 5.25
20 110 5.5
21 115 5.75
22 120 6
23 125 6.25
24 130 6.5
25 135 6.75
26 140 7
27 145 7.25
28 150 7.5
29 155 7.75
30 160 8
31 165 8.25
32 170 8.5
33 175 8.75
34 180 9
35 185 9.25
36 190 9.5
37 195 9.75
38 200 10
39 205 10.25
40 210 10.~
41 215 10.75
42 220 11
43 225 11.25
44 230 11.5
45 235 11.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
Example 18: High-Throughput Screening Assay Identif~g Changes in Small
Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of
small
molecules, such as calcium, potassium, sodium, and pH, as well as alter
membrane
potential. These alterations can be measured in an assay to identify
supernatants
which bind to receptors of a particular cell. Although the following protocol
describes an assay for calcium, this protocol can easily be modified to detect
changes
in potassium, sodium, pH, membrane potential, or any other small molecule
which is
detectable by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to
measure changes in fluorescent molecules (Molecular Probes) that bind small
molecules. Clearly, any fluorescent molecule detecting a small molecule can be
used
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instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog
no. F-14202), used here.
For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star
black 96-well plate with clear bottom. The plate is incubated in a CO~
incubator for
20 hours. The adherent cells are washed two times in Biotek washer with 200 ul
of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final
wash.
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To
load the cells with fluo-4 , 50 ul of 12 ug/ml fluo-4 is added to each well.
The plate
is incubated at 37 degrees C in a CO~ incubator for 60 min. The plate is
washed four
times in the Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are
re-suspended to 2-5x106 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1
mg/ml
fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell
suspension.
The tube is then placed in a 37 degrees C water bath for 30-60 min. The cells
are
washed twice with HBSS, resuspended to 1x106 cells/ml, and dispensed into a
microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The
plate is
then washed once in Denley CellWash with 200 ul, followed by an aspiration
step to
100 ul final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as
fluo-4 . The supernatant is added to the well, and a change in fluorescence is
detected.
To measure the fluorescence of intracellular calcium, the FLIPR is set for the
following parameters: ( 1 ) System gain is 300-800 mW; (2) Exposure time is
0.4
second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is
530 nm;
and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event which has resulted in an increase in the
intracellular
Ca++ concentration.
Example 19: High-Throughput Screening Assay Identif ins Tyrosine Kinase
Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of
transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine
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Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth
factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor
subfamilies.
In addition there are a large family of RPTKs for which the corresponding
ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins, but also
membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor
dimerization, resulting in transphosphorylation of the receptor subunits and
activation
of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include
receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and
non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak
family,
members of which mediate signal transduction triggered by the cytokine
superfamily
of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine
kinase activity, the identification of novel human secreted proteins capable
of
activating tyrosine kinase signal transduction pathways are of interest.
Therefore, the
following protocol is designed to identify those novel human secreted proteins
capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately
25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased
from
Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute
rinses with
100% ethanol, rinsed with water and dried overnight. Some plates are coated
for 2 hr
with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or
polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St.
Louis, MO) or 10% Matrigel purchased from Becton Dickinson (Bedford,MA), or
calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is
assayed by seeding 5,000 cells/well in growth medium and indirect quantitation
of
cell number through use of alamarBlue as described by the manufacturer Alamar
Biosciences, Inc. (Sacramento, CA) after 48 hr. Falcon plate covers #3071 from
Becton Dickinson (Bedford,MA) are used to cover the Loprodyne Silent Screen
Plates. Falcon Microtest III cell culture plates can also be used in some
proliferation
experiments.
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To prepare extracts, A431 cells are seeded onto the nylon membranes of
Loprodyne plates (20,000/ZOOmI/well) and cultured overnight in complete
medium.
Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-
20
minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in
Example 1 l, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCI, 1 % Triton X-100, 0.1 % SDS, 2 mM Na3V04, 2 mM
Na4P2O7 and a cocktail of protease inhibitors (# 1836170) obtained from
Boeheringer Mannheim (Indianapolis, IN) is added to each well and the plate is
shaken on a rotating shaker for 5 minutes at 4 degrees C. The plate is then
placed in a
vacuum transfer manifold and the extract filtered through the 0.45 mm membrane
bottoms of each well using house vacuum. Extracts are collected in a 96-well
catch/assay plate in the bottom of the vacuum manifold and immediately placed
on
ice. To obtain extracts clarified by centrifugation, the content of each well,
after
detergent solubilization for 5 minutes, is removed and centrifuged for 15
minutes at 4
degrees C at 16,000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. Although
many
methods of detecting tyrosine kinase activity are known, one method is
described
here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by
determining its ability to phosphorylate a tyrosine residue on a specific
substrate (a
biotinylated peptide). Biotinylated peptides that can be used for this purpose
include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)
and
PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are
substrates for
a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in
order. First, add 10u1 of SuM Biotinylated Peptide, then 10u1 ATP/Mg2+ (SmM
ATP/SOmM MgCl2), then 10u1 of Sx Assay Buffer (40mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1mM EGTA, 100mM MgCl2, 5 mM MnCl2,
0.5 mg/ml BSA), then Sul of Sodium Vanadate(1mM), and then Sul of water. Mix
the
components gently and preincubate the reaction mix at 30 degrees C for 2 min.
Initial
the reaction by adding l0ul of the control enzyme or the filtered supernatant.
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The tyrosine kinase assay reaction is then terminated by adding 10 ul of
120mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of
reaction
mixture to a microtiter plate (MTP) module and incubating at 37 degrees C for
20
min. This allows the streptavadin coated 96 well plate to associate with the
biotinylated peptide. Wash the MTP module with 300u1/well of PBS four times.
Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish
peroxidase(anti-P-Tyr-POD(O.Su/ml)) to each well and incubate at 37 degrees C
for
one hour. Wash the well as above.
Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and
incubate at room temperature for at least 5 mins (up to 30 min). Measure the
absorbance of the sample at 405 nm by using ELISA reader. The level of bound
peroxidase activity is quantitated using an ELISA reader and reflects the
level of
tyrosine kinase activity.
Example 20: High-Throu~hnut Screening Assav Identifvin~ Phosnhorvlation
Activity
As a potential alternative and/or compliment to the assay of protein tyrosine
kinase activity described in Example 19, an assay which detects activation
(phosphorylation) of major intracellular signal transduction intermediates can
also be
used. For example, as described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of
other
molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase,
Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other
phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected
by
substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with O.lml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates
are
then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G
plates are then treated with 2 commercial monoclonal antibodies ( 100ng/well)
against
Erk-land Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other
molecules,
this step can easily be modified by substituting a monoclonal antibody
detecting any
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of the above described molecules.) After 3-5 rinses with PBS, the plates are
stored at
4 degrees C until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and
cultured overnight in growth medium. The cells are then starved for 48 hr in
basal
medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the
supernatants
obtained in Example 11 for 5-20 minutes. The cells are then solubilized and
extracts
filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed.
As
a positive control, a commercial preparation of MAP kinase ( l Ong/well) is
used in
place of A431 extract. Plates are then treated with a commercial polyclonal
(rabbit)
antibody (lug/ml) which specifically recognizes the phosphorylated epitope of
the
Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by
standard
procedures. The bound polyclonal antibody is then quantitated by successive
incubations with Europium-streptavidin and Europium fluorescence enhancing
reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased
fluorescent signal over background indicates a phosphorylation.
Example 21: Method of Determining Alterations in a Gene Corresponding: to a
Pol~rnucleotide
RNA isolated from entire families or individual patients presenting with a
phenotype of interest (such as a disease) is be isolated. cDNA is then
generated from
these RNA samples using protocols 'known in the art. (See, Sambrook.) The cDNA
is then used as a template for PCR, employing primers surrounding regions of
interest
in SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95 degrees C
for
30 seconds; 60-120 seconds at 52-58 degrees C; and 60-120 seconds at 70
degrees C,
using buffer solutions described in Sidransky et al., Science 252:706 (1991).
PCR products are then sequenced using primers labeled at their 5' end with T4
polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre
Technologies). The intron-exon borders of selected exons is also determined
and
genomic PCR products analyzed to confirm the results. PCR products harboring
suspected mutations is then cloned and sequenced to validate the results of
the direct
sequencing.
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PCR products is cloned into T-tailed vectors as described in Holton et al.,
Nucleic Acids Research, 19:1156 ( 1991 ) and sequenced with T7 polymerase
(United
States Biochemical). Affected individuals are identified by mutations not
present in
unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations in a gene corresponding to a polynucleotide. Genomic clones
isolated
according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5'-
triphosphate (Boehringer Manheim), and FISH performed as described in Johnson
et
al., Methods Cell Biol. 35:73-99 ( 1991 ). Hybridization with the labeled
probe is
carried out using a vast excess of human cot-1 DNA for specific hybridization
to the
corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and
propidium iodide, producing a combination of C- and R-bands. Aligned images
for
precise mapping are obtained using a triple-band filter set (Chroma
Technology,
Brattleboro, VT) in combination with a cooled charge-coupled device camera
(Photometrics, Tucson, AZ) and variable excitation wavelength filters.
(Johnson et
al., Genet. Anal. Tech. Appl., 8:75 ( 1991 ).) Image collection, analysis and
chromosomal fractional length measurements are performed using the ISee
Graphical
Program System. (Inovision Corporation, Durham, NC.) Chromosome alterations of
the genomic region hybridized by the probe are identified as insertions,
deletions, and
translocations. These alterations are used as a diagnostic marker for an
associated
disease.
Example 22: Method of Detecting Abnormal Levels of a Pol~~eptide in a
Biological Sample
A polypeptide of the present invention can be detected in a biological sample,
and if an increased or decreased level of the polypeptide is detected, this
polypeptide
is a marker for a particular phenotype. Methods of detection are numerous, and
thus,
it is understood that one skilled in the art can modify the following assay to
fit their
particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a
sample, preferably a biological sample. Wells of a microtiter plate are coated
with
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specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The
antibodies are
either monoclonal or polyclonal and are produced by the method described in
Example 10. The wells are blocked so that non-specific binding of the
polypeptide to
the well is reduced.
The coated wells are then incubated for > 2 hours at RT with a sample
containing the polypeptide. Preferably, serial dilutions of the sample should
be used
to validate results. The plates are then washed three times with deionized or
distilled
water to remove unbounded polypeptide.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a
concentration of 25-400 ng,. is added and incubated for 2 hours at room
temperature.
The plates are again washed three times with deionized or distilled water to
remove
unbounded conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl
phosphate (NPP) substrate solution to each well and incubate 1 hour at room
temperature. Measure the reaction by a microtiter plate reader. Prepare a
standard
curve, using serial dilutions of a control sample, and plot polypeptide
concentration
on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear
scale).
Interpolate the concentration of the polypeptide in the sample using the
standard
curve.
Example 23: Formulation
The invention also provides methods of treatment and/or prevention of
diseases or disorders (such as, for example, any one or more of the diseases
or
disorders disclosed herein) by administration to a subject of an effective
amount of a
Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the
invention (including fragments and variants), agonists or antagonists thereof,
and/or
antibodies thereto, in combination with a pharmaceutically acceptable carrier
type
(e.g., a sterile carrier).
The Therapeutic will be formulated and dosed in a fashion consistent with
good medical practice, taking into account the clinical condition of the
individual
patient (especially the side effects of treatment with the Therapeutic alone),
the site of
delivery, the method of administration, the scheduling of administration, and
other
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250
factors known to practitioners. The "effective amount" for purposes herein is
thus
determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the
Therapeutic administered parenterally per dose will be in the range of about
lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above,
this
will be subject to therapeutic discretion. More preferably, this dose is at
least 0.01
mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day
for
the hormone. If given continuously, the Therapeutic is typically administered
at a
dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4
injections per
day or by continuous subcutaneous infusions, for example, using a mini-pump.
An
intravenous bag solution may also be employed. The length of treatment needed
to
observe changes and the interval following treatment for responses to occur
appears
to vary depending on the desired effect.
Therapeutics can be are administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as by powders,
ointments,
gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid
or liquid
filler, diluent, encapsulating material or formulation auxiliary of any. The
term
"parenteral" as used herein refers to modes of administration which include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-
release systems. Suitable examples of sustained-release Therapeutics are
administered orally, rectally, parenterally, intracistemally, intravaginally,
intraperitoneally, topically (as by powders, ointments, gels, drops or
transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable
carrier"
refers to a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or
formulation auxiliary of any type. The term "parenteral" as used herein refers
to
modes of administration which include intravenous, intramuscular,
intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and infusion.
Therapeutics of the invention are also suitably administered by sustained-
release systems. Suitable examples of sustained-release Therapeutics include
suitable
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polymeric materials (such as, for example, semi-permeable polymer matrices in
the
form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic
materials
(for example as an emulsion in an acceptable oil) or ion exchange resins, and
sparingly soluble derivatives (such as, for example, a sparingly soluble
salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et
al.,
Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et
al.,
J. Biomed. Mater. Res. 15:167-277 ( 1981 ), and Langer, Chem. Tech. 12:98-105
(1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988).
Sustained-release Therapeutics also include liposomally entrapped
Therapeutics of the invention (see generally, Langer, Science 249:1527-1533
(1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-
Berestein and Fidler (eds.), Liss, New York, pp. 317 -327 and 353-365 (
1989)).
Liposomes containing the Therapeutic are prepared by methods known per se: DE
3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang
et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;
EP
88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small
(about 200-800 Angstroms) unilamellar type in which the lipid content is
greater than
about 30 mol. percent cholesterol, the selected proportion being adjusted for
the
optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are
delivered by way of a pump (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)).
Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
For parenteral administration, in one embodiment, the Therapeutic is
formulated generally by mixing it at the desired degree of purity, in a unit
dosage
injectable form (solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at the dosages
and
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concentrations employed and is compatible with other ingredients of the
formulation.
For example, the formulation preferably does not include oxidizing agents and
other
compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the Therapeutic
uniformly and intimately with liquid carriers or finely divided solid carriers
or both.
Then, if necessary, the product is shaped into the desired formulation.
Preferably the
carrier is a parenteral carrier, more preferably a solution that is isotonic
with the blood
of the recipient. Examples of such carrier vehicles include water, saline,
Ringer's
solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl
oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances
that enhance isotonicity and chemical stability. Such materials are non-toxic
to
recipients at the dosages and concentrations employed, and include buffers
such as
phosphate, citrate, succinate, acetic acid, and other organic acids or their
salts;
antioxidants such as ascorbic acid; low molecular weight (less than about ten
residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as
serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic
acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates including
cellulose
or its derivatives, glucose, manose, or dextrins; chelating agents such as
EDTA; sugar
alcohols such as mannitol or sorbitol; counterions such as sodium; andJor
nonionic
surfactants such as polysorbates, poloxamers, or PEG.
The Therapeutic is typically formulated in such vehicles at a concentration of
about 0.1 mglml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8.
It will
be understood that the use of certain of the foregoing excipients, carriers,
or
stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile filtration
membranes
(e.g., 0.2 micron membranes). Therapeutics generally are placed into a
container
having a sterile access port, for example, an intravenous solution bag or vial
having a
stopper pierceable by a hypodermic injection needle.
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Therapeutics ordinarily will be stored in unit or mufti-dose containers, for
example, sealed ampoules or vials, as an aqueous solution or as a lyophilized
formulation for reconstitution. As an example of a lyophilized formulation, 10-
ml
vials are filled with 5 ml of sterile-filtered 1 % (w/v) aqueous Therapeutic
solution,
and the resulting mixture is lyophilized. The infusion solution is prepared by
reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-
Injection.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the Therapeutics
of the
invention. 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. In addition, the Therapeutics may be
employed in
conjunction with other therapeutic compounds.
The Therapeutics of the invention may be administered alone or in
combination with adjuvants. Adjuvants that may be administered with the
Therapeutics of the invention include, but are not limited to, alum, alum plus
deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG,
and MPL. In a specific embodiment, Therapeutics of the invention are
administered
in combination with alum. In another specific embodiment, Therapeutics of the
invention are administered in combination with QS-21. Further adjuvants that
may be
administered with the Therapeutics of the invention include, but are not
limited to,
Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005,
Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be
administered with the Therapeutics of the invention include, but are not
limited to,
vaccines directed toward protection against MMR (measles, mumps, rubella),
polio,
varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B,
whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera,
yellow
fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis.
Combinations may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
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separately but simultaneously, e.g., as through separate intravenous lines
into the
same individual. Administration "in combination" further includes the separate
administration of one of the compounds or agents given first, followed by the
second.
The Therapeutics of the invention may be administered alone or in
combination with other therapeutic agents. Therapeutic agents that may be
administered in combination with the Therapeutics of the invention, include
but not
limited to, other members of the TNF family, chemotherapeutic agents,
antibiotics,
steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic
agents, cytokines and/or growth factors. Combinations'may be administered
either
concomitantly, e.g., as an admixture, separately but simultaneously or
concurrently;
or sequentially. This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures in which
the
combined agents are administered separately but simultaneously, e.g., as
through
separate intravenous lines into the same individual. Administration "in
combination"
further includes the separate administration of one of the compounds or agents
given
first, followed by the second.
In one embodiment, the Therapeutics of the invention are administered in
combination with members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the Therapeutics of the invention
include,
but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-
alpha,
also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-
beta),
OPGL, Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma
(International Publication No. WO 96/14328), AIM-I (International Publication
No.
WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6
(International Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921, OX40, and nerve growth factor
(NGF),
and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International
Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904),
DR4 (International Publication No. WO 98/32856), TRS (International
Publication
No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7
(International Publication No. WO 98/41629), TRANK, TR9 (International
Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202),
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312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms
CD 154, CD70, and CD 153.
In certain embodiments, Therapeutics of the invention are administered in
combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors,
non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
Nucleoside reverse transcriptase inhibitors that may be administered in
combination
with the Therapeutics of the invention, include, but are not limited to,
RETROVIRT""
(zidovudine/AZT), VIDEXT"~ (didanosine/ddI), HIVIDT"" (zalcitabine/ddC),
ZERITT"~
(stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRT"~
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that
may
be administered in combination with the Therapeutics of the invention,
include, but
are not limited to, VIRAMUNET"" (nevirapine), RESCRIPTORT"" (delavirdine), and
SUSTIVAT"" (efavirenz). Protease inhibitors that may be administered in
combination with the Therapeutics of the invention, include, but are not
limited to,
CRIXIVANT~~ (indinavir), NORVIRT"" (ritonavir), INVIRASET"" (saquinavir), and
VIRACEPTT"~ (nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase
inhibitors, and/or protease inhibitors may be used in any combination with
Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV
infection.
In other embodiments, Therapeutics of the invention may be administered in
combination with anti-opportunistic infection agents. Anti-opportunistic
agents that
may be administered in combination with the Therapeutics of the invention,
include,
but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET"",
DAPSONET"", PENTAMIDINET"", ATOVAQUONETM, ISONIAZIDT"",
RIFAMPINT"", PYRAZINAMIDETM, ETHAMBUTOLT"~, RIFABUTINTM,
CLARITHROMYCINTM, AZITHROMYCINT"~, GANCICLOVIRT"',
FOSCARNETT"", CIDOFOVIRT"', FLUCONAZOLET"", ITRACONAZOLET"",
KETOCONAZOLET"", ACYCLOVIRT"", FAMCICOLVIRT"', PYRIMETHAMINETM,
LEUCOVORINT"", NEUPOGENTM (filgrastim/G-CSF), and LEUKINET"'
(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention
are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLET"",
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DAPSONET"", PENTAMIDINET"', and/or ATOVAQUONET"" to prophylactically
treat or prevent an opportunistic Pneacmocystis carinii pneumonia infection.
In
another specific embodiment, Therapeutics of the invention are used in any
combination with ISONIAZIDT"~, RIFAMPINT"", PYRAZINAMIDET"~, and/or
ETHAMBUTOLT"" to prophylactically treat or prevent an opportunistic
Mycobacterium avium complex infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with RIFABUTINT"",
CLARITHROMYCINT"~, and/or AZITHROMYCINT"~ to prophylactically treat or
prevent an opportunistic Mycobacterium tuberculosis infection. In another
specific
embodiment, Therapeutics of the invention are used in any combination with
GANCICLOVIRT"~, FOSCARNETTM, and/or CIDOFOVIRT"~ to prophylactically treat
or prevent an opportunistic cytomegalovirus infection. In another specific
embodiment, Therapeutics of the invention are used in any combination with
FLUCONAZOLET"", ITRACONAZOLET"", and/or KETOCONAZOLET"~ to
prophylactically treat or prevent an opportunistic fungal infection. In
another
specific embodiment, Therapeutics of the invention are used in any combination
with
ACYCLOVIRT"" and/or FAMCICOLVIRT"~ to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection. In another
specific
embodiment, Therapeutics of the invention are used in any combination with
PYRIMETHAMINET"" and/or LEUCOVORINT"" to prophylactically treat or prevent
an opportunistic Toxoplasma gondii infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with LEUCOVORINT"'
and/or NEUPOGENT"~ to prophylactically treat or prevent an opportunistic
bacterial
infection.
In a further embodiment, the Therapeutics of the invention are administered
in combination with an antiviral agent. Antiviral agents that may be
administered
with the Therapeutics of the invention include, but are not limited to,
acyclovir,
ribavirin, amantadine, and remantidine.
In a further embodiment, the Therapeutics of the invention are administered
in combination with an antibiotic agent. Antibiotic agents that may be
administered
with the Therapeutics of the invention include, but are not limited to,
amoxicillin,
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beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,
erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,
quinolones,
rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-
sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be
administered in combination with the Therapeutics of the invention include,
but are
not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and
other
immunosuppressive agents that act by suppressing the function of responding T
cells.
In specific embodiments, Therapeutics of the invention are administered in
combination with immunosuppressants. Immunosuppressants preparations that may
be administered with the Therapeutics of the invention include, but are not
limited to,
ORTHOCLONET"" (OKT3), SANDIMMUNET"~/NEORALT"~/SANGDYAT~~
(cyclosporin), PROGRAFT"" (tacrolimus), CELLCEPTT"" (mycophenolate),
Azathioprine, glucorticosteroids, and RAPAMUNET"" (sirolimus). In a specific
embodiment, immunosuppressants may be used to prevent rejection of organ or
bone
marrow transplantation.
In an additional embodiment, Therapeutics of the invention are administered
alone or in combination with one or more intravenous immune globulin
preparations.
Intravenous immune globulin preparations that may be administered with the
Therapeutics of the invention include, but not limited to, GAMMART"',
IVEEGAMT"", SANDOGLOBULINTM, GAMMAGARD S/DT"~, and GAMIMUNET"~.
In a specific embodiment, Therapeutics of the invention are administered in
combination with intravenous immune globulin preparations in transplantation
therapy (e.g., bone marrow transplant).
In an additional embodiment, the Therapeutics of the invention are
administered alone or in combination with an anti-inflammatory agent. Anti-
inflammatory agents that may be administered with the Therapeutics of the
invention
include, but are not limited to, glucocorticoids and the nonsteroidal anti-
inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid
derivatives,
arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid
derivatives,
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pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-
acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid,
amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol,
emorfazone,
guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline,
perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
In another embodiment, compostions of the invention are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the Therapeutics of the invention include, but are not
limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and
dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,
fluorouracil, 5-
FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin,
mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine,
hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine
sulfate);
hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol
diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives
(e.g.,
mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids
and combinations (e.g., bethamethasone sodium phosphate); and others (e.g.,
dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate,
and
etoposide).
In a specific embodiment, Therapeutics of the invention are administered in
combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or any combination of the components of CHOP. In another
embodiment, Therapeutics of the invention are administered in combination with
Rituximab. In a further embodiment, Therapeutics of the invention are
administered
with Rituxmab and CHOP, or Rituxmab and any combination of the components of
CHOP.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with cytokines. Cytokines that may be administered
with the Therapeutics of the invention include, but are not limited to. IL2,
IL3, IL4,
ILS, IL6, IL7, IL 10, IL 12, IL 13, IL 15, anti-CD40, CD40L, IFN-gamma and TNF-
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alpha. In another embodiment, Therapeutics of the invention may be
administered
with any interleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-
2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16,
IL-17, IL-18, IL-19, IL-20, and IL-21.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with angiogenic proteins. Angiogenic proteins that
may
be administered with the Therapeutics of the invention include, but are not
limited to,
Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European
Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as
disclosed in European Patent Number EP-282317; Placental Growth Factor (P1GF),
as
disclosed in International Publication Number WO 92/06194; Placental Growth
Factor-2 (P1GF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 (
1993);
Vascular Endothelial Growth Factor (VEGF), as disclosed in International
Publication
Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as
disclosed in European Patent Number EP-506477; Vascular Endothelial Growth
Factor-2 (VEGF-2), as disclosed in International Publication Number WO
96/39515;
Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth
Factor B-186 (VEGF-B 186), as disclosed in International Publication Number WO
96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in
International Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D), as disclosed in International Publication Number WO
98/07832;
and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German
Patent
Number DE 19639601. The above mentioned references are incorporated herein by
reference herein.
In an additional embodiment, the Therapeutics of the invention are
administered in combination with hematopoietic growth factors. Hematopoietic
growth factors that may be administered with the Therapeutics of the invention
include, but are not limited to, LEUKINET"~ (SARGRAMOSTIMT"~) and
NEUPOGENT~" (FILGRASTIMT"")
In an additional embodiment, the Therapeutics of the invention are
administered in combination with Fibroblast Growth Factors. Fibroblast Growth
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Factors that may be administered with the Therapeutics of the invention
include, but
are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,
FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In additional embodiments, the Therapeutics of the invention are administered
in
combination with other therapeutic or prophylactic regimens, such as, for
example,
radiation therapy.
Example 24: Method of Treating Decreased Levels of the Polypeptide
The present invention relates to a method for treating an individual in need
of
an increased level of a polypeptide of the invention in the body comprising
administering to such an individual a composition comprising a therapeutically
effective amount of an agonist of the invention (including polypeptides of the
invention). Moreover, it will be appreciated that conditions caused by a
decrease in
the standard or normal expression level of a secreted protein in an individual
can be
treated by administering the polypeptide of the present invention, preferably
in the
secreted form. Thus, the invention also provides a method of treatment of an
individual in need of an increased level of the polypeptide comprising
administering
to such an individual a Therapeutic comprising an amount of the polypeptide to
increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily
dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably,
the
polypeptide is in the secreted form. The exact details of the dosing scheme,
based on
administration and formulation, are provided in Example 23.
Example 25: Method of Treating Increased Levels of the Polvneptide
The present invention also relates to a method of treating an individual in
need
of a decreased level of a polypeptide of the invention in the body comprising
administering to such an individual a composition comprising a therapeutically
effective amount of an antagonist of the invention (including polypeptides and
antibodies of the invention).
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In one example, antisense technology is used to inhibit production of a
polypeptide of the present invention. This technology is one example of a
method of
decreasing levels of a polypeptide, preferably a secreted form, due to a
variety of
etiologies, such as cancer. For example, a patient diagnosed with abnormally
increased levels of a polypeptide is administered intravenously antisense
polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This
treatment is
repeated after a 7-day rest period if the treatment was well tolerated. The
formulation
of the antisense polynucleotide is provided in Example 23.
Example 26: Method of Treatment Using Gene Therapy-Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of
expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained
from a
subject by skin biopsy. The resulting tissue is placed in tissue-culture
medium and
separated into small pieces. Small chunks of the tissue are placed on a wet
surface of
a tissue culture flask, approximately ten pieces are placed in each flask. The
flask is
turned upside down, closed tight and left at room temperature over night.
After 24
hours at room temperature, the flask is inverted and the chunks of tissue
remain fixed
to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10%
FBS,
penicillin and streptomycin) is added. The flasks are then incubated at 37
degree C
for approximately one week.
At this time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of fibroblasts
emerge.
The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified
using PCR primers which correspond to the 5' and 3' end sequences respectively
as set
forth in Example 1 using primers and having appropriate restriction sites and
initiation/stop codons, if necessary. Preferably, the 5' primer contains an
EcoRI site
and the 3' primer includes a HindIII site. Equal quantities of the Moloney
murine
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sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are
added together, in the presence of T4 DNA ligase. The resulting mixture is
maintained under conditions appropriate for ligation of the two fragments. The
ligation mixture is then used to transform bacteria HB101, which are then
plated onto
agar containing kanamycin for the purpose of confirming that the vector has
the gene
of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10% calf serum (CS), penicillin and streptomycin. The MSV vector containing
the
gene is then added to the media and the packaging cells transduced with the
vector.
The packaging cells now produce infectious viral particles containing the gene
(the
packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the
media from the producer cells. This media is removed and replaced with fresh
media.
If the titer of virus is high, then virtually all fibroblasts will be infected
and no
selection is required. If the titer is very low, then it is necessary to use a
retroviral
vector that has a selectable marker, such as neo or his. Once the fibroblasts
have been
efficiently infected, the fibroblasts are analyzed to determine whether
protein is
produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or
after having been grown to confluence on cytodex 3 microcarrier beads.
Example 27~ Gene Theranv Using Endogenous Genes Corresponding To
Polynucleotides of the Invention
Another method of gene therapy according to the present invention involves
operably associating the endogenous polynucleotide sequence of the invention
with a
promoter via homologous recombination as described, for example, in U.S.
Patent
NO: 5,641,670, issued June 24, 1997; International Publication NO: WO
96/29411,
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published September 26, 1996; International Publication NO: WO 94/12650,
published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-
8935
(1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves
the
activation of a gene which is present in the target cells, but which is not
expressed in
the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and targeting
sequences, which are homologous to the 5' non-coding sequence of endogenous
polynucleotide sequence, flanking the promoter. The targeting sequence will be
sufficiently near the 5' end of the 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 the amplified targeting sequences are digested
with the appropriate restriction enzymes and subsequently treated with calf
intestinal
phosphatase. The digested promoter and digested targeting sequences are added
together in the presence of T4 DNA ligase. The resulting mixture is maintained
under
conditions appropriate for ligation of the two fragments. The construct is
size
fractionated on an agarose gel then purified by phenol extraction and ethanol
precipitation.
In this Example, the polynucleotide constructs are administered as naked
polynucleotides via electroporation. However, the polynucleotide constructs
may also
be administered with transfection-facilitating agents, such as liposomes,
viral
sequences, viral particles, precipitating agents, etc. Such methods of
delivery are
known in the art.
Once the cells are transfected, homologous recombination will take place
which results in the promoter being operably linked to the endogenous
polynucleotide
sequence. This results in the expression of polynucleotide corresponding to
the
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polynucleotide in the cell. Expression may be detected by immunological
staining, or
any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in DMEM + 10°lo fetal calf serum. Exponentially growing or early
stationary
phase fibroblasts are trypsinized and rinsed from the plastic surface with
nutrient
medium. An aliquot of the cell suspension is removed for counting, and the
remaining
cells are subjected to centrifugation. The supernatant is aspirated and the
pellet is
resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM
NaCI,
5 mM KCI, 0.7 mM Naz HP04, 6 mM dextrose). The cells are recentrifuged, the
supernatant aspirated, and the cells resuspended in electroporation buffer
containing 1
mg/ml acetylated bovine serum albumin. The final cell suspension contains
approximately 3X106 cells/ml..Electroporation should be performed immediately
following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to
construct a plasmid for targeting to the locus corresponding to the
polynucleotide of
the invention, plasmid pUCl8 (MBI Fermentas, Amherst, NY) is digested with
HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5' end
and
a BamHI site on the 3'end. Two non-coding sequences are amplified via PCR: one
non-coding sequence (fragment 1) is amplified with a HindIII site at the 5'
end and an
Xba site at the 3'end; the other non-coding sequence (fragment 2) is amplified
with a
BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promoter and
the
fragments ( 1 and 2) are digested with the appropriate enzymes (CMV promoter -
XbaI
and BamHI; fragment 1 - XbaI; fragment 2 - BamHI) and ligated together. The
resulting ligation product is digested with HindIII, and ligated with the
HindIII-
digested pUC 18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap
(Bio-Rad). The final DNA concentration is generally at least 120 ~glml. 0.5 ml
of the
cell suspension (containing approximately 1.5.X 106 cells) is then added to
the cuvette,
and the cell suspension and DNA solutions are gently mixed. Electroporation is
performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are
set at
960 ~F and 250-300 V, respectively. As voltage increases, cell survival
decreases, but
the percentage of surviving cells that stably incorporate the introduced DNA
into their
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genome increases dramatically. Given these parameters, a pulse time of
approximately 14-20 mSec should be observed.
Electroporated cells are maintained at room temperature for approximately 5
min, and the contents of the cuvette are then gently removed with a sterile
transfer
pipette. The cells are added directly to 10 ml of prewarmed nutrient media
(DMEM
with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The
following
day, the media is aspirated and replaced with 10 ml of fresh media and
incubated for a
further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts
now produce the protein product. The fibroblasts can then be introduced into a
patient as described above.
Example 28: Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods
to treat disorders, diseases and conditions. The gene therapy method relates
to the
introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA)
sequences into an animal to increase or decrease the expression of the
polypeptide.
The polynucleotide of the present invention may be operatively linked to a
promoter
or any other genetic elements necessary for the expression of the polypeptide
by the
target tissue. Such gene therapy and delivery techniques and methods are known
in
the art, see, for example, W090/11092, W098/11779; U.S. Patent NO. 5693622,
5705151, 5580859; Tabata et al., Cardiovasc. Res. 35(3):470-479 ( 1997); Chao
et al.,
Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318
(1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al.,
Circulation
94( 12):3281-3290 ( 1996) (incorporated herein by reference).
The polynucleotide constructs may 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, intestine and the like).
The
polynucleotide constructs can be delivered in a pharmaceutically acceptable
liquid or
aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences that are
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free from any delivery vehicle that acts to assist, promote, or facilitate
entry into the
cell, including viral sequences, viral particles, liposome formulations,
lipofectin or
precipitating agents and the like. However, the polynucleotides of the present
invention may also be delivered in liposome formulations (such as those taught
in
Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et
al.
(1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to
those
skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Any strong promoter known to those
skilled in
the art can be used for driving the expression of DNA. Unlike other gene
therapies
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 can be delivered to the interstitial space of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and
connective tissue. Interstitial space of the tissues comprises the
intercellular fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers
in the walls of vessels or chambers, collagen fibers of fibrous tissues, or
that same
matrix within connective tissue ensheathing muscle cells or in the lacunae of
bone. It
is similarly the space occupied by the plasma of the circulation and the lymph
fluid of
the lymphatic channels. Delivery to the interstitial space of muscle tissue is
preferred
for the reasons discussed below. They may be conveniently delivered by
injection
into the tissues comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or skin
fibroblasts. In
vivo muscle cells are particularly competent in their ability to take up and
express
polynucleotides.
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For the naked polynucleotide injection, an effective dosage amount of DNA or
RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg
body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 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 polynucleotide constructs can
be
delivered to arteries during angioplasty by the catheter used in the
procedure.
The dose response effects of injected polynucleotide in muscle in vivo is
determined as follows. Suitable template DNA for production of mRNA coding for
polypeptide of the present invention is prepared in accordance with a standard
recombinant DNA methodology. The template DNA, which may be either circular or
linear, is either used as naked DNA or complexed with liposomes. The
quadriceps
muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is
made on
the anterior thigh, and the quadriceps muscle is directly visualized. The
template
DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over
one minute, approximately 0.5 cm from the distal insertion site of the muscle
into the
knee and about 0.2 cm deep. A suture is placed over the injection site for
future
localization, and the skin is closed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared by excising the entire quadriceps. Every fifth 15 um cross-section of
the
individual quadriceps muscles is histochemically stained for protein
expression. A
time course for protein expression may be done in a similar fashion except
that
quadriceps from different mice are harvested at different times. Persistence
of DNA
in muscle following injection may be determined by Southern blot analysis
after
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preparing total cellular DNA and HIRT supernatants from injected and control
mice.
The results of the above experimentation in mice can be use to extrapolate
proper
dosages and other treatment parameters in humans and other animals using naked
DNA.
Example 29: Transgenic Animals.
The polypeptides of the invention can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g.,
baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a
specific embodiment, techniques described herein or otherwise known in the
art, are
used to express polypeptides of the invention in humans, as part of a gene
therapy
protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
polynucleotides of the invention) into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698
(1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology
(NY) 9:830-834 ( 1991 ); and Hoppe et al., U.S. Pat. No. 4,873,191 ( 1989));
retrovirus
mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl.
Acad. Sci.,
USA 82:6148-6152 ( 1985)), blastocysts or embryos; gene targeting in embryonic
stem cells (Thompson et al., Cell 56:313-321 ( 1989)); electroporation of
cells or
embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the
polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al.,
Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent
stem cells and transferring the stem cells back into the blastocyst; and sperm-
mediated gene transfer (Lavitrano et al., Cell 57:717-723 ( 1989); etc. For a
review of
such techniques, see Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-
229
( 1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced to
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quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-
813 ( 1997)).
The present invention provides for transgenic animals that carry the transgene
in all their cells, as well as animals which carry the transgene in some, but
not all their
cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a
single
transgene or as multiple copies such as in concatamers, e.g., head-to-head
tandems or
head-to-tail tandems. The transgene may also be selectively introduced into
and
activated in a particular cell type by following, for example, the teaching of
Lasko et
al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory
sequences required for such a cell-type specific activation will depend upon
the
particular cell type of interest, and will be apparent to those of skill in
the art: When
it is desired that the polynucleotide transgene be integrated into the
chromosomal site
of the endogenous gene, gene targeting is preferred. Briefly, when such a
technique is
to be utilized, vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and disrupting the function of
the
nucleotide sequence of the endogenous gene. The transgene may also be
selectively
introduced into a particular cell type, thus inactivating the endogenous gene
in only
that cell type, by following, for example, the teaching of Gu et al. (Gu et
al., Science
265:103-106 (1994)). The regulatory sequences required for such a cell-type
specific
inactivation will depend upon the particular cell type of interest, and will
be apparent
to those of skill in the art.
Once transgenie animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening
may be accomplished by Southern blot analysis or PCR techniques to analyze
animal
tissues to verify that integration of the transgene has taken place. The level
of mRNA
expression of the transgene in the tissues of the transgenic animals may also
be
assessed using techniques which include, but are not limited to, Northern blot
analysis
of tissue samples obtained from the animal, in situ hybridization analysis,
and reverse
transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may
also
be evaluated immunocytochemically or immunohistochemically using antibodies
specific for the transgene product.
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Once the founder animals are produced, they may be bred, inbred, outbred, or
crossbred to produce colonies of the particular animal. Examples of such
breeding
strategies include, but are not limited to: outbreeding of founder animals
with more
than one integration site in order to establish separate lines; inbreeding of
separate
lines in order to produce compound transgenics that express the transgene at
higher
levels because of the effects of additive expression of each transgene;
crossing of
heterozygous transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate the need
for
screening of animals by DNA analysis; crossing of separate homozygous lines to
produce compound heterozygous or homozygous lines; and breeding to place the
transgene on a distinct background that is appropriate for an experimental
model of
interest.
Transgenic animals of the invention have uses which include, but are not
limited to, animal model systems useful in elaborating the biological function
of
polypeptides of the present invention, studying conditions and/or disorders
associated
with aberrant expression, and in screening for compounds effective in
ameliorating
such conditions and/or disorders.
Example 30: Knock-Out Animals.
Endogenous gene expression can also be reduced by inactivating or "knocking
out" the gene and/or its promoter using targeted homologous recombination.
(E.g.,
see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-
512 ( 1987); Thompson et al., Cell 5:313-321 ( 1989); each of which is
incorporated by
reference herein in its entirety). For example, a mutant, non-functional
polynucleotide of the invention (or a completely unrelated DNA sequence)
flanked by
DNA homologous to the endogenous polynucleotide sequence (either the coding
regions or regulatory regions of the gene) can be used, with or without a
selectable
marker and/or a negative selectable marker, to transfect cells that express
polypeptides of the invention in vivo. In another embodiment, techniques known
in
the art are used to generate knockouts in cells that contain, but do not
express the gene
of interest. Insertion of the DNA construct, via targeted homologous
recombination,
results in inactivation of the targeted gene. Such approaches are particularly
suited in
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research and agricultural fields where modifications to embryonic stem cells
can be
used to generate animal offspring with an inactive targeted gene (e.g., see
Thomas &
Capecchi 1987 and Thompson 1989, supra). However this approach can be
routinely
adapted for use in humans provided the recombinant DNA constructs are directly
administered or targeted to the required site in vivo using appropriate viral
vectors that
will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered
to express the polypeptides of the invention, or alternatively, that are
genetically
engineered not to express the polypeptides of the invention (e.g., knockouts)
are
administered to a patient in vivo. Such cells may be obtained from the patient
(i.e.,
animal, including human) or an MHC compatible donor and can include, but are
not
limited to fibroblasts, bone marrow cells, blood cells (~, lymphocytes),
adipocytes,
muscle cells, endothelial cells etc. The cells are genetically engineered in
vitro using
recombinant DNA techniques to introduce the coding sequence of polypeptides of
the
invention into the cells, or alternatively, to disrupt the coding sequence
and/or
endogenous regulatory sequence associated with the polypeptides of the
invention,
e_.g_, by transduction (using viral vectors, and preferably vectors that
integrate the
transgene into the cell genome) or transfection procedures, including, but not
limited
to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes,
etc.
The coding sequence of the polypeptides of the invention can be placed under
the
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve
expression, and preferably secretion, of the polypeptides of the invention.
The
engineered cells which express and preferably secrete the polypeptides of the
invention can be introduced into the patient systemically, e.g., in the
circulation, or
intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in
the
body, ~, genetically engineered fibroblasts can be implanted as part of a skin
graft;
genetically engineered endothelial cells can be implanted as part of a
lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349;
and
Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by
reference herein in its entirety).
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When the cells to be administered are non-autologous or non-MHC
compatible cells, they can be administered using well known techniques which
prevent the development of a host immune response against the introduced
cells. For
example, the cells may be introduced in an encapsulated form which, while
allowing
for an exchange of components with the immediate extracellular environment,
does
not allow the introduced cells to be recognized by the host immune system.
Transgenic and "knock-out" animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating the
biological
function of polypeptides of the present invention, studying conditions and/or
disorders
associated with aberrant expression, and in screening for compounds effective
in
ameliorating such conditions and/or disorders.
Example 31: Production of an Antibody
a) Hybridoma Technology
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 polypeptide(s) of the invention are administered to an animal to
induce the
production of sera containing polyclonal antibodies. In a preferred method, a
preparation of polypeptide(s) of the invention is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into
an animal in order to produce polyclonal antisera of greater specific
activity.
Monoclonal antibodies specific for polypeptide(s) of the invention are
prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975);
Kohler
et al., Eur. J. Immunol. 6:511 ( 1976); Kohler et al., Eur. J. Immunol. 6:292
( 1976);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier,
N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is
immunized
with polypeptide(s) of the invention or, more preferably, with a secreted
polypeptide-
expressing cell. Such polypeptide-expressing cells are cultured in any
suitable tissue
culture medium, preferably in Earle's modified Eagle's medium supplemented
with
10% fetal bovine serum (inactivated at about 56°C), and supplemented
with about 10
g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100
pg/ml
of streptomycin.
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The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma
cells obtained through such a selection are then assayed to identify clones
which
secrete antibodies capable of binding the polypeptide(s) of the invention.
Alternatively, additional antibodies capable of binding to polypeptide(s) of
the
invention 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 polypeptide(s) of the invention. Such antibodies comprise anti-
idiotypic antibodies to the protein-specific antibody and are used to immunize
an
animal to induce formation of further protein-specific antibodies.
For in vivo use of antibodies in humans, an antibody is "humanized". Such
antibodies can be produced using genetic constructs derived from hybridoma
cells
producing the monoclonal antibodies described above. Methods for producing
chimeric and humanized antibodies are known in the art and are discussed
herein.
(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 et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268
(1985).)
b) Isolation Of Antibody Fragments Directed Against
Polypeptide(s) From A Library Of scFvs
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Naturally occurring V-genes isolated from human PBLs are constructed into a
library of antibody fragments which contain reactivities against
polypeptide(s) of the
invention to which the donor may or may not have been exposed (see e.g., U.S.
Patent
5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library.
A library of scFvs is constructed from the RNA of human PBLs as described
in PCT publication WO 92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml
of
2xTY containing 1 % glucose and 100 ~g/ml of ampicillin (2xTY-AMP-GLU) and
grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to
innoculate 50
ml of 2xTY-AMP-GLU, 2 x 108 TU of delta gene 3 helper (M13 delta gene III, see
PCT publication WO 92/01047) are added and the culture incubated at
37°C for 45
minutes without shaking and then at 37°C for 45 minutes with shaking.
The culture is
centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters
of 2xTY
containing 100 pg/ml ampicillin and 50 ug/ml kanamycin and grown overnight.
Phage are prepared as described in PCT publication WO 92/01047.
M13 delta gene III is prepared as follows: M13 delta gene III helper phage
does not encode gene III protein, hence the phage(mid) displaying antibody
fragments have a greater avidity of binding to antigen. Infectious M 13 delta
gene III
particles are made by growing the helper phage in cells harboring a pUC 19
derivative
supplying the wild type gene III protein during phage morphogenesis. The
culture is
incubated for 1 hour at 37° C without shaking and then for a further
hour at 37°C with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended
in
300 ml 2xTY broth containing 100 pg ampicillin/ml and 25 pg kanamycin/ml (2xTY-
AMP-KAN) and grown overnight, shaking at 37°C. Phage particles are
purified and
concentrated from the culture medium by two PEG-precipitations (Sambrook et
al.,
1990), resuspended in 2 ml PBS and passed through a 0.45 pm filter (Minisart
NML;
Sartorius) to give a final concentration of approximately 1013 transducing
units/ml
(ampicillin-resistant clones).
Panning of the Library.
Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100
~tg/ml or 10 ~tg/ml of a polypeptide of the present invention. Tubes are
blocked with
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2% Marvel-PBS for 2 hours at 37°C and then washed 3 times in PBS.
Approximately
1013 TU of phage is applied to the tube and incubated for 30 minutes at room
temperature tumbling on an over and under turntable and then left to stand for
another
1.5 hours. Tubes are washed 10 times with PBS 0.1 % Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15
minutes on an under and over turntable after which the solution is immediately
neutralized with 0.5 ml of 1.OM Tris-HCI, pH 7.4. Phage are then used to
infect 10
ml of mid-log E. coli TG 1 by incubating eluted phage with bacteria for 30
minutes at
37°C. The E. coli are then plated on TYE plates containing 1 % glucose
and 100
pg/ml ampicillin. The resulting bacterial library is then rescued with delta
gene 3
helper phage as described above to prepare phage for a subsequent round of
selection.
This process is then repeated for a total of 4 rounds of affinity purification
with tube-
washing increased to 20 times with PBS, 0.1 % Tween-20 and 20 times with PBS
for
rounds 3 and 4.
Characterization of Binders.
Eluted phage from the 3rd and 4th rounds of selection are used to infect E.
coli
HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single
colonies for
assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml
of
the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones
positive in ELISA are further characterized by PCR fingerprinting (see, e.g.,
PCT
publication WO 92/01047) and then by sequencing. These ELISA positive clones
may also be further characterized by techniques known in the art, such as, for
example, epitope mapping, binding affinity, receptor signal transduction,
ability to
block or competitively inhibit antibody/antigen binding, and competitive
agonistic or
antagonistic activity.
Example 32: Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
Generation of functional humoral immune responses requires both soluble and
cognate signaling between B-lineage cells and their microenvironment. Signals
may
impart a positive stimulus that allows a B-lineage cell to continue its
programmed
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development, or a negative stimulus that instructs the cell to arrest its
current
developmental pathway. To date, numerous stimulatory and inhibitory signals
have been
found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-
7, IL10, IL-
13, IL-14 and IL-15. Interestingly, these signals are by themselves weak
effectors but can,
in combination with various co-stimulatory proteins, induce activation,
proliferation,
differentiation, homing, tolerance and death among B cell populations.
One of the best studied classes of B-cell co-stimulatory proteins is the TNF-
superfamily. Within this family CD40, CD27, and CD30 along with their
respective
ligands CD 154, CD70, and CD 153 have been found to regulate a variety of
immune
responses. Assays which allow for the detection and/or observation of the
proliferation
and differentiation of these B-cell populations and their precursors are
valuable tools in
determining the effects various proteins may have on these B-cell populations
in terms of
proliferation and differentiation. Listed below are two assays designed to
allow for the
detection of the differentiation, proliferation, or inhibition of B-cell
populations and their
precursors.
In Vitro Assav- Purified polypeptides of the invention, or truncated forms
thereof,
is assessed for its ability to induce activation, proliferation,
differentiation or inhibition
andlor death in B-cell populations and their precursors. The activity of the
polypeptides of
the invention on purified human tonsillar B cells, measured qualitatively over
the dose
range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-
stimulation
assay in which purified tonsillar B cells are cultured in the presence of
either formalin-
fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM
antibody as
the priming agent. Second signals such as lL-2 and IL-15 synergize with SAC
and IgM
crosslinking to elicit B cell proliferation as measured by tritiated-thymidine
incorporation.
Novel synergizing agents can be readily identified using this assay. The assay
involves
isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-
positive
cells. The resulting cell population is greater than 95% B cells as assessed
by expression
of CD45R(B220).
Various dilutions of each sample are placed into individual wells of a 96-well
plate
to which are added 105 B-cells suspended in culture medium (RPMI 1640
containing 10%
FBS, 5 X 10-SM 2ME, 100U/ml penicillin, l0ug/ml streptomycin, and 10-5
dilution of
SAC) in a total volume of 150u1. Proliferation or inhibition is quantitated by
a 20h pulse
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(luCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72h post factor addition.
The
positive and negative controls are IL2 and medium respectively.
In Vivo Assav- BALB/c mice are injected (i.p.) twice per day with buffer only,
or
2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice
receive this
treatment for 4 consecutive days, at which time they are sacrificed and
various tissues and
serum collected for analyses. Comparison of H&E sections from normal spleens
and
spleens treated with polypeptides of the invention identify the results of the
activity of the
polypeptides on spleen cells, such as the diffusion of peri-arterial lymphatic
sheaths,
andlor significant increases in the nucleated cellularity of the red pulp
regions, which may
indicate the activation of the differentiation and proliferation of B=cell
populations.
Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used
to
determine whether any physiological changes to splenic cells, such as splenic
disorganization, are due to increased B-cell representation within loosely
defined B-cell
zones that infiltrate established T-cell regions.
Flow cytometric analyses of the spleens from mice treated with polypeptide is
used
to indicate whether the polypeptide specifically increases the proportion of
ThB+,
CD45R(B220)dull B cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell representation in
vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA
levels are
compared between buffer and polypeptide-treated mice.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides of the invention (e.g., gene therapy),
agonists, and/or
antagonists of polynucleotides or polypeptides of the invention.
Example 33: T Cell Proliferation Assay
A CD3-induced proliferation assay is performed on PBMCs and is measured by
the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well
plates are
coated with 100 ~,1/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control
mAb (B33.1) overnight at 4 degrees C (1 ~g/ml in .OSM bicarbonate buffer, pH
9.5), then
washed three times with PBS. PBMC are isolated by F/H gradient centrifugation
from
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human peripheral blood and added to quadruplicate wells (5 x 10'~/well) of mAb
coated
plates in RPMI containing 10% FCS and P/S in the presence of varying
concentrations of
polypeptides of the invention (total volume 200 ul). Relevant protein buffer
and medium
alone are controls. After 48 hr. culture at 37 degrees C, plates are spun for
2 min. at 1000
rpm and 100 ~.l of supernatant is removed and stored -20 degrees C for
measurement of
IL-2 (or other cytokines) if effect on proliferation is observed. Wells are
supplemented
with 100 ul of medium containing 0.5 uCi of 3H-thymidine and cultured at 37
degrees C
for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a
measure of
proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2
( 100 U/ml) is
also used as a control which enhances proliferation. Control antibody which
does not
induce proliferation of T cells is used as the negative controls for the
effects of
polypeptides of the invention.
The studies described in this example tested activity of polypeptides of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides of the invention (e.g., gene therapy),
agonists, and/or
antagonists of polynucleotides or polypeptides of the invention.
Example 34~ Effect of Polynentides of the Invention on the Expression of MHC
Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of
Monocytes and Monocvte-Derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors
found in
the peripheral blood: adherent PBMC or elutriated monocytic fractions are
cultured for 7-
10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have
the
characteristic phenotype of immature cells (expression of CD1, CD80, CD86,
CD40 and
MHC class II antigens). Treatment with activating factors, such as TNF-a,
causes a rapid
change in surface phenotype (increased expression of MHC class I and II,
costimulatory
and adhesion molecules, downregulation of FC~yRII, upregulation of CD83).
These
changes correlate with increased antigen-presenting capacity and with
functional
maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-
3
days with increasing concentrations of polypeptides of the invention or LPS
(positive
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control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then
incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal
antibodies
for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are
analyzed by
flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cytokines. Cytokines generated by dendritic cells,
in
particular IL-12, are important in the initiation of T-cell dependent immune
responses. IL-
12 strongly influences the development of Thl helper T-cell immune response,
and
induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-
12 release
as follows. Dendritic cells ( 106/ml) are treated with increasing
concentrations of
polypeptides of the invention for 24 hours. LPS ( 100 ng/ml) is added to the
cell culture as
positive control. Supernatants from the cell cultures are then collected and
analyzed for
IL-12 content using commercial ELISA kit (e..g, R & D Systems (Minneapolis,
MN)).
The standard protocols provided with the kits are used.
Effect on the expression of MHC Class II, costimulatory and adhesion
molecules.
Three major families of cell surface antigens can be identified on monocytes:
adhesion
molecules, molecules involved in antigen presentation, and Fc receptor.
Modulation of
the expression of MHC class II antigens and other costimulatory molecules,
such as B7
and ICAM-1, may result in changes in the antigen presenting capacity of
monocytes and
ability to induce T cell activation. Increase expression of Fc receptors may
correlate with
improved monocyte cytotoxic activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes
are
treated 1-5 days with increasing concentrations of polypeptides of the
invention or LPS
(positive control), washed with PBS containing 1 % BSA and 0.02 mM sodium
azide, and
then incubated with 1:20 dilution of appropriate FITC- or PE-labeled
monoclonal
antibodies for 30 minutes at 4 degreesC. After an additional wash, the labeled
cells are
analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival Assays for molecules that
activate
(or alternatively, inactivate) monocytes and/or increase monocyte survival (or
alternatively, decrease monocyte survival) are known in the art and may
routinely be
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applied to determine whether a molecule of the invention functions as an
inhibitor or
activator of monocytes. Polypeptides, agonists, or antagonists of the
invention can be
screened using the three assays described below. For each of these assays,
Peripheral
blood mononuclear cells (PBMC) are purified from single donor leukopacks
(American
Red Cross, Baltimore, MD) by centrifugation through a Histopaque gradient
(Sigma).
Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
Monocyte Survival Assav. Human peripheral blood monocytes progressively lose
viability when cultured in absence of serum or other stimuli. Their death
results from
internally regulated process (apoptosis). Addition to the culture of
activating factors, such
as TNF-alpha dramatically improves cell survival and prevents DNA
fragmentation.
Propidium iodide (PI) staining is used to measure apoptosis as follows.
Monocytes are
cultured for 48 hours in polypropylene tubes in serum-free medium (positive
control), in
the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of
varying
concentrations of the compound to be tested. Cells are suspended at a
concentration of 2 x
106/ml in PBS containing PI at a final concentration of 5 ~.g/ml, and then
incubaed at room
temperature for 5 minutes before FACScan analysis. PI uptake has been
demonstrated to
correlate with DNA fragmentation in this experimental paradigm.
Effect on cytokine release. An important function of monocytes/macrophages is
their regulatory activity on other cellular populations of the immune system
through the
release of cytokines after stimulation. An ELISA to measure cytokine release
is
performed as follows. Human monocytes are incubated at a density of 5x105
cells/ml with
increasing concentrations of the a polypeptide of the invention and under the
same
conditions, but in the absence of the polypeptide. For IL-12 production, the
cells are
primed overnight with IFN ( 100 U/ml) in presence of a polypeptide of the
invention. LPS
(10 ng/ml) is then added. Conditioned media are collected after 24h and kept
frozen until
use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a
commercially available ELISA kit (e..g, R & D Systems (Minneapolis, MN)) and
applying
the standard protocols provided with the kit.
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Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1x105
cell/well.
Increasing concentrations of polypeptides of the invention are added to the
wells in a total
volume of 0.2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and
antibiotics).
After 3 days incubation, the plates are centrifuged and the medium is removed
from the
wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (
140 mM
NaCI, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol
red
and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The
plates
are incubated at 37°C for 2 hours and the reaction is stopped by adding
20 ~l 1N NaOH
per well. The absorbance is read at 610 nm. To calculate the amount of H202
produced by
the macrophages, a standard curve of a H~OZ solution of known molarity is
performed for
each experiment.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polypeptides, polynucleotides (e.g., gene therapy),
agonists, and/or
antagonists of the invention.
Example 35: Biological Effects of Polxpeptides of the Invention
Astrocyte and Neuronal Assts
Recombinant polypeptides of the invention, expressed in Escherichia coli and
purified as described above, can be tested for activity in promoting the
survival, neurite
outgrowth, or phenotypic differentiation of cortical neuronal cells and for
inducing the
proliferation of glial fibrillary acidic protein immunopositive cells,
astrocytes. The
selection of cortical cells for the bioassay is based on the prevalent
expression of FGF-1
and FGF-2 in cortical structures and on the previously reported enhancement of
cortical
neuronal survival resulting from FGF-2 treatment. A thymidine incorporation
assay, for
example, can be used to elucidate a polypeptide of the invention's activity on
these cells.
Moreover, previous reports describing the biological effects of FGF-2 (basic
FGF)
on cortical or hippocampal neurons in vitro have demonstrated increases in
both neuron
survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor
promotes
survival of dissociated hippocampal neurons and enhances neurite extension."
Proc. Natl.
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Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in
its
entirety). However, reports from experiments done on PC-12 cells suggest that
these two
responses are not necessarily synonymous and may depend on not only which FGF
is
being tested but also on which receptors) are expressed on the target cells.
Using the
primary cortical neuronal culture paradigm, the ability of a polypeptide of
the invention to
induce neurite outgrowth can be compared to the response achieved with FGF-2
using, for
example, a thymidine incorporation assay.
Fibroblast and endothelial cell assavs~
Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and
maintained in growth media from Clonetics. Dermal microvascular endothelial
cells are
obtained from Cell Applications (San Diego, CA). For proliferation assays, the
human
lung fibroblasts and dermal microvascular endothelial cells can be cultured at
5,000
cells/well in a 96-well plate for one day in growth medium. The cells are then
incubated
for one day in 0.1 % BSA basal medium. After replacing the medium with fresh
0.1 % BSA
medium, the cells are incubated with the test proteins for 3 days. Alamar Blue
(Alamar
Biosciences, Sacramento, CA) is added to each well to a final concentration of
10%. The
cells are incubated for 4 hr. Cell viability is measured by reading in a
CytoFluor
fluorescence reader. For the PGEZ assays, the human lung fibroblasts are
cultured at
5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1%
BSA
basal medium, the cells are incubated with FGF-2 or polypeptides of the
invention with or
without IL-la for 24 hours. The supernatants are collected and assayed for
PGEZ by EIA
kit (Cayman, Ann Arbor, MI). For the IL-6 assays, the human lung fibroblasts
are
cultured at 5,000 cells/well in a 96-well plate for one day. After a medium
change to
0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without
polypeptides of the invention IL-la for 24 hours. The supernatants are
collected and
assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA).
Human lung fibroblasts are cultured with FGF-2 or polypeptides of the
invention
for 3 days in basal medium before the addition of Alamar Blue to assess
effects on growth
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of the fibroblasts. FGF-2 should show a stimulation at 10 - 2500 ng/ml which
can be used
to compare stimulation with polypeptides of the invention.
Parkinson Models.
The loss of motor function in Parkinson's disease is attributed to a
deficiency of
striatal dopamine resulting from the degeneration of the nigrostriatal
dopaminergic
projection neurons. An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4 phenyl
1,2,3,6-
tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and
catabolized
by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.
Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-
affinity
reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria
by the
electrochemical gradient and selectively inhibits nicotidamide adenine
disphosphate:
ubiquinone oxidoreductionase (complex I), thereby interfering with electron
transport and
eventually generating oxygen radicals.
It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF)
has
trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev.
Biol. 1989).
Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in
gel foam
implants in the striatum results in the near complete protection of nigral
dopaminergic
neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J.
Neuroscience, 1990).
Based on the data with FGF-2, polypeptides of the invention can be evaluated
to
determine whether it has an action similar to that of FGF-2 in enhancing
dopaminergic
neuronal survival in vitro and it can also be tested in vivo for protection of
dopaminergic
neurons in the striatum from the damage associated with MPTP treatment. The
potential
effect of a polypeptide of the invention is first examined in vitro in a
dopaminergic
neuronal cell culture paradigm. The cultures are prepared by dissecting the
midbrain floor
plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with
trypsin and
seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass
coverslips.
The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N 1 ). The cultures are fixed with
paraformaldehyde
after 8 days in vitro and are processed for tyrosine hydroxylase, a specific
marker for
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dopminergic neurons, immunohistochemical staining. Dissociated cell cultures
are
prepared from embryonic rats. The culture medium is changed every third day
and the
factors are also added at that time.
Since the dopaminergic neurons are isolated from animals at gestation day 14,
a
developmental time which is past the stage when the dopaminergic precursor
cells are
proliferating, an increase in the number of tyrosine hydroxylase
immunopositive neurons
would represent an increase in the number of dopaminergic neurons surviving in
vitro.
Therefore, if a polypeptide of the invention acts to prolong the survival of
dopaminergic
neurons, it would suggest that the polypeptide may be involved in Parkinson's
Disease.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 36: The Effect of Poly~eptides of the Invention on the rowth of
Vascular Endothelial Cells
On day l, human umbilical vein endothelial cells (HUVEC) are seeded at 2-Sx104
cells/35 mm dish density in M199 medium containing 4% fetal bovine serum
(FBS), 16
units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10%
FBS, 8
units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NO:Y,
and
positive controls, such as VEGF and basic FGF (bFGF) are added, at varying
concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number
is
determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the polypeptide of the
invention may proliferate vascular endothelial cells.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 37~ Stimulatory Effect of Polypeptides of the Invention on the
Proliferation of Vascular Endothelial Cells
For evaluation of mitogenic activity of growth factors, the colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-
tetrazolium) assay with the electron coupling reagent PMS (phenazine
methosulfate) was
performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate
(5,000
cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach
overnight.
After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF,65 or
a
polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are
added to
wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and
allowed
to incubate for 1 hour at 37°C before measuring the absorbance at 490
nm in an ELISA
plate reader. Background absorbance from control wells (some media, no cells)
is
subtracted, and seven wells are performed in parallel for each condition. See,
Leak et al.
In Vitro Cell. Dev. Biol. 30A: 512-518 ( 1994).
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 38~ Inhibition of PDGF-induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
HAoSMC proliferation can be measured, for example, by BrdUrd incorporation.
Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are
transfected with
CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum
and 6
mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd
Staining
Kit (Zymed Laboratories). In brief, the cells are incubated with the
biotinylated mouse
anti-BrdUrd antibody at 4 degrees C for 2 h after being exposed to denaturing
solution and
then incubated with the streptavidin-peroxidase and diaminobenzidine. After
counterstaining with hematoxylin, the cells are mounted for microscopic
examination, and
the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a
percent of the
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BrdUrd-positive cells to the total cell number. In addition, the simultaneous
detection of
the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for
individual cells by the concomitant use of bright field illumination and dark
field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271
(36):21985-21992
( 1996).
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 39: Stimulation of Endothelial Migration
This example will be used to explore the possibility that a polypeptide of the
invention may stimulate lymphatic endothelial cell migration.
Endothelial cell migration assays are performed using a 48 well
microchemotaxis
chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., et al., J. Immunological
Methods
1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore
size of 8
um (Nucleopore Corp. Cambridge, MA) are coated with 0.1 % gelatin for at least
6 hours
at room temperature and dried under sterile air. Test substances are diluted
to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and
25
ul of the final dilution is placed in the lower chamber of the modified Boyden
apparatus.
Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and
trypsinized
for the minimum time required to achieve cell detachment. After placing the
filter
between lower and upper chamber, 2.5 x 105 cells suspended in 50 ul M 199
containing 1 %
FBS are seeded in the upper compartment. The apparatus is then incubated for 5
hours at
37°C in a humidified chamber with 5% C02 to allow cell migration. After
the incubation
period, the filter is removed and the upper side of the filter with the non-
migrated cells is
scraped with a rubber policeman. The filters are fixed with methanol and
stained with a
Giemsa solution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantified
by
counting cells of three random high-power fields (40x) in each well, and all
groups are
performed in quadruplicate.
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The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 40~ Stimulation of Nitric Oxide Production b~~ Endothelial Cells
Nitric oxide released by the vascular endothelium is believed to be a mediator
of
vascular endothelium relaxation. Thus, activity of a polypeptide of the
invention can be
assayed by determining nitric oxide production by endothelial cells in
response to the
polypeptide.
Nitric oxide is measured in 96-well plates of confluent microvascular
endothelial
cells after 24 hours starvation and a subsequent 4 hr exposure to various
levels of a
positive control (such as VEGF-1) and the polypeptide of the invention. Nitric
oxide in
the medium is determined by use of the Griess reagent to measure total nitrite
after
reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of
the polypeptide
of the invention on nitric oxide release is examined on HUVEC.
Briefly, NO release from cultured HUVEC monolayer is measured with a NO-
specific polarographic electrode connected to a NO meter (Iso-NO, World
Precision
Instruments Inc.) ( 1049). Calibration of the NO elements is performed
according to the
following equation:
2KN02+2KI+2HZS0462N0+Iz+2H20+2KZS04
The standard calibration curve is obtained by adding graded concentrations of
KNOZ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration
solution containing
KI and HZS04. The specificity of the Iso-NO electrode to NO is previously
determined by
measurement of NO from authentic NO gas ( 1050). The culture medium is removed
and
HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells
are then
bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the
cell plates are
kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature
at 37°C.
The NO sensor probe is inserted vertically into the wells, keeping the tip of
the electrode 2
mm under the surface of the solution, before addition of the different
conditions.
S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount
of
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released NO is expressed as picomoles per 1x106 endothelial cells. All values
reported are
means of four to six measurements in each group (number of cell culture
wells). See,
Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).
The studies described in this example tested activity of polypeptides of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 41: Effect of PolYpepides of the Invention on Cord Formation in
Angio~,~ enesis
Another step in angiogenesis is cord formation, marked by differentiation of
endothelial cells. This bioassay measures the ability of microvascular
endothelial cells to
form capillary-like structures (hollow structures) when cultured in vitro.
CADMEC (microvascular endothelial cells) are purchased from Cell Applications,
Inc. as proliferating (passage 2) cells and are cultured in Cell Applications'
CADMEC
Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the
wells of a
48-well cell culture plate are coated with Cell Applications' Attachment
Factor Medium
(200 ml/well) for 30 min. at 37°C. CADMEC are seeded onto the coated
wells at 7,500
cells/well and cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium containing
control
buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells-are
cultured for an
additional 48 hr. The numbers and lengths of the capillary-like chords are
quantitated
through use of the Boeckeler VIA-170 video image analyzer. All assays are done
in
triplicate.
Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-esteradiol (
1
ng/ml) is used as a negative control. The appropriate buffer (without protein)
is also
utilized as a control.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 42: Angio,genic Effect on Chick Chorioallantoic Membrane
Chick chorioallantoic membrane (CAM) is a well-established system to examine
angiogenesis. Blood vessel formation on CAM is easily visible and
quantifiable. The
ability of polypeptides of the invention to stimulate angiogenesis in CAM can
be
examined.
Fertilized eggs of the White Leghorn chick (callus gallus) and the Japanese
qual
(Coturnix coturnix) are incubated at 37.8°C and 80% humidity.
Differentiated CAM of
16-day-old chick and 13-day-old qual embryos is studied with the following
methods.
On Day 4 of development, a window is made into the egg shell of chick eggs.
The
embryos are checked for normal development and the eggs sealed with cellotape.
They
are further incubated until Day 13. Thermanox coverslips (Nunc, Naperville,
IL) are cut
into disks of about 5 mm in diameter. Sterile and salt-free growth factors are
dissolved in
distilled water and about 3.3 mg/ 5 ml are pipetted on the disks. After air-
drying, the
inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3%
glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate
buffer.
They are photographed with a stereo microscope [Wild M8] and embedded for semi-
and
ultrathin sectioning as described above. Controls are performed with carrier
disks alone.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 43: An;~iogenesis Assa Using a Matrigel Implant in Mouse
In vivo angiogenesis assay of a polypeptide of the invention measures the
ability of
an existing capillary network to form new vessels in an implanted capsule of
murine
extracellular matrix material (Matrigel). The protein is mixed with the liquid
Matrigel at 4
degree C and the mixture is then injected subcutaneously in mice where it
solidifies. After
7 days, the solid "plug" of Matrigel is removed and examined for the presence
of new
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blood vessels. Matrigel is purchased from Becton Dickinson
Labware/Collaborative
Biomedical Products.
When thawed at 4 degree C the Matrigel material is a liquid. The Matrigel is
mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C and
drawn into cold
3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with
the
mixture of Matrigel and experimental protein at 2 sites at the midventral
aspect of the
abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical
dislocation, the
Matrigel plugs are removed and cleaned (i.e., all clinging membranes and
fibrous tissue is
removed). Replicate whole plugs are fixed in neutral buffered 10%
formaldehyde,
embedded in paraffin and used to produce sections for histological examination
after
staining with Masson's Trichrome. Cross sections from 3 different regions of
each plug
are processed. Selected sections are stained for the presence of vWF. The
positive control
for this assay is bovine basic FGF ( 150 ng/ml). Matrigel alone is used to
determine basal
levels of angiogenesis.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 44: Rescue of Ischemia in Rabbit Lower Limb Model
To study the in vivo effects of polynucleotides and polypeptides of the
invention
on ischemia, a rabbit hindlimb ischemia model is created by surgical removal
of one
femoral arteries as described previously (Takeshita et al., Am J. Pathol
147:1649-1660
(1995)). The excision of the femoral artery results in retrograde propagation
of thrombus
and occlusion of the external iliac artery. Consequently, blood flow to the
ischemic limb
is dependent upon collateral vessels originating from the internal iliac
artery (Takeshitaet
al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for
post-
operative recovery of rabbits and development of endogenous collateral
vessels. At 10
day post-operatively (day 0), after performing a baseline angiogram, the
internal iliac
artery of the ischemic limb is transfected with 500 mg naked expression
plasmid
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containing a polynucleotide of the invention by arterial gene transfer
technology using a
hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther.
4:749-758
(1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a
polypeptide of the
invention is used in the treatment, a single bolus of 500 mg polypeptide of
the invention or
control is delivered into the internal iliac artery of the ischemic limb over
a period of 1
min. through an infusion catheter. On day 30, various parameters are measured
in these
rabbits: (a) BP ratio - The blood pressure ratio of systolic pressure of the
ischemic limb to
that of normal limb; (b) Blood Flow and Flow Reserve - Resting FL: the blood
flow
during undilated condition and Max FL: the blood flow during fully dilated
condition (also
an indirect measure of the blood vessel amount) and Flow Reserve is reflected
by the ratio
of max FL: resting FL; (c) Angiographic Score - This is measured by the
angiogram of
collateral vessels. A score is determined by the percentage of circles in an
overlaying grid
that with crossing opacified arteries divided by the total number m the rabbit
thigh; (d)
Capillary density - The number of collateral capillaries determined in light
microscopic
sections taken from hindlimbs.
The studies described in this example tested activity of polynucleotides and
polypeptides of the invention. However, one skilled in the art could easily
modify the
exemplified studies to test the agonists, and/or antagonists of the invention.
Example 45: Effect of PolYpeptides of the Invention on Vasodilation
Since dilation of vascular endothelium is important in reducing blood
pressure, the
ability of polypeptides of the invention to affect the blood pressure in
spontaneously
hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300,
and 900
mg/kg) of the polypeptides of the invention are administered to 13-14 week old
spontaneously hypertensive rats (SHR). Data are expressed as the mean +/- SEM.
Statistical analysis are performed with a paired t-test and statistical
significance is defined
as p<0.05 vs. the response to buffer alone.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 46: Rat Ischemic Skin Flap Model
The evaluation parameters include skin blood flow, skin temperature, and
factor
VIII immunohistochemistry or endothelial alkaline phosphatase reaction.
Expression of
polypeptides of the invention, during the skin ischemia, is studied using in
situ
hybridization.
The study in this model is divided into three parts as follows:
a) Ischemic skin
b) Ischemic skin wounds
c) Normal wounds
The experimental protocol includes:
a) Raising a 3x4 cm, single pedicle full-thickness random skin flap
(myocutaneous
flap over the lower back of the animal).
b) An excisional wounding (4-6 mm in diameter) in the ischemic skin (skin-
flap).
c) Topical treatment with a polypeptide of the invention of the excisional
wounds
(day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges: lmg
to 100 mg.
d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding
for
histological, immunohistochemical, and in situ studies.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, andlor
antagonists of the
invention.
Example 47: Peripheral Arterial Disease Model
Angiogenic therapy using a polypeptide of the invention is a novel therapeutic
strategy to obtain restoration of blood flow around the ischemia in case of
peripheral
arterial diseases. The experimental protocol includes:
a) One side of the femoral artery is ligated to create ischemic muscle of
the hindlimb, the other side of hindlimb serves as a control.
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b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is
delivered intravenously and/or intramuscularly 3 times (perhaps more) per week
for 2-3
weeks.
c) The ischemic muscle tissue is collected after ligation of the femoral
artery at l, 2, and 3 weeks for the analysis of expression of a polypeptide of
the invention
and histology. Biopsy is also performed on the other side of normal muscle of
the
contralateral hindlimb.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 48: Ischemic Myocardial Disease Model
A polypeptide of the invention is evaluated as a potent mitogen capable of
stimulating the development of collateral vessels, and restructuring new
vessels after
coronary artery occlusion. Alteration of expression of the polypeptide is
investigated in
situ. The experimental protocol includes:
a) The heart is exposed through a left-side thoracotomy in the rat.
Immediately,
the left coronary artery is occluded with a thin suture (6-0) and the thorax
is closed.
b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is
delivered intravenously and/or intramuscularly 3 times (perhaps more) per week
for 2-4
weeks.
c) Thirty days after the surgery, the heart is removed and cross-sectioned
for morphometric and in situ analyzes.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 49: Rat Corneal Wound Healing Model
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This animal model shows the effect of a polypeptide of the invention on
neovascularization. The experimental protocol includes:
a) Making a 1-1.5 mm long incision from the center of cornea into the stromal
layer.
b) Inserting a spatula below the lip of the incision facing the outer corner
of
the eye.
c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).
d) Positioning a pellet, containing SOng- Sug of a polypeptide of the
invention,
within the pocket.
e) Treatment with a polypeptide of the invention can also be applied topically
to the corneal wounds in a dosage range of 20mg - SOOmg (daily treatment for
five days).
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example 50: Diabetic Mouse and Glucocorticoid-Impaired Wound Healing
Models
A. Diabetic db+ldb+ Mouse Model.
To demonstrate that a polypeptide of the invention accelerates the healing
process,
the genetically diabetic mouse model of wound healing is used. The full
thickness wound
healing model in the db+/db+ mouse is a well characterized, clinically
relevant and
reproducible model of impaired wound healing. Healing of the diabetic wound is
dependent on formation of granulation tissue and re-epithelialization rather
than
contraction (Gartner, M.H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh,
D.G. et al.,
Am. J. Pathol. 136:1235 ( 1990)).
The diabetic animals have many of the characteristic features observed in Type
II
diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their
normal
heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a
single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl.
Acad.
Sci. USA 77:283-293 ( 1982)). Animals show polyphagia, polydipsia and
polyuria.
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Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or
normal insulin
levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375
(1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51 (1):l-7 (1983); Leiter
et al., Am.
J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial
complications, and
microvascular lesions, basement membrane thickening and glomerular filtration
abnormalities have been described in these animals (Norido, F. et al., Exp.
Neurol.
83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980);
Giacomelli et al.,
Lab Invest. 40(4):460-473 ( 1979); Coleman, D.L., Diabetes 31 (Supply: l-6 (
1982)). These
homozygous diabetic mice develop hyperglycemia that is resistant to insulin
analogous to
human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this
model
may be similar to the healing observed in human diabetes (Greenhalgh, et al.,
Am. J. of
Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic
(db+/+m) heterozygous littermates are used in this study (Jackson
Laboratories). The
animals are purchased at 6 weeks of age and are 8 weeks old at the beginning
of the study.
Animals are individually housed and received food and water ad libitum. All
manipulations are performed using aseptic techniques. The experiments are
conducted
according to the rules and guidelines of Human Genome Sciences, Inc.
Institutional
Animal Care and Use Committee and the Guidelines for the Care and Use of
Laboratory
Animals.
Wounding protocol is performed according to previously reported methods
(Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172:245-251 (1990)). Briefly, on
the day of
wounding, animals are anesthetized with an intraperitoneal injection of
Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized
water. The
dorsal region of the animal is shaved and the skin washed with 70% ethanol
solution and
iodine. The surgical area is dried with sterile gauze prior to wounding. An 8
mm full-
thickness wound is then created using a Keyes tissue punch. Immediately
following
wounding, the surrounding skin is gently stretched to eliminate wound
expansion. The
wounds are left open for the duration of the experiment. Application of the
treatment is
given topically for 5 consecutive days commencing on the day of wounding.
Prior to
treatment, wounds are gently cleansed with sterile saline and gauze sponges.
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Wounds are visually examined and photographed at a fixed distance at the day
of
surgery and at two day intervals thereafter. Wound closure is determined by
daily
measurement on days 1-5 and on day 8. Wounds are measured horizontally and
vertically
using a calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is
no longer visible and the wound is covered by a continuous epithelium.
A polypeptide of the invention is administered using at a range different
doses,
from 4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control
groups
received 50mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for
histology and immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for further
processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are
evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated
group.
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total square area of the wound. Contraction is then
estimated by
establishing the differences between the initial wound area (day 0) and that
of post
treatment (day 8). The wound area on day 1 is 64mm2, the corresponding size of
the
dermal punch. Calculations are made using the following formula:
[Open area on day 8] - [Open area on day 1 ] / [Open area on day 1 ]
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are
sectioned perpendicular to the wound surface (5mm) and cut using a Reichert-
Jung
microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-
sections of
bisected wounds. Histologic examination of the wounds are used to assess
whether the
healing process and the morphologic appearance of the repaired skin is altered
by
treatment with a polypeptide of the invention. This assessment included
verification of
the presence of cell accumulation, inflammatory cells, capillaries,
fibroblasts, re-
epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J.
Pathol. 136:1235
(1990)). A calibrated lens micrometer is used by a blinded observer.
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Tissue sections are also stained immunohistochemically with a polyclonal
rabbit
anti-human keratin antibody using ABC Elite detection system. Human skin is
used as a
positive tissue control while non-immune IgG is used as a negative control.
Keratinocyte
growth is determined by evaluating the extent of reepithelialization of the
wound using a
calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is
demonstrated
by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human
colon
cancer can serve as a positive tissue control and human brain tissue can be
used as a
negative tissue control. Each specimen includes a section with omission of the
primary
antibody and substitution with non-immune mouse IgG. Ranking of these sections
is
based on the extent of proliferation on a scale of 0-8, the lower side of the
scale reflecting
slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of < 0.05
is
considered significant.
B. Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in
various
in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In:
Anti-
Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989);
Wahlet al., J.
Immunol. I15: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)).
Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing
vascular
permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast
proliferation,
and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes
et al.,
J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of
circulating
monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 ( 1978); Wahl,
"Glucocorticoids and
wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical
Aspects,
Academic Press, New York, pp. 280-302 ( 1989)). The systemic administration of
steroids
to impaired wound healing is a well establish phenomenon in rats (Beck et al.,
Growth
Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797
(1978); Wahl,
'°Glucocorticoids and wound healing", In: Antiinflammatory Steroid
Action: Basic and
Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al.,
Proc.
Natl. Acad. Sci. USA 86: 2229-2233 ( 1989)).
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To demonstrate that a polypeptide of the invention can accelerate the healing
process, the effects of multiple topical applications of the polypeptide on
full thickness
excisional skin wounds in rats in which healing has been impaired by the
systemic
administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River
Laboratories) are used in this example. The animals are purchased at 8 weeks
of age and
are 9 weeks old at the beginning of the study. The healing response of rats is
impaired by
the systemic administration of methylprednisolone ( l7mg/kg/rat
intramuscularly) at the
time of wounding. Animals are individually housed and received food and water
ad
libitum. All manipulations are performed using aseptic techniques. This study
is
conducted according to the rules and guidelines of Human Genome Sciences, Inc.
Institutional Animal Care and Use Committee and the Guidelines for the Care
and Use of
Laboratory Animals.
The wounding protocol is followed according to section A, above. On the day of
wounding, animals are anesthetized with an intramuscular injection of ketamine
(50
mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and
the skin
washed with 70% ethanol and iodine solutions. The surgical area is dried with
sterile
gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes
tissue
punch. The wounds are left open for the duration of the experiment.
Applications of the
testing materials are given topically once a day for 7 consecutive days
commencing on the
day of wounding and subsequent to methylprednisolone administration. Prior to
treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day
of
wounding and at the end of treatment. Wound closure is determined by daily
measurement
on days 1-5 and on day 8. Wounds are measured horizontally and vertically
using a
calibrated Jameson caliper. Wounds are considered healed if granulation tissue
is no
longer visible and the wound is covered by a continuous epithelium.
The polypeptide of the invention is administered using at a range different
doses,
from 4mg to SOOmg per wound per day for 8 days in vehicle. Vehicle control
groups
received SOmL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium
pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested
for
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histology. Tissue specimens are placed in 10% neutral buffered formalin in
tissue
cassettes between biopsy sponges for further processing.
Four groups of 10 animals each (5 with methylprednisolone and 5 without
glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control
3) treated
groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal
axis and obtaining the total area of the wound. Closure is then estimated by
establishing
the differences between the initial wound area (day 0) and that of post
treatment (day 8).
The wound area on day 1 is 64mm', the corresponding size of the dermal punch.
Calculations are made using the following formula:
[Open area on day 8] - [Open area on day 1] / [Open area on day 1)
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are
sectioned perpendicular to the wound surface (Smm) and cut using an Olympus
microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-
sections of
bisected wounds. Histologic examination of the wounds allows assessment of
whether the
healing process and the morphologic appearance of the repaired skin is
improved by
treatment with a polypeptide of the invention. A calibrated lens micrometer is
used by a
blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using an unpaired t test. A p value of < 0.05
is
considered significant.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
Example Sl' L~~hadema Animal Model
or The purpose of this experimental approach is to create an appropriate and
consistent lymphedema model for testing the therapeutic effects of a
polypeptide of the
invention in lymphangiogenesis and re-establishment of the lymphatic
circulatory system
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in the rat hind limb. Effectiveness is measured by swelling volume of the
affected limb,
quantification of the amount of lymphatic vasculature, total blood plasma
protein, and
histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more
importantly,
the chronic progress of the edema is followed for up to 3-4 weeks.
Prior to beginning surgery, blood sample is drawn for protein concentration
analysis. Male rats weighing approximately --350g are dosed with
Pentobarbital.
Subsequently, the right legs are shaved from knee to hip. The shaved area is
swabbed
with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing.
Circumference and volumetric measurements are made prior to injecting dye into
paws
after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal
paw). The
intradermal dorsum of both right and left paws are injected with 0.05 ml of 1%
Evan's
Blue. Circumference and volumetric measurements are then made .following
injection of
dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made
circumferentially allowing the femoral vessels to be located. Forceps and
hemostats are
used to dissect and separate the skin flaps. After locating the femoral
vessels, the
lymphatic vessel that runs along side and underneath the vessels) is located.
The main
lymphatic vessels in this area are then electrically coagulated suture
ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and
adductors) are bluntly dissected. The popliteal lymph node is then located.
The 2
proximal and 2 distal lymphatic vessels and distal blood supply of the
popliteal node are
then and ligated by suturing. The popliteal lymph node, and any accompanying
adipose
tissue, is then removed by cutting connective tissues.
Care is taken to control any mild bleeding resulting from this procedure.
After
lymphatics are occluded, the skin flaps are sealed by using liquid skin
(Vetbond) (AJ
Buck). The separated skin edges are sealed to the underlying muscle tissue
while leaving
a gap of -0.5 cm around the leg. Skin also may be anchored by suturing to
underlying
muscle when necessary.
To avoid infection, animals are housed individually with mesh (no bedding).
Recovering animals are checked daily through the optimal edematous peak, which
typically occurred by day 5-7. The plateau edematous peak are then observed.
To
evaluate the intensity of the lymphedema, the circumference and volumes of 2
designated
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places on each paw before operation and daily for 7 days are measured. The
effect plasma
proteins on lymphedema is determined and whether protein analysis is a useful
testing
perimeter is also investigated. The weights of both control and edematous
limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
Circumference Measurements: Under brief gas anesthetic to prevent limb
movement, a cloth tape is used to measure limb circumference. Measurements are
done at
the ankle bone and dorsal paw by 2 different people then those 2 readings are
averaged.
Readings are taken from both control and edematous limbs.
Volumetric Measurements: On the day of surgery, animals are anesthetized with
Pentobarbital and are tested prior to surgery. For daily volumetrics animals
are under
brief halothane anesthetic (rapid immobilization and quick recovery), both
legs are shaved
and equally marked using waterproof marker on legs. Legs are first dipped in
water, then
dipped into instrument to each marked level then measured by Buxco edema
software(Chen/Victor). Data is recorded by one person, while the other is
dipping the
limb to marked area.
Blood-plasma protein measurements: Blood is drawn, spun, and serum separated
prior to surgery and then at conclusion for total protein and Ca2+ comparison.
Limb Weight Comparison: After drawing blood, the animal is prepared for tissue
collection. The limbs are amputated using a quillitine, then both experimental
and control
legs are cut at the ligature and weighed. A second weighing is done as the
tibio-cacaneal
joint is disarticulated and the foot is weighed.
Histological Preparations: The transverse muscle located behind the knee
(popliteal) area is dissected and arranged in a metal mold, filled with
freezeGel, dipped
into cold methylbutane, placed into labeled sample bags at - 80EC until
sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics..
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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Example 52: Suppression of TNF alpha-induced adhesion molecule expression
by a Poly~eptide of the Invention
The recruitment of lymphocytes to areas of inflammation and angiogenesis
involves specific receptor-ligand interactions between cell surface adhesion
molecules
(CAMS) on lymphocytes and the vascular endothelium. The adhesion process, in
both
normal and pathological settings, follows a mufti-step cascade that involves
intercellular
adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and
endothelial leukocyte adhesion molecule-1 (E-selectin) expression on
endothelial cells
(EC). The expression of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the local
vasculature and
extravasate into the local tissue during the development of an inflammatory
response. The
local concentration of cytokines and growth factor participate in the
modulation of the
expression of these CAMs.
Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a
stimulator of all three CAMS on endothelial cells and may be involved in a
wide variety of
inflammatory responses, often resulting in a pathological outcome.
The potential of a polypeptide of the invention to mediate a suppression of
TNF-a
induced CAM expression can be examined. A modified ELISA assay which uses ECs
as a
solid phase absorbent is employed to measure the amount of CAM expression on
TNF-a
treated ECs when co-stimulated with a member of the FGF family of proteins.
To perform the experiment, human umbilical vein endothelial cell (HUVEC)
cultures are obtained from pooled cord harvests and maintained in growth
medium (EGM-
2; Clonetics, San Diego, CA) supplemented with 10% FCS and 1 %
penicillin/streptomycin in a 37 degree C humidified incubator containing 5%
C02.
HUVECs are seeded in 96-well plates at concentrations of 1 x 104 cells/well in
EGM
medium at 37 degree C for 18-24 hrs or until confluent. The monolayers are
subsequently
washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100
U/ml
penicillin and 100 mg/ml streptomycin, and treated with a given cytokine
and/or growth
factors) for 24 h at 37 degree C. Following incubation, the cells are then
evaluated for
CAM expression.
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Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96
well plate to confluence. Growth medium is removed from the cells and replaced
with 90
ul of 199 Medium (10% FBS). Samples for testing and positive or negative
controls are
added to the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C for
either 5 h (selectin and integrin expression) or 24 h (integrin expression
only). Plates are
aspirated to remove medium and 100 pl of 0.1 % paraformaldehyde-PBS(with Ca++
and
Mg++) is added to each well. Plates are held at 4°C for 30 min.
Fixative is then removed from the wells and wells are washed 1X with
PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 ~1 of
diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin,
Anti-VCAM-
1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 pg/ml
{1:10 dilution
of 0.1 mg/ml stock antibody). Cells are incubated at 37°C for 30 min.
in a humidified
environment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.
Then add 20 pl of diluted ExtrAvidin-Alkaline Phosphotase ( 1:5,000 dilution)
to
each well and incubated at 37°C for 30 min. Wells are washed X3 with
PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in
5 ml
of glycine buffer (pH 10.4). 100 pl of pNPP substrate in glycine buffer is
added to each
test well. Standard wells in triplicate are prepared from the working dilution
of the
ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 ( 10°) > 10-
° 5 > 10-' > 10-' ~5. 5
pl of each dilution is added to triplicate wells and the resulting AP content
in each well is
5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 ~1 of pNNP reagent must then be added
to each of
the standard wells. The plate must be incubated at 37°C for 4h. A
volume of 50 pl of 3M
NaOH is added to all wells. The results are quantified on a plate reader at
405 nm. The
background subtraction option is used on blank wells filled with glycine
buffer only. The
template is set up to indicate the concentration of AP-conjugate in each
standard well [
5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound
AP-
conjugate in each sample.
The studies described in this example tested activity of a polypeptide of the
invention. However, one skilled in the art could easily modify the exemplified
studies to
test the activity of polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of the
invention.
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It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible in light of
the above
teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the Background of the Invention, Detailed Description, and
Examples
is hereby incorporated herein by reference. Further, the hard copy of the
sequence
listing submitted herewith and the corresponding computer readable form are
both
incorporated herein by reference in their entireties.
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305
Applicant's or agent's file pF503PCT IntemationalapplicationNo. UNASSIGNED
reference number
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below
relate to the microorganism
referred to in the description
on page 56 , line NfA .
B. IDENTIF'ICATIONOFDEPOSTT
Further deposits are identified
on an additional sheet
Nameofdepositaryinstitution
American Type Culture Collection
Address of depositary institution
(including postal code and
countn)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date ofdeposit Accession Number
08 April 1999 203917
C. ADDITIONALINDICATIONS(leaveblankijnotapplicable)
This information is continued
on anadditiona(sheet
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INDICATIONS ARE MADE (ijtheindicationsarenotjoralldesignatedStates)
Europe
In respect to those designations
in which a European Patent
is sought a sample of the
deposited
microorganism will be made
available until the publication
of the mention of the grant
of the European patent
or until the date on which
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or withdrawn or is deemed
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the issue of such a sample
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sample (Rule 28 (4) EPC).
E. SEPARATE FURNISHING OF INDICATIONS(lem~eblankifnotapplicable)
The indications listed below
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Bureau later Ispec~thegeneralnatureofrheindicationseg.,'Accession
Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with theintemationalapplication ~ This sheet was
received by theIntemationalBureauon:
Authorized officer ~ ~ .4uthorizedufficer
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306
ATCC Deposit No.: 203917
CANADA
The applicant requests that, until either a Canadian patent has been issued on
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the furnishing of
a sample of the deposited biological material referred to in the application
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the International Bureau accordingly before completion of technical
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The applicant hereby requests that the application has been laid open to
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Norwegian Patent
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shall only be
effected to an expert in the art. The request to this effect shall be filed by
the applicant with
the Norwegian Patent Office not later than at the time when the application is
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to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If
such a request has
been filed by the applicant, any request made by a third party for the
furnishing of a sample
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recognized experts drawn up by the Norwegian Patent Office or any person
approved by the
applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a
microorganism shall
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The applicant hereby requests that, until the application has been laid open
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The applicant hereby requests that the furnishing of a sample of a
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307
ATCC Deposit No.: 203917
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The applicant hereby requests that, until the application has been laid open
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furnishing of a sample
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applicant with the Danish Patent Office not later that at the time when the
application is made
available to the public under Sections 22 and 33(3) of the Danish Patents Act.
If such a
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The applicant hereby requests that, until the application has been laid open
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The applicant hereby requests that until the date of a grant of a Netherlands
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the Netherlands
Industrial Property Office before the date on which the application is made
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Netherlands, whichever of the two dates occurs earlier.
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<110> Komatsoulis et al.
<120> 49 Human Secreted Proteins
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<141> 2000-03-09
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<213> Homo Sapiens
<400> 6
gcgctcgagg gatgacagcg atagaacccc gg 32
<210> 7
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 7
gcgaagcttc gcgactcccc ggatccgcct c 31
<210> 8
<211> 12
<212> DNA
<213> Homo Sapiens
<400> 8
ggggactttc cc 12
<210> 9
<211> 73
<212> DNA
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
3
<213> Homo Sapiens
<400> 9
gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60
ccatctcaat tag 73
<210> 10
<211> 256
<212> DNA
<213> Homo sapiens
<400> 10
ctcgaggggactttcccggggactttccggggactttccgggactttccatctgccatct 60
caattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcc 120
cagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccga 180
ggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagg 240
cttttgcaaaaagctt 256
<210> 11
<211> 1205
<212> DNA
<213> Homo Sapiens
<400> 11
tgcaggaattcggcacgagcccgagtggtcgccaacagtggctttgatgtgctttgccat 60
gccctggagtcatacaccaccctgccctaccacctgcggacccctgcccttcaaatccca 120
tcacacggcctgcgtaccagggcagcaacccaatcagtgacatttgggctatccacgcgc 180
tgcggatcgtggctaagtatctgaagagggctgtcagaaatcccgatgatcttgaagcaa 240
ggtctcatatgcacttggcaagtgcttttgctggcatcggctttggaaatgctggtgttc 300
atctgtgccatggaatgtcttacccaatttcaggtttagtgaagatgtataaagcaaagg 360
attacaatgtggatcacccactggtgccccatggcctttctgtggtgctcacgtccccag 420
cggtgttcactttctcggcccagatgtttccagagcgacacctggagatggcagaaatac 480
tgggagccgacacccgcactgccaggatccaagatgcagggctggtgttggcagacacgc 540
tccggaaattcttattcgatctggatgttgatgatggcctagcagctgttggttactcca 600
aagctgatatccccgcactagtgaaaggaacgctgccccaggaaagggtcaccaagcttg 660
caccccgtccccagtcagaagaggatctggctgctctgtttgaagcttcaatgaaactgt 720
attaattgtcattttaactgaaagaattaccgctggccattgtagtgctgagagcaagag 780
ctgatctagctagggctttgtcttttcatctttgtgcataacttacctgttaccagtata 840
ggtgggatatacatttatcttgcaggaaattccccaaagctcagagtccagttccttcca 900
taaaacaggctggacaaatgaccactatgttagacccccaggctcgacttcaggggtcag 960
tgttcctgtcccaaaccccacacagaatactctgcctctgcttcatgtagcaaatgagca 1020
aaaactcagtatctatcaaaagtgtaaattatatttcctatgcctagtaattcacttcat 1080
gtctaaaaatttatctgatagaaacactagcaccagtacatacagaagcatggcaaggat 1140
gtttctggcagcacttttctaataataaaagatttgaaacaaaaaaaaaaaaaaaaaaac 1200
tcgag 1205
<210> 12
<211> 1844
<212> DNA
<213> Homo Sapiens
<400>
12
gactaactgtaataaatgtatgacattattttgattgatacattaaaaaagagtttttag 60
aacaaatatggcatttagctttattatttatttgcttttaagaaatattctttgtggaat 120
tgttgaataaactataaaatattattttgtattgcagctttaaagtggcacactccataa 180
taatctacttactagaaatagtggtgctaccacaaaaaatgttaaccatcagtaccattg 240
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
~1
tttgggagaaagaaacagatcaagaatgcatattattcagtgaccgctttcctagagtta300
aaatacctcctctttgtaaggtttgtaggtaaattgaggtataaactatggatgaaccaa360
ataattagttcaaagtgttgtcatgattccaaatttgtggagtctggtgtttttaccata420
gaatgtggacagaagtacagtcatagctcagtagctatatgtatttccctttatgttaga480
agagactttcttgagtgacatttttaaatagaggaggtattcactatgtttttctgtatc540
acagcagcattcctagtccttaggccctcggacagagtgaaatcatgagtatttatgagt600
tcaatattgtcaaataaggctacagtatttgcttttttgtgtgaatgtattgcatataat660
gttcaagtagatgattttacatttatggacatataaaatgtctgattaccccattttatc720
agtcctgactgtacaagattgttgcattttcagaatagcagttttataaattgatttatc780
ttttaatctataacaatttgtgttagctgttcatttcaggattatattttctacaagttc840
cacttgtgggactccttttgttgcccctatttttttttaaagaaggaagaaagaaaaata900
agtagcagtttaaaaatgagaatggagagaaaagaaaaagaatgaaaaggaaaggcagta960
aagagggaaaaaaaaggaaggatggaaggaatgaaggaaggaagggaggaaggggagaag1020
gtaggaagaaagaaaggatgagagggaaggaagaatcagagtattagggtagttaactta1080
cacatttgcattcttagtttaactgcaagtggtgtaactatgtttttcaatgatcgcatt1140
tgaaacataagtcctattataccattaagttcctattatgcagcaattatataataaaaa1200
gtactgcccaagttatagtaatgtgggtgtttttgagacactaaaagatttgagagggag1260
aatttcaaacttaaagccacttttggggggtttataacttaactgaaaaattaatgcttc1320
atcataacatttaagctatatctagaaagtagactggagaactgagaaaattacccaggt1380
aattcagggaaaaaaaaaaatatatatatatataaatacccctacatttgaagtcagaaa1440
actctgaaaaactgaattatcaaagtcaatcatctataatgatcaaatttactgaacaat1500
tgttaatttatccattgtgcttagctttgtgacacagccaaaagttacctatttaatctt1560
ttcaataaaaattgttttttgaaatccagaaatgatttaaaaagaggtcaggtttttaac1620
tatttattgaagtatgtggatgtacagtatttcaatagatatgaatatgaataaatggta1680
tgccttaagattctttgaatatgtatttactttaaagactggaaaaagctcttcctgtct1740
tttagtaaaacatccatatttcataacctgatgtaaaatatgttgtactgtttacaatag1800
gtgaatataaactcagtttatcaattaaaaaaaaaaaaaaaaaa 1844
<210> 13
<211> 1270
<212> DNA
<213> Homo sapiens
<400>
13
aggaattcggcacgagcagaattcctttctcatatttcaagtgtccctgtgaattatgag60
gggaaaaaaatctttattaaagaaaaaagtgaaaataaatatgcatggatacttggattt120
ttcttttagtaacaaagatatttaaattatttgtatacacacacacacacacacacacac180
acacacacgtatctgtacctagaaatgtttataggggaggtcagttttctgaagattaaa240
tgcagccctaatgtcagattaatgttataaacacatcgtttaatcacaagttttcagaga300
gcaggctccacagatagtctctaactttctatcattacaaatcgctatttttatatcatt360
gctaatttaaataataaagtaaattatgaagaggaatcattggttgcaagtcaccatggg420
agtttagtccctgtgaaaatataaagcatttaaataatttgtattcttttaccatttttt480
attacatctctttaatttttgtcacttgaatatattaggatgatgatgatactataatca540
ctggaacaaagacatttgcttggacatcttttcttttttcccccatttttgttctgttaa600
taatttttaactatagcttttcctttcttgtccttatctgtcccttatcgatcatagata660
gtttcactactatttttaagtttttattgttaaattgaagatgaatctgtacagttactt720
gtgaattaagatgcagctaagttaaaatcaagtataattttgaagctgattttacattta780
actagatgattaaatatattttttcaggtgcttcttcaatttaaatcaagttttatggtt840
tcagcaaaatttagaaaatatgtactttacctaaaaacttttcttttagtgctttggata900
tatacagaagcttaaatgagtagagtatcccaaacatccagatgcttctcaaaatagcat960
ttccggccgggcgcggtggctcacgcctgtaatcccagcactttgggaggccgaggcggg1020
cggatcacgaggtcaggagatcgagaccatcccggctaaaacggtgaaaccccgtctcta1080
ctaaaaatacaaaaaattagccgggcgtagtggcgggcgcctgtagtcccagctacttgg1140
gaggctgaggcaggagaatagcgtgaacccgggaggcggagcttgcagtgagccgagatc1200
ccgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaa1260
aaaactcgag 1270
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
<210> 14
<211> 1237
<212> DNA
<213> Homo sapiens
<400>
14
tcgacccacgcgtccgggcagccatggagtctctgggatatttatgaaatatgatctcag60
ttctcttatggtgacagttactgaggagcacatgccattctggcagttttttgtaagact120
ctgtggtattgttggaggaatcttttcaacaacaggttaacaaccatttcctttttgtct180
aatttctgaaagtgttgcttatacttaagttgcttcttctcaaaggggcaagaagtatac240
agattttcatgtttcggtgtttaatagcttttgctttaattacaaaactccaaaattata300
tggactaaccagtaaacataactaatttcattgtattcaatgatgtaaggtagattatat360
aatggtgagttggaaagatcctgggtgttaacatcagattgaagtagaattgaatgtaag420
ttctgtcatttacttggtggtagcttggaatagatcacctaccttttctgaggttttcta480
atcagtaaaaatagcaataataatacctaatttgcaagtgtgctataggattaaatatga540
aaatgcctagaaggcatttaagacatttttaactgtacagtatgtaaaatcatatgttat600
agtactttgaattatgcaatacatgaaactagatttgtaatcagtaacatgtttatgatt660
tttgtcctgagatcctggaattcgctaagattatttgtcgaattgtaatgatgaaaattg720
ggagttaagtccatagagaaaaaggaaacaatcctattttcaccttcagtccttattcag780
tattaatggagctggggtatttattatacttagaatcatatttttaaatgaatgaatagc840
tttcttcgaaattcttgttagtgctcatttcatctatttaaatttgtgtggttgactcat900
tattccaaatgtttgggtggaactgaaaagattgtgtttaggcccggtgcagtggctcat960
gcctatatcccaacactttgggaggccaaggctggcggatcaacttgaggccagaagttc1020
cagatcagcctggccaacatggctaaaccccgtctctactaaaaatacaaaaattacccg1080
gttatggtgtgcgcatttgtactgttagctactcgggaggctgaggttggggaatcgctt1140
gaacctgggaggtagagattgcagtgagccaagatcatgccactgtactccagcctgggc1200
aacagggtgagactctgtctcaaaaaaaaaaaaaaaa 1237
<210> 15
<211> 2182
<212> DNA
<213> Homo Sapiens
<400>
ccacgcgtccgaaagaaggcccctggctgtggcgccatgtgaaaaggatgaagacctcca 60
ggcccttctccctctgagtctccctccccctgatgaccctctggaagcctgctggctcct 120
cctcaccctcactcacacttcaactcccagttggattggcctgtggacctacctgctgcg 180
tctcagtaggagagaaataatccagacctcaggaacttgacctcacagctccagggaatt 240
caccacgtgggcgaacgggtagagtagaagatgctcagtgaacatgcgcactgaggtggg 300
cgcccaaagagcatgcgcagtgagatgtgtgcttgccttaggggctgtggtgtccctcct 360
tgttccccctacccctgctctttcctccatcccttcctaggaccccactaaggactttgg 420
aatccatcctcccttgtgtgttttttttatgcctctgagactcaaatatctcaatatcac 480
cgttcaaaactcactctgtgttcacctgaccctcctgccctctatggcccttgtcctccc 540
ttgtctgggcccctcaagggagcaagtgcatggtggtgttgctgacttctagtagaagga 600
tgaggatctctatcaggccgtggaccaagtggcccaacccatcgaagtctgctcactttc 660
cctcatgttcactcacactcaacttctgggaggactgatctatggacctatctgatgtgt 720
tatagttagcaagaaagaagtcacaacatttcctcccacagctctgcagggacagaaggc 780
agagagcatggcccacagggaacaggaatttgcataggaagggacatgcacattgagcca 840
gggactttcagggaatgccagagttttccctctgtcaccatgatagcagcagccactgcc 900
atcatactggctgcagcagggaagcccagccagagctgcatgctctgtggagccagcaaa 960
agccagggacaagtgggatccccaccttttacaagttggggagggagcccctgggtgccc 1020
ctgcagccacccaaactgcagttgaagacccagacttctggctctatggagcaggcagga 1080
tccctgccctcctgggtgcagctgtagccactggatccatggctgcagacccaggagctg 1140
gggacaagtgggagccccaacccttcagagttggcgggtcaggagctccttgggtgcagc 1200
tgcaacttcccttcaaggtgcaggacccaggtgtgtctgtagcctgcaatctctgaagcc 1260
tgggaaggcccccactgtccctggggagcagggaacaggggacactgtccctggggagca 1320
ggctcagaggtgtctgctcccactacctggtctctccctgctcccagcacccactatgat 1380
ttcagagcaggttgtgggctgagcccctgcactctcacagctcagatgggcttgcacatg 1440
CA 02361277 2001-08-28
WO fl0/55177 PCT/US00/06058
6
ggcaggtcagccctggaatgccagccccctgccacctcagacccctccagaatttgggtg 1500
ccaagaaacatgagaggggaagccaagggggtgctgagggcagctgggtagtgctctgca 1560
ggtccgagcagcctgggcaccatggactgcagtgggaggcagacagggtccagagcagaa 1620
gggggtggtcctaggtaaggtcccaccttcaggccagggatgggccaggctgccaatccc 1680
agagaccagagtatggacttgtggtgccttttctaggcccacccatggactaatgtacat 1740
gcactttctcccctctgaggtccgtaaatgcctgggactcagccagagcagggcagagga 1800
tgggagcaatgggacaaccagctgcagagaggagctaccctctccagggcctcctctctg 1860
ctgagagctgcagatgttggaaagacctgcctgcagagaggagacacactcttcagggcc 1920
tcctctctgctgagagctgaacacttgacctgacgacctgcctaaagaggagctactgac 1980
tgtggatctcctctgagctgttctaacactcaataaagctccacttgtttgcatacctta 2040
ttcttcccagatgcattaacaagaacttgaacaaaggtgccaccagacacagaggtttcc 2100
agccagaaaattgactgcccaaagatcccatataacccgtacccacatccacagggcatt 2160
ttttagtaaaaaaaaaaaaaas 2182
<210> 16
<211> 1585
<212> DNA
<213> Homo Sapiens
<400>
16
ggcacgagggtgggcgctctttctttttctcttagaagagggtttagcacaggttttttc60
gttctcacttccacaccaccttaccgcctcccgaccccccctctccccctccccacctat120
cgtcatgacggcctctccggattacttggtggtgctttttgggatcactgctggggccac180
cggggccaagctaggctcggatgagaaggagttgatcctgctgttctggaaagtcgtgga240
tctggccaacaagaaggtgggacagttgcacgaatctagttagaccggatcagttggaac300
tgacggaggactgcaaagaagaaactaaaatagacgtcgaaagcctgtcctcggcgtcgc360
agctggaccaagccctccgacagtttaaccagtcagtgagcaatgaactgaatattggag420
tagggacttccttctgtctctgtactgatggggcagcttcatgtcaggcaaatcctgcat480
cctgaggcttccaagaagaatgtactattacctgaatgcttctattccttttttgatctt540
cgaaaagaattcaagaaatgttgccctggttcacctgatattgacaaactggacgttgcc600
acaatgacagagtatttaaattttgagaagagtagttcagtctctcgatatggagcctct660
caagttgaagatatggggaatataattttagcaatgatttcagagccttataatcacagg720
ttttcagatccagagagagtgaattacaagtttgaaagtggaacttggtaagtgcttgag780
tactatttatttgagctttttaacttgttttttttttttttgtctgtttgtttgtttttg840
agacagaatcttgctctgtcgcccaggctggagtgcagtggcacgatctcagctcactgc900
aagctccgcctcccgggttcatgccattctcctgcctcagcctcccgagtagctgggact960
acaggctcccgctaccatacctggctaatttttttgtagttttagtagagatggggtttc1020
accatattagccaggatggtctcaatctcctgacctcgtgatctgcccgcctcgcctccc1080
aaagtgctgggattacaggcgtgagccacctcgcccggcctttaacttgtttttgttcca1140
taattgtgcctcttaattcttgttatatttgagaaactttactaatccttttagctatcc1200
tttcttgctgttcggtcatctgtaaaagaaggttgtattacttagagcttgatattggga1260
ataaaatgtaatgacttactggcatctttagtcaccctgaccttaagtatattatatgct1320
catatctgtgattgttattttggtctccccaaactatccaatctttcatttagaaaattt1380
ccaaaggagaaattgtaggctttaatagagttcacaatttttactgagataaaattttac1440
gtacattctataagtacatttatttatttatttattttagttttgagacacttgctctgt1500
tgcccagctggaatatcgcagtgagctgagatcgcgccactgcactccagcctggtgaga1560
gagcaagactccatcttaaaaaaaa 1585
<210> 17
<211> 1888
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (37)
<223> n equals a,t,g, or c
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
7
<220>
<221> SITE
<222> (1475)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1477)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (1879)
<223> n equals a,t,g, or c
<400>
17
gggggggaaatttgagggcccccggcccgtttagganaaattttgggggagttttggaat 60
ttttttttttttttttaaaaaaaagctggagttggtggcttaggccatcacccttccctt 120
ggctggaactactggacagacccttttgagatgtgcctgtggtgctgtggagatgtgtgt 180
agtggtcttagctctttgttgagcttgtgtgtgcgttgtgtagtcttagctgtatgctga 240
aattgggcgtgtgttggagggcttcttagctctttggtgagattgtatttctatgtgttt 300
gtatcagctgaatgttgctggaaataaaaccttggtttgtcaaggctcytttttgtggga 360
agtaagtaggggaaaaggtctttgagggttcctaggctcctttgtacaacaggaaaatgc 420
ctcaaagccttgcttcccagcaacctggggctggttcccagtgcctggtcctgccccttc 480
ctggttcttatctcaaggcagagcttctgaatttcaggccttcattccagagccctcttg 540
tggccaggccttcctttgctggaggaaggtacacagggtgaagctgatgctgtacttggg 600
ggatctccttggcctgttccaccaagtgagagaaggtacttactcttgtacctcctgttc 660
agccaggtgcattaacagacctccctacagctgtaggaactactgtcccagagctgaggc 720
aaggggatttctcaggtcatttggagaacaagtgctttagtagtagtttaaagtagtaac 780
tgctactgtatttagtggggtggaattcagaagaaatttgaagaccagatcatgggtggt 840
ctgcatgtgaatgaacaggaatgagccggacagcctggctgtcattgctttcttcctccc 900
catttggacccttctctgcccttacatttttgtttctccatctaccaccatccaccagtc 960
tatttattaacttagcaagaggacaagtaaagggccctcttggcttgattttgcttcttt 1020
ctttctgtggaggatatactaagtgcgactttgccctatcctatttggaaatccctaaca 1080
gaattgagttttctattaaggatccaaaaagaaaaacaaaatgctaatgaagccatcagt 1140
caagggtcacatgccaataaacaataaattttccagaagaaatgaaatccaactagacaa 1200
ataaagtagagcttatgaaatggttcagtaargatgagtttgttgttttttgttttgttt 1260
tgttttgtttttttaaagacggagtctcgctctgtcacycaggctggagtgcagtggtat 1320
gatcttggctcactgtaacctccgcctcccgggttcaagccattctcctgcctcagtctc 1380
ctgagtagctgggattgcgggtgcgtgccaccatgcctggctaatttttgtgtttttagt 1440
agagacagggtttcaccatgkggtcgggctggtcnanaactccygaccycytgatccgcc 1500
tgccytggcctcccaaagtgatgggattacagatgtgagccacccgtgcctagccaagga 1560
tgagatttttaaagtatgtttcagttctgtgtcatggttggaagacagagtaggaaggat 1620
atggaaaaggtcatggggaagcagaggtgattcatggctctgtgaatttgaggtgaatgg 1680
ttccttattgtctaggccacttgtgaagaatatgagtcagttattgccagccttggaatt 1740
tacttctctagcttacaatggaccttttgaactggaaaacaccttgtctgcattcacttt 1800
aaaatgtcaaaactaatttttataataaatgtttattttcacattgaaaaaaaaaaaaaa 1860
tttaaaaactcgggggggncccggtacc
1888
<210> 18
<211> 1380
<212> DNA
<213> Homo sapiens
<400> 18
gggaaaggaa gggatggtgg gccaggtggc tctctagatg ggaggtgtcc gggaacttgc 60
aggtctcaag cctgggtgac taggaggatg gtgcgcaaaa caggaaaggc aggacgcggc 120
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
S
tgctgggttcctttctcgagtgcacgcagggctgaaaggaggggagctgagatgagaacg 180
gcccttttccccactgaatgctgcctgcccatgtgtgtcgtcttagctgttttctatttg 240
cccatcgtgtttagtagaataattgaatc~gcagacagttttgactagtccat~ttgcct 300
ctcactccaccccgctgcctctcgtgctttctctctctcttccttacctgcctgctgcca 360
gctccttgcctcccctctgattactgcgcccccgcaataactcactgtttgatcttccac 420
atacgggaactcgaaacccagctactgtc=gcccagcaaggctgcagtgtccagggtgac 480
ccaggccatgtcctgcactgattgtaatcctcctctgcgcctaacacagagccttacgtg 540
tataccgagtagaggagaaggaaggatgtcaaacatgtcactaccaccccccagtgcatg 600
ttggtctccctcacgctggactgtggcaggagtcttccaaaactgctctcagtgccccat 660
cccctctgtgtgcaatcatatcgttgcttacttaaccaaccattcatctcctagggcctg 720
agttctgttacccagcctcctcagaatcttcagcagttcctcatttccataggggaaagc 780
cagctgtttatttcctttacttatgcaacgaataagtcagtgtcagatagtatttttgtt 840
gataaagccwcagcggmgagcaaaacattttggtcctgcttcctgtgagctgactttctg 900
caagggagagagatgataaatgcacatataatatagttgagagtgatcagtgctgcaagg 960
aaaagcagagctgaggaaggagcaaatggtggagagggaggagagggagtggtgctggcc 1020
tagacggtcaaaggttgctgtggtaaggggacactggagttaagatctgagtgaggctgg 1080
gtgcggtggctcatgcctgtaatcccagcactttgggaggatgaggtgggcggatcactt 1140
gaggtcaggagttcaagaccagctcgggcaacatggctaatccccatctctactaaaagt 1200
acaaaaattagcctggcatggtggtatgcacctgtaattccagctacttgggaggctgag 1260
gcaggagaattgcttaagcccgggaggggaaggttgcagtgagccgagatcacaccgctg 1320
cactccagcctgggcaacagagtgagactctgtcacaaaataaaatataaataaaaaaaa 1380
<210> 19
<211> 1048
<212> DNA
<213> Homo sapiens
<400>
19
gccgtcctgcaggtggttgccatcgcggccttcaccaggtagctacggacacccgggaat60
accccacactggggccctcctcctgggcctgaccagtcccccagctgtcacctccccatt120
cctggacaggaagggcacttttcctagtgaactggccatagatggttttggatggttcca180
tctgttctggcaggagtgggagcaggagccagggcagaacaaactgctggaggccctggt240
gttgggaacagctgcggggagggtagggaccagacagaactgccttcaagatgagtccca300
ggagcgcacactcagccctgtcagtggggtctggctttagcagccaggcctccacagacc360
cccatgggcccccagggccgagagggaggacagagcccttcagaacagaggcctcatctc420
actgcatcccccatcaccccctagttccccaatggtcctaatttgtgttctgagatccca480
gtttactccgtggccaggccccacctgtgtttccaagtcgggctggagacgcaggatggg540
gtaggccttgtgctctgagcaaccccagctctgcctcacaggcaggcaggcccggtgcaa600
gagtggactctgggttcctaaagcaataaatgcaaacaagccaacagctctgctgcctag660
caatttccatcttagccacacttctcccttcaggggcttcggaggagaggtcagggctaa720
ggccggggatgagactgcaggagagagagcagcggagggccacattcggagcctccgtcc780
actccagttttatcagcttttgccttttgcacggagtgctaaacaaattctagctctgtg840
tttttttcccattcccagatttactatcagttctccttaaaaagtatctaagctgttaca900
gtagctttcccttcacttgattctattgtgtgttttctatgtttggaataattacaccca960
aatatctagatattttctcttcaccgcattttgtaaataaagagatgtgtatgccwmmmw1020
raaaaaaaaaaaaaaaaagggcggccgc 1048
<210> 20
<211> 1882
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (1407)
<223> n equals a,t,g, or c
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
9
<220>
<221> SITE
<222> (1412)
<223> n equals a,t,g, or c
<400>
20
gaattcggcacgaggtaaatgtggtgtttctcctagctgtgaactgtgtcctaatcagga 60
tggaattttcaaggagacagatgctggaagatgggttcatattgtttgtgccctgtatgt 120
tcctggagtagcctttggagatattgacaaattacgaccagtaacactaacggaaatgaa 180
ctattccaaatatggtgccaaggagtgtagcttttgtgaagaccctcgctttgctagaac 240
tggggtttgcattagctgtgatgcagggatgtgcagagcctatttccatgtgacctgtgc 300
tcaaaaggaaggtctgctttcagaggcagcggcggaagaggatatagcagatccattctt 360
gcttattgtaagcaacatgcagataggttagacagaaagtggaagagaaaaaactacttg 420
gctctacagtcctattgtaaaatgtctttgcaagagagagagaagcaacta~caccagaa 480
gcacaggcaaggatcaatgcccggcttcagcagtatcgtgccaaagcagaactagctcga 540
tctaccagaccccaggcctgggttccaagggaaaaattgcccagaccactcaccagcagt 600
gcttcagctattcgtaaacttatgcggaaagcagaactcatggggatcagtacagatatc 660
tttccagtggacaattcagatactagttctagtgtggatggaaggagaaaacataagcaa 720
ccagctctcactgcagattttgtgaattattattttgagagaaatatgcgca~gattcaa 780
attcaggaaaatatggctgaacaaaagaatataaaagataaattagagaatgaacaagaa 840
aagcttcatgtagaatataataagctatgtgaatctttagaagaactacaaaacctgaat 900
ggaaaacttcgaagtgaaggacaaggaatatgggctttactaggcagaatcacagggcag 960
aagttgaatataccggcaattttgcgagcacccaaggagagaaaaccaagtaaaaaagaa 1020
ggaggcacacaaaagacatctactcttcctgcagtactttatagttgtgggatttgtaag 1080
aagaaccatgatcagcatcttcttttattgtgtgatacctgtaaactacattaccatctt 1140
ggatgtctggatcctcctcttacaaggatgccaagaaagaccaaaaacagttattggcag 1200
tgctcggaatgtgaccaggcagggagcagtgacatggaagcagatatggccatggaaacc 1260
ctaccagatg.gaaccaaacgatcaaggaggcagattaaggaaccagtgaaatttgttcca 1320
caggatgtgccaccagaacccaagaagattccgataagaaacacgagaaccagaggacga 1380
aaacgaagcttcgttcctgaggaaganaaacntgaggaaagagttcctagagagagaaga 1440
caaagacagtctgtgttgcaaaagaagcccaaggctgaagatttaagaactgaatgtgca 1500
acttgcaagggaactggagacaatgaaaatcttgtcaggtgtgatgaatgcagactctgc 1560
taccattttggctgtttggatcctcctttgaaaaagtctcctaaacagacaggctacgga 1620
tggatatgtcaggaatgtgattcttcatcttccaaggaagatgaaaatgaagctgaaaga 1680
aaaaatatatctcaggagctcaacatggaacagaaaaatccaaagaaataaaagattttc 1740
tgtagtgtttttgaaaagtttgcagcttatgtaatagcagataaaatttctaattgtaaa 1800
atgttaaattgtaaaatctaatttgcaaaatgttctcaataaagtcattcaaaatgaaaa 1860
aaaaaaaaaaaaaaaactcgto 1882
<210> 21
<211> 1245
<212> DNA
<213> Homo Sapiens
<400> 21
gtagaatgccatggagttaatagcttctgtgatcctacatgttccaagataactgcagca 60
gtccaaaaagccagtttcctttctggtgtttgtgggcatacactttgttctgctcctgag 120
taagtcacactttgtcttccaggtcctctaggatccctgcaatgttccatttgttgcctg 180
ggcattggagtaatagcagtgcttggtgctacagaaatagatggatggaagttgtggtta 240
ctgtgacaccaaaaacttgtcccctgagctccctactcctctttctcctatattttttgg 300
taattggctctgtcattcacctcactgcaggattcagaatcctggtactgggtcttgtat 360
ttctctttttcccttaccctccttatccaaattgtcaccaagttctgcttcatgctctca 420
tgatttctcacctgagttaccctagtagcttccaaatagggccctctgatttcaaccttg 480
gacactcccactatcttttatattatggaaagatctaatcatgttaccccccatttaaaa 540
tccctcagagagcactgcagatacaattcaagccagttagcttagtgttaaggttttaca 600
tgcatttctgcagccatctgcccctattcttgcaccctaataatgccaaacttcttttag 660
gccttaatttcatcatctgcaaaatgaggataataaatagtacctacttcatatgattgt 720
aataaggsccctcaaaactgtttcatgcctcttttgcccactgcattaggctgttctcac 780
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
attgctataaagaaatacctgaggctgcgtaatttataaagaaaagaagtttaattgact840
cacatttmtacaggctatacaggaagcatgatggtggcatctgcttggctgctggggagg900
gctcagaaaacttacagtcagccgggcacagtggctcactcctgtaatcccagcacttgg960
ggaggccgaggtgggcagatcacctaaggtcagaagttcgagaccagcctgtccaacatg1020
gtgaaacctcatccctactaaaaatacaaaaattagccgggtgtggtggggcggcacctg1080
taactccagctactcagaaggytgaggcaggagaatcacttgaacccaggaggtggaggt1140
tgcagtgagccaagattgcgccactgcactccagcctggacaacagagtgggactctgtc1200
tcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcga 1245
<210> 22
<211> 810
<212> DNA
<213> Homo Sapiens
<400>
22
gattactaacatttttaatatgacacaaattctgttttctgttttgaagattagcaccac 60
agacaggtgatcattaatgaaatatggcccttaaaatacacattacaaaagagaaactga 120
tggtaaaattgctggtgaagttaacttttatcatttctccactaattaaaagttcagatt 180
ctgggatcacatctctaagctgttcatatcaaagagctattttttaaagatcctgattat 240
aggcaacaaaagcaaaaataaacaaataggattacatcaaaactgaaaagcctctgcaca 300
gcaaaggaaacaacagaatgaagagccaacctacataatgggagaaaatatttgtaaatt 360
atacatctgataaggggttaataatcaaaatatataaggaactcaaagaatgcaatagta 420
agaaaacaacccaattaaaaaacaggcaattccagcctgggcaacatggtgaaaccctgt 480
ctctacaaaaaatagataaaattggccgggcacggtgctcacgcctgtaaccccagcact 540
ttggttggccgaggtgggtggatcgcctgaggtcaggagatcgagaccagcctggccaac 600
atggcaaaaccccacctgtactgraaatacaaaaaattagccgggtgcagtggtgggcgc 660
ctgtaatcccagctactcgggaggctaaggcaggagaattgcttgaacccgggaggcgga 720
ggttgccatgagccgagtttgcaccattgcactccagcctgggcaacaagagcaaaattc 780
tgtttcaaaaaaaaaaaaaaaaaactcgag 810
<210> 23
<211> 1675
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (465)
<223> n equals a,t,g, or c
<400>
23
caccgcggtgcggcgcgcctctagaactagtggatcccccgggctgcaggaattcggcac 60
gagctgaaataagtattaatacctaacatttaatattaattctgtcctatgtgcacttac 120
aaatacgtcatataagtatataaattgactttacctattatatgcagaaattgaaggggg 180
gaatttctgtgtttctagctttcttgttaatgtgaagggtggggcattttatgttttttc 240
aggattgccttattcaatgaagcaagctgggtttcctttgggaatattgcttttattctg 300
ggtttcatatgttacaggtaaaatactatataatttcttatttctgcaacttcaactgtg 360
caataactacacacatacacacacacacacacacacacatgctgcaactgaatttcctag 420
ttaattaaaaagtgcatcaaagaatcctaaagaatttaagcatynatgcctgaggasaty 480
cacttttcttactaatcctttaaacagattwgaaaaagtagaaaagcattatatattacc 540
aagcctaaatccttttcatgtgcctttttcactttttgcagaaagtagtagatggggaag 600
tcattttaaagcttcattaaggctaagagacgactgtgctctgaaagtgcagatagcagg 660
gcttcgtgggcaggtgcgtgtgaatgagcaaccttattcagctgttgwttgtggaggtag 720
aaatgaagctctggtgatcaaccagcagggctcatcaagcctctccgcctggcttatgcc 780
tgcccaaactcgctgcctgctactgtctttaaatgcagcaataagttgcttctgtatcac 840
atcttctcttctgcttccccttcttcatgtaaatagttcattcacacccacgttttgtca 900
ctctcccatttaaatgcaggctttacaamtcataatcatctcctctttttgttgtcctcg 960
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
tccccgccttg~ca~cccaccatctcaccttatgacaaacatggcgtctcctctacctta 1020
ggatcactgtgaaagaggtggcaatgatcaaaaacagacaccactcacaggagttgctgc 1080
tgtggtggattagatgcccttttaccagc~agtgcaaaacacctcagctgccctttgctc 1140
tggagtgttgccc~gggcctccagggggtctccttgcytccacgggaccaatgtgtggta 1200
cagttggagctccagggctccaccactgcagtacttgctgaactctttcccctgccctgt 1260
cctctcattccccttcttctggagcatttcatccacagaccccttgcccaagaatgtctg 1320
tctcagctctgcttctagagatcctccaagatgccatccctcgtaaaacctcaggcttcc 1380
aagacctgcccaacactcagctattttttctcaggctgacgaagacagagtaaagcatga 1440
aatagttttcaaattcaaagctagagaggcattgagctccctgccttcttcctaccacct 1500
ccacccttaagacctaaactgcttctagttattacagctcttaatttgtaacttaggacc 1560
cataaattagcagcatgagacctaagtgtgcttattaagagttgctggtgtttaaaaatt 1620
gcagtctacttattggggcatttattacactaaaaaaaaaaaaaaaaaactcgag 1675
<210> 24
<211> 1069
<212> DNA
<213> Homo Sapiens
<400>
24
gtttaaaagttaccttgatgacagagtcattgctatacttgcaacttattttattgtggg 60
ggatcagtgaaataccctctagtaatactgagatgtatagaaaatgcccctaaactaaat 120
gcccccactgttttaagttaaactgtattgttggcctgggagcaagggtaatgggtagtt 180
tttccttgtagttaagctttgagctgtcatttcttattttagttattgtactgcctatga 240
aggctcttctaatttttaaattcttaactcgttttggtgaagtagatgaacaaccaagtt 300
tattgaagctttaatcatagttgaacatgtaattgagtcactgggatctgcataaaagtt 360
tgcagatyctgatatacagtctttataggagttagaatatgaagattgtagtagctattt 420
tcatcacccatcagacttatatgtttattcagtagagctgaaattattaaatgtatgaat 480
attactggctttttcctgaccattcacacttcatggtgatttcacaagagaaggtatatg 540
attctggtttccttgcagacttgttttttataatcacrtttattgaggcttaattttcat 600
acaataaattttatatatattttaagtttacaatttgccactcagatcaagatacggaaa 660
attctcatctattggagaaagtttcctcatacttctttgcagccatcctcccsracctgc 720
cacttggcccaggcaatcactaatctaatttttaacactacagattagtttgccacttca 780
tctaaatagaaacatattttatgtagttgtttgtctggcttttttccttcaacaatgtct 840
gccaataatattgcatttattgaaagttacttcatttttattgctgagtagtaagtagtc 900
tattgtgtaatggataccacagtttatccattcgtctgtggatggacatttggggttttt 960
acgcttttaagttgcaatgaacatgcatacaagtattgctgtgatcattgtttttccttc 1020
tcttgaataaataactagaagtggaaaaaaaaaaaaaaaaaaactcgag 1069
<210> 25
<211> 1475
<212> DNA
<213> Homo Sapiens
<400> 25
cacaggccaggctccaccctctagtaaaggggaagaggctgctggttatgcccagragtc 60
tcaaagggaggaggccagctgagtaggcagcctggtgagggggggcaggggatgggcagg 120
agggtgggagagtggatgaggggcttctcactgtacatagagtcactggcatgatgccct 180
cgctcccccatgcccccacatcccagtggggcataactaggggtcacgggagagcagtct 240
cgtctcctgtgtgtatgtgtgtgagtggtgggcaggccagtggcagggccggccccagcc 300
cctgcatggattccttgtggcttttctgtcttttgctagcttcaccagtttctgttcctt 360
gtgggatgctgctctagggatactcagggggctcctgctctccttccccttcccttcttg 420
cctcaccattcccctaggcaggccctgcaggtcccacactctcccaggccctaaacttgg 480
gcggccttgccctgagagctggtcctccagcgaggccctgtcagcggtcttaggctcctg 540
cacatgaaggtgtgtgcctgtggtgtgtgggctgctctaggagcagatacaggctggtat 600
agaggatgcagaaaggtagggcagtatgtttaagtccagacttggcacatggctagggat 660
actgctcactagctgtggaggtcctcaggagtggagagaatgagtaggagggcagaagct 720
tccatttttgtccttcctaagaccctgttatttgtgttatttcctgcctttccgagtcct 7g0
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
1?
gcagtgggctgccctgtaccctgaacctcatgagcctctaagggaaaggaggaacaatta 840
ggacgtggcaatgagacctggcagggcagagtacaagcccagcacccagtgtcccagcct 900
tactgggtccttaccctgggccaaacagggagggctgatacctccttgctct~cctagat 960
gcccacctcctacaatctcagcccacaagtcctctccaccctagggggcttgc~gcatgg 1020
caataactcataatctgatttggaggtttgccctttacaggggcagattttctgctcagt 1080
tcaacaatgaaatgaagaggaactccctctttctacagctcacttctatcagaggcccag 1140
gtgcctcagagccacattgagttgctttttctgggatgaggaagtagggttaaactcccc 1200
agtttcctgagggaggctcctgacaggtgccctttgtcagaccctaccacagcctggata 1260
ggcagccacattggtcctcgcccttgctcggcactccgtggtggtcctgcccttctccct 1320
gcatgcctgtgggtctgctctggtgtgtgaaggtcggtgggttaactgtgtgcctactga 1380
acctggcaaataaacatcaccctgcaaagccaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1440
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1475
<210> 26
<211> 931
<212> DNA
<213> Homo Sapiens
<400>
26
ggcacgagctgcctcggcctcctgagtagctgggattacaggtgtgcgccactacaccca 60
gctaatttttgtatttttattagagacgtggtttcaccatgtcagccaggatgttctcaa 120
tctcttgacacgtgtctccctccagcttctgcccttatttacctgcctgttggctcttaa 180
aggcatttttggttgagacttttgcatgtaaaataagaactttgccatctttggattccc 240
ccatgtttgtttgtttgtttttaataaataatatttaatattaaaaggagctgtggtttt 300
tttctctctaaagagcaggaaaggacagagactggtcgctctctgcagtcacacttgarg 360
tgaagaaaccatgggatagaattagcygttagttgagtcccyaggttcmgaggragaaag 420
aagaccataccacctggtagggagtgcgaaggaggttccagggactgatccatagaaggg 480
tktcagamcaacttcmttaatwaaggaktgccttaatttycttggtcaaggggaaacttc 540
ctgtgtgcctttctatcctgatgacaccccctcactgaaatcctggtgttgtttccacag 600
agctgtctggccttttgtccttgatccttggttaaggaaatgaccaaccaggtaagacct 660
gtgttgaagggaaaaacatcaattggtctctttgccaagttcaagtccagtaggttttct 720
ggatgtgtagtcaagaaactagcatctcattcttgcccggctcctaaagagctgggtgac 780
cctgggtacctagtcaccacagtgttaggtgggactagtgaaatgcaaatactgagagga 840
ggattggttcatccaagtctttaccacaatttagtgagtaatagaagaagcttaaaagat 900
gttgtacaaaaaaaaaaaaaaaaaactcgag 931
<210> 27
<211> 911
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (265)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (327)
<223> n equals a,t,g, or c
<400> 27
ggcacgagcc acgcccggct aattttttgt atttttggtg gagactgggt ttcaccgtgt 60
tggccgggat ggtcacgatc tcctgatctc atgatccgcc cgcctcagcc tcccaaagtg 120
ctgggattac aggcttgagc caccgcgccc ggcctgtcag atgctttttt gcatctgttg 180
tgatgatcat atggtttttg cccttcactt tgcttgtatg gtttatcaca tttatagatt 240
tgtttatgtt gaaccaccct tgtantcctg ggataaatct cacttgatca tggtgaatgc 300
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
13
ttcttttatgggctgttgaattggttngttagtattttgktgaggattttkgcatattkg 360
ttcatcaggatattgacctgtaattttcttgtagtgtccttgtctcgctttggtatcag 420
g
ggtaatgccgacgtcgtaaaatgagtttggaagtgtatctggaagtattcgtcgtctttg 480
atttttgggaagagtttgagaagaattgg-actagttttaccttaagtgtttggcagaat 540
tcttctggtctcttgatgttttcattgccttgctgagcatagatccaggtacttatgagc 600
aaaattctactcagtctgacaactctgcttagaattatgaatgtgattaaatgcagtggt 660
cactctacctttctaaggtagttcagctcaagttacagcgatccagtcctgatagagctt 720
tgaaacctcaagtctattatataacgtaatgaaaaattggcttggtgaggtggttcacac 780
ctataatcccagcactttgggaggctgaggtgggtggatcacttgagttcggagttcagg 840
accaggctgggcaacatggcaaaaccccacttctaccaaaaaaaaaaaaaaaaaaaaaaa 900
ctcgtgccgaa 911
<210> 28
<211> 1697
<212> DNA
<213> Homo Sapiens
<400>
28
ggaagaaaacaatgctggccccttcatctaaattctgagaattatttaacacttagtacc60
caactctcaagttgttatgaagattaaatcacataatgtgatgttcccggcacagtgctc120
tgtaacatactccggagcacatagtacctgcttaataagcattgcataagtatatgtgta180
catgttgtttttcagtgcagacttactcagacgttgttgccttctcctgtctctgaagtt240
aaagagctagcaaagaatgtggtttttcaggatagagatcgattttttatttgatcagaa300
gtatttgtgttgtgatgagtgatgagtgtaagagtttgttccatgcctgctttctctagc360
taaatgctactaatgatgggtctggggaagctacatcagcattgacaggagatgtgttta420
aaatgcactttcatggatgcttttaaaacctgcagawtcacattmcatagtgtaggscca480
gggttaccaaatgatttatatgcacactgaagtttgagagscaatgtttagctaagtggt540
tcttggcccaggcttctaattaagattccatggccaggttgcagaaatcttttcacttgt600
gcccttcccgcaggctctgtatattggtgtgggtgggagcatccttgttgatataattaa660
gtgcctcatgtgactccaggttgaggccagggtcaaacatgaggaattcaaaatacattc720
atgagagttgagttcaaactttattccaaagggaggtccattccaatggttggatttggt780
agggacaagttagtgtggcccatatccccattgctgtagcagaaattgtggcatctgtgg840
caggaaaagagaaagataaattttgatctttgtggaggagctcactgtccttgaatctca900
cctgttataaagaacataaatgagtggacattttctgcatgcctggatctttctacctgt960
gtttgtggtggtagcaggtgaagagattgtgctgattcctttaaaggcatattcccaaga1020
tgcaggtgtgacttgtccagagaatatcacctgagaataaatcctagagaaggacgatga1080
agagaaaaatggctttttctcaggtgactgtgtctcagaccaggaacagtgttcactctg1140
cctyctggaatgccatatgtttagaacttacaaacctgtacttcttgactttatgctgkt1200
tctccctataagtttgkttaaacactttttcttctcatgatagtcaaacaactctgaaaa1260
aatattattttctatacactagagtcttctcgacattctcttcatcttggcttcttctct1320
gctatgcagaattctcatcattaatttatgactcataatattaaaaatattccctttggc1380
tgggcgcggtggctcatgcttattatcccagcattttgagaggccgatgtgtgtggatga1440
cctgaggtcaggagttcgagaccagcctggccaacatggtgaaaccctgtcactactaaa1500
aatagaaaaaatagctgggcatggtggtgggtgcctgtaatcccagctacttgggaggct1560
gaggcaagagaatcacttgaacccaggaggtggaggttctggaggttgcagtgagcctac1620
atcgtgccgctgcactccagcctgggcaacagagagagactccatctccaaagaaaaaaa1680
aaaaaaaaaaaactcga 1697
<210> 29
<211> 1142
<212> DNA
<213> Homo sapiens
<400> 29
gaggcaatag gtcggggaag gtgatgaatg ttctgtgggg catgtcaaat tgggtggaac 60
ctctggggcc ttctgtggga ratgcccagg gagcacagat ttaggagatg ggagcaacta 120
gtgtgatggt ggtgagggct ggttgaaacc ctggggagta tgtggagctc atctgtgttt 180
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
14
ccacagagcttatctcccaggagatagccatcgggagtgccttgcctggcatgttcccct 240
gctgaggtctgttacccaggagcctgcagacacaaagagcaggctggtaatgctgagaag 300
cgaacattcagtacctgtcaccagaacccagcatgggtgttcaacactatctggtgactc 360
tgtgagaagaccctatgctcaggggatgaagtgtgttgcttgtgcaagagggatggagag 420
agagtgttttccaagtatatgtgtgtgtgcatgtgtgtgtacccaggtggaaccctcctg 480
catgctcacatatgcctttatgaatactggaatctctaaacctaccatcatgcatctcgt 540
cttagcttcctacctctctctttctacccctgcaacagccatgttattgccagtaacaca 600
tgagaagagtgagggagacctgtctgtagacaagctcagtgtgctgctaaggaagcaggc 660
agcagttctgtcctcatgcgttcccattgtgccctgtcctgggatggcaaatgcaaggcc 720
agacaggctctgggstctctggtctgaccactaatagcatcttctcctcccgctaggctg 780
accagcctccaaggcaggactctgacaccagggttataaatgcatctgtctgggcacatt 840
atctaaattgttatgtatcaccctgggtaatggcaaaagtaaaaaccgctgttagctcag 900
tgaataaatccttggtgctgatcaatcattgcacgacatagactcttttaataggcacaa 960
tttacacagaggcttggcagactgcttcgtcttctaattgctgatggaaaatggatgccg 1020
agctctgctgtgccgtaaaaatgtaaactataaatgacttaaaaacttgtgtgctccctt 1080
ctccacccagcactcattttaaccttttatttagaaacaaaaaaaaaaaaaaaaaactcg 1140
ag
1142
<210> 30
<211> 2322
<212> DNA
<213> Homo sapiens
<400>
30
ctcgtgccgaattcggcacgagcggcacgagggagacggttgggagaaccgttgtggcga 60
gcgctacacgaggcaaacgacttctcccttctttgaactggaccccgcgagcaccagagt 120
cggcgtaactatcgcctgacaggcatttaaatcaaacggtattgagatggattgggttat 180
gaaacataatggtccaaatgacgctagtgatgggacagtacgacttcgtggactaccatt 240
tggttgcagcaaagaggaaatagttcagttctttcaagggttggaaatcgtgccaaatgg 300
gataacattgacgatggactaccaggggagaagcacaggggaggccttcgtgcagtttgc 360
ttcaaaggagatagcagaaaatgctctggggaaacacaaggaaagaatagggcacaggta 420
tattgagatcttcagaagtagcaggagtgaaatcaaaggattttatgatccaccaagaag 480
attgctgggacagcgaccgggaccatatgatagaccaataggaggaagagggggttatta 540
tggagctgggcgtggaagttatggaggttttratgactatggtggctataataattacgg 600
ctatgggaatgatggctttgatgacagaatgagagatggaagaggtatgggaggacatgg 660
ctatggtggagctggtgatgcaagttcaggttttcatggtggtcatttcgtacatatgag 720
agggttgccttttcgtgcaactgaaaatgacattgctaatttcttctcaccactaaatcc 780
aatacgagttcatattgatattggagctgatggcagagcacaggagaagcagatgtagag 840
tttgtgacacatgaagatgcagtagctgccatgtctaaagataaaaataacatgcaacat 900
cgatatattgaactcttcttgaattctactcctggaggcggctctggcatgggaggttct 960
ggaatgggaggctacggaagagatggaatggataatcagggaggctatggatcagttgga 1020
agaatgggaatggggaacaattacagtggaggatatggtactcctgatggtttgggtggt 1080
tatggccgtggtggtggaggcagtggaggttactatgggcaaggcggcatgagtggaggt 1140
ggatggcgtgggatgtactgaaagcaaaaacaccaacatacaagtcttgacaacagcatc 1200
tggtctactagactttcttacagatttaatttcttttgtattttaagaactttataatga 1260
ctgaaggaatgtgttttcaaaatattatttggtaaagcaacagattgtgatgggaaaatg 1320
ttttctgtaggtttatttgttgcatactttgacttaaaaataaatttttatattcaaacc 1380
actgatgttgatactttttatatactagttactcctaaagatgtgctgccttcataagat 1440
ttgggttgatgtattttactattagttctacaagaagtagtgtggtgtaattttagagga 1500
taatggttcacctctgcgtaaactgcaagtcttaagcagacatctggaatagagcttgac 1560
aaataattagtgtaacttttttctttagttcctcctggacaacactgtaaatataaagcc 1620
taaagatgaagtggcttcaggagtataaattcagctaattatttctatattattattttt 1680
caaatgtcatttatcaggcatagctctgaaacattgatgatctaagaggtattgatttct 1740
gaatattcataattgtgttacctgggtatgagagtgttggaagctgaattctagccctag 1800
attttggagtaaaaccccttcagcacttgaccgaaataccaaaaatgtctccaaaaaatt 1860
gatagttgcaggttatcgcaagatgtcttagagtagggttaaggttctcagtgacacaag 1920
aattcagtattaagtacataggtatttactatggagtataattctcacaattgtattttc 1980
agttttctgcccaatagagtttaaataactgtataaatgatgactttaaaaaaatgtaag 2040
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
is
caacaagtcc atgtcatagtcaataaaaacaatcctgcagttgggttttgtatctgatcc 2100
ctgcttggag ttttagtttaaagaatctatatgtagcaaggaaaaggtgctttttaattt 2160
taatcccttt gatcaatatggcttttttccaaattggctaatggatcaaaatgaaacctg 2220
ttgatgtgaa ttcagttattgaacttgttacttgtttttgccagaaatgttattaataaa 2280
tgtcaatgtg ggagataataaaaaaaaaaaaaaaaaaaaaas 2322
<210> 31
<211> 658
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (565)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (571)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (589)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (596)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (621)
<223> n equals a,t,g,or c
<220>
<221> SITE
<222> (643)
<223> n equals a,t,g,or c
<400> 31
ttcggcacga gtatacacaacattttgttttttttttgcaaatactctactgaatatttc 60
cccattctat ttaatatattatgatctttcatactttatgaaactctaaaactgtaaaat 120
aatttttggg agggagagaagagttctattgcaaaaaaaaatgctttatgcacataattt 180
ttttatgcta aacaatttccttggaataaacttgcaggcatggacatctagattaagtwt 240
gcaatttctt ttgacatttgctacatattgctatgctaatttccaaaagaactgtactca 300
gtaagacaat tacacatttcagcctccgcactgtaactctgtcagcatttgtgtgtgcac 360
acataattac acatagagttgacacagacattttgccaagataaaaaaaaaaaaaaaaaa 420
aactcgaggg ggggcccggtacccaattcgccctatagtgagtcgtattacaattcactg 480
gccgtcgttt tacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcctt 540
gcagcacatc cccctttcgccagcnggcgtnaatagcgaagaggcccgnaccgatngccc 600
ttcccaacag ttgcgcacctngaaatggcgaatggcaaattgnaagcgttaaaaattt 658
<210> 32
<211> 1437
<212> DNA
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
16
<213> Homo sapiens
<400>
32
ggcacgagaaatattcttcctgaggttaatggaacataattttccacagccaatagcatt 60
caatagtcaagactgcttttaatgattttccagtgcgtgccgcttcttaatttgcaaata 120
tggtggatgttataattatataaccaatatgtatgtattagttaaacagatattggaggg 180
ataactagggaatctaaccttaacctctaataccatgagaaaatcaattgcaaattaagt 240
tctgagatagaagctgtttgcaaatctgtatatggagagagcaacatgaaaacacaaaat 300
cactgtattttttgcattgttattttattttctacttcagtgccacctttaatttggtcg 360
tggcagtgtgtttccgtccattctttattttaaatagagaagtcatttatttggctcatg 420
gttctggaggctgagcagtccaagatgtagtggctgcatctggtgagggcctttgtgctg 480
caccatagcatggcagaagggccagcaagcccacgagacagagaaaatgggagccaaact 540
tacccttttgtcaggaacccactcccttgataactaacacgctcctatgatagtggtatt 600
aatccagtcataagggctctgctctcaagatctaatcacctcttaaaggcccaacctccc 660
aacctcctttttggcctgtcttttgtgtgtgcaggcgcatgtgtgcaaataaaatcacat 720
caggttaagcttgtcaaagtagggagcagctggctttaagataaactctctcatccacac 780
atattttccccaccccaagcacagctgcctcttaactgctgttggaccataactccaagt 840
gaagtggtcctccctcccccatctccacaaatagcaggaaaagtaatgttgtaatttaca 900
aagttacttactccctgccatttcagagagaagatggagtataggaaagccctcccaccc 960
actgtccttgtgtgatgtgacacaacttgctccctcagtcttggaatacagcctcatgtg 1020
gtccttggccagaggcagagaaagggccttcatctctctcacagctgaagggtgggaaac 1080
atggtcttcattccgtgtaaccacgtgctcagctagagatcagaggttctgctcataaat 1140
tataaaggagaagagagatttggggatgattagcagcctctgacataggggcatctcccc 1200
cagtgaggcgtgggcagattattaaaggaatggtactgggaataaaagtggctgaggata 1260
aattttaaattctcatattacttcttacaaaaattctaagttttaaatcaagtcgtataa 1320
tttttaagaataggtcttcaattgatttctagatggcaaataatttatataaacaaaaac 1380
aatggagccttctccaaaaatgaatactcaattaaaaaaaaaaaaaaaaaactcgag 1437
<210> 33
<211> 1968
<212> DNA
<213> Homo Sapiens
<400> 33
gcgggtctgcgaggtggggtaggcgggcaaggcgggcgccgaggtttgcaaaggctcgca 60
gcggccagaaacccggctccgagcggcggcggcccggcttccgctgcccgtgagctaagg 120
acggtccgctccctctagccagctccgaatcctgatccaggcgggggccaggggcccytc 180
gcctcccctctgaggaccgaagatgagcttcctcttcagcagccgctcttctaaaacatt 240
caaaccaaagaagaatatccctgaaggatctcatcagtatgaactcttaaaacatgcaga 300
agcaactctaggaagtgggaatctgagacaagctgttatgttgcctgagggagaggatct 360
caatgaatggattgctgtgaacactgtggatttctttaaccagatcaacatgttatatgg 420
aactattacagaattctgcactgaagcaagctgtccagtcatgtctgcaggtccgagata 480
tgaatatcactgggcagatggtactaatattaaaaagccaatcaaatgttctgcaccaaa 540
atacattgactatttgatgacttgggttcaagatcagcttgatgatgaaactctttttcc 600
ttctaagattggtgtcccatttcccaaaaactttatgtctgtggcaaagactattctaaa 660
gcgtctgttcagggtttatgcccatatttatcaccagcactttgattctgtgatgcagct 720
gcaagaggaggcccacctcaacacctcctttaagcactttattttctttgttcaggagtt 780
taatctgattgataggcgtgagctggcacctcttcaagaattaatagagaaacttggatc 840
aaaagacagataaatgtttcttctagaacacagttaccccettgcttcatctattgctag 900
aactatctcattgctatctgttatagactagtgatacaaactttaagaaaacaggataaa 960
aagatacccattgcctgtgtctactgataaaattatcccaaaggtaggttggtgtgatag 1020
tttccgagtaagaccttaaggacacagccaaatcttaagtactgtgtgaccactcttgtt 1080
gttatcacatagtcatacttggttgtaatatgtgatggttaacctgtagcttataaattt 1140
acttattattcttttactcatttactcagtcatttctttacaagaaaatgattgaatctg 1200
ttttaggtgacagcacaatggacattaagaatttccatcaataatttatgaataagtttc 1260
cagaacaaatttcctaataacacaatcagattggttttattcttttattttacgaataaa 1320
aaatgtatttttcagtatccttgagatttagaacatctgt-gtcacttcagataacatttt 1380
agtttcaagtttgtatggtagtgtttttatagataagatacgtctattttttcaaaattc 1440
CA 02361277 2001-08-28 pCT/US00/06058
WO.00/55177
17
atgattgcagtttaaatcatcatatgacgtgtgggtgggagcaaccaaagttatttttac 1500
agggactttattttttgatctttatttgagattgttttcatatctatctaaattattagg 1560
agtgtgtgtatcagaagtaattttttaatgtcttctaaggatggtcttccaggcttttaa 1620
actgaaaagcttaattcagatagtagcttttggctgagaaaaggaatccaaaatattaat 1680
aaatttagatctcaaaaccactatttttattatttcattatttttcagaggccttaaaat 1740
tctggataagagaatggaggraaatactcagagtacttgattawtttawttccttttatt 1800
aaaaaattacttctatgtttttattgtctcttgagccttagttaagagtagtgtagaaat 1860
gcatgaacttmatcctaataaggataaaacttaaggaaaaccacaataaaccatgaaggt 1920
gtacacatcttaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1968
<210> 34
<211> 754
<212> DNA
<213> Homo sapiens
<400> 34
ggcacgaggaaatttgggggaacggtgttccaggcatagggaatagcagatgtaaaggcc 60
gtgatttgggaataaacttggtgtgatggaggaatggcagagagggcagaacagagcaag 120
agggcaagatttgtaggagatgaggtcatcaggggcctggcaggccatggtgggggtgtg 180
gattctatttttaagtgctgtggaaagtcaaggcagggttttagcagagcaaagatgcaa 240
cctggcttgggctttataaagctccccctggctgcatggagagaaataaaatgtaggcag 300
caaaagtggaagaggagaggcagctggtgcactaatccaggtgagaggtaaagatggatg 360
ggtagggctgaaggatggtggggcaggtggtgagaagtgacttggttctggagacatatg 420
aaggaagatggtcaggcaggccgggcacagtggctcatgcctgtaatcccagcaatttta 480
gaggctgaggtgggaggattgcttgagctcaggagcttgagaccagcctgggctacatag 540
caaaacctcctctctacaaataaaaaaaagtagtcgattgtggtggcatatgcctgtagt 600
cccagctactgggaggcagagataggaggattgcttgaccccaggaggtcgaggctgcag 660
tgagccaagattgcaccactgcactccagcctgggtgacagagccagaccctgtcttaaa 720
aagctaaagcaaaaaaaaaaaaaaaaaaaaaaaa 754
<210> 35
<211> 1324
<212> DNA
<213> Homo sapiens
<400> 35
cccccgggctgcaggaattcggcacgagctttgctatgaagtggcaaattacatgtagag 60
tgtctccttccttttcagagaacagttaatcaaggcaaatcagcaagcccccaaagtgct 120
gtaatttaacatcatgattaccaccttcgaagctatatattttgcatactttaaaatcac 180
ctaacttggactgcttgaattacattggcttttagaaccgaaattgtaactatgtattgt 240
atttcatgggaggtatattttatgagctttttggctttcttttttcccacagcaactgcc 300
aatgaaggatgaatcccctttttaaaaagttgttgttgttgttgttatttgattttgagt 360
taggagggataatagagaagtccatttaaaaattatttttagaagctaaagaaagtaatt 420
atgcttcctgtgaattgtcttttactggcatctttgttttcctctttgatgttagtaaat 480
ttggtgtaatacgtggggcttccatatttcaaagtggaagctttcttctctgaagtcgat 540
atatggttttgaattactagagctttggtcagtatttccttccctatatgtcacagaggg 600
caccactgagaactgcgtgcataggacctcaaaatacaaaattagcagggcctcacagtc 660
agcttcctcatggctagtttttcccccttatattacaatttttgttttataagtcatttt 720
tttcctgatatttccaccacttttcagagtcatctacaaaatttttctttcctcaagaaa 780
agagttccttttgccttattccttatgccttccccactggtattgaggttttgataataa 840
attggtaggaaaaaaaagtacctcctagaaggaagccttccccaccatttccaggtgcca 900
actgctaagcagatatattccaaaaatggtaactgtcatgtgcacactgttggttatttt 960
taataagcctcttcctactagaacattttattttccttgttcaccatacaatcatgtact 1020
ctttaacagaaattgcttttaaaaaatatctggaactatctttaaaaaaactttattaat 1080
aatcatgtatttttactgatcacattttgaaatgcctaaaagactttattgttctaatta 1140
tccagatgtacctttgtaaaatagctcttttatgaattagctgataaggctgtatgtttc 1200
tggaacaaaatattggtcatctaaaaactttctgttttctggggtctgggaaaatagaaa 1260
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
iH
ataagatttc aaata~taaa taagcttaaa ggaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320
aaac 1324
<210> 36
<211> 1099
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (1051)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1073)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1076)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1087)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1093)
<223> n equals or c
a,t,g,
<400> 36
ggagtatatt tcctgaataatatctgttttggagacttggacaaagttaacttttttgct 60
tttttaaaag ttgctgtttaaattaacttcctgcatttctaatgtagaaaaaatatggca 120
cagtgatttg aattttgtttatggtttattatcttcctaaattatcagctctttaaaata 180
tgggtctctg gcctgtataccatctgataaattaggttgggtaaatttttcacctgaaaa 240
aattcattct tttgacccaggttgtttggcttggaaaaaaaaaatcacatactcattgca 300
tgattaaact tgtccatcagattgtgattttgtgcgttatgaggattgtagctggggtaa 360
ttttaaaatg ttggtatttggatagaactgccagcccaggattttgataaagatcaaaat 420
atatactgta tctaaagtttcttttttaaaagttatttcacttataatcaaaaggtttaa 480
ataagtttag tgtatattttatgattagtattattttgtatcgattcagagagaggaaaa 540
ctgcattccc atagacagggagacttggtgcattaatatccaaagcttagtttaagctgg 600
tgatgattag aaaagtggctttcatttgtgttctctggaagattctcagtagtcctgtgg 660
gcatgtgaac aagagcatgtgtaatcttcataatgtatgtcttgttagcagaaatgtctg 720
aggcattgct gagatgtaggatgagctgaggtactggttttgtctttcatggttccctat 780
tatgtttttt gagaaactgagcttttcctgacagtgtttagtttttcttggctatttact 840
aatgctatta atagtaacagtttccattatggagcctcagtaggttcttgacatacatta 900
acccattgct attatgtaattttctaataaaatgatgtgataaaacatattttctgtaat 960
atactcatag aatatgtgattataagtaaataaatagatgttggggtaaaaaaaaaaaaa 1020
aaaaaaaaaa aagggcggccgcccaaagganccccgaggggccagctaagcgncancgaa 1080
gcaagcnccc ccnaaaggg 1099
<210> 37
<211> 1379
<212> DNA
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
1 ~)
<213> Homo Sapiens
<400>
37
cccgggtcgacccacgcgtccgcttcctcaggtccaagagaatgggaggagggtcatgtt60
gaagtgtagccactggaactttcttgcatttatggagttgctttttctttcccaaggaaa120
ctgatgtttgcctgtccgctttatctttgtagtacatgaacagttcagccttagactatg180
taactgttttctcatccttagctgaaaatgaggggcctactgtattgcgagactgttcag240
gggtaggggtggagggggtggtcccttaatggcccctgaagatgttggatgtgtttctca300
aaagctgctttgtctctttcctgagtttaatagtgaaactactaaacattaatagatttg360
cacagccacaaagaatgagagttgataataccgaagaagtgatgcaaaaacaaaaaatca420
cattactgatcattgatagtattacaaataaatgtctcttcctttctcttcctcccttcc480
tccccctgccttcctcttaacctatgtgctcatccttgctacttgaggctgttatttccc540
tccgagtatttaaggagcccaaacaccttggtcttcctgggtgggggcataattaaggga600
agggagtgtataaggaaggaaacctcataccctccattcccatccctggatatattgcct660
tttcagcagcctcgggttcagtgtgcttggggtcaggagtgccaggttcccaagagcagc720
gtaaaacatccctgtaccccttgacagttataagcatttctgcgttaaacttgaagattc780
cagagattccccacgacctaatgatctaagaatgcagattggattctttgatgttcaaat840
ttctcatttacttatgaaaatccctaattatatagttttatataatgtgtatacagagta900
ggattgttgatgaaattgagcggttggaacaccctcatggaacactggcaacatggaatg960
tagagagctgatgtcttccccctcatagaggccattggcttcctttgtataaggagaggg1020
agaagtgattctggaaggagaagttggtatgtctagcttcattgcggcccctcgggctac1080
ccagcaagaggattctgcatcaaggaactggacgagccgctacccaaactgccaccatct1140
ttactctttacaaaatggacccatcgagaatgttgaaaagcttggaagtatcacttttga1200
aaaaaaaaaaaattattttgcattcagcatggactcgaccaggcatctatggagattatt1260
tttttgcttcattttataaagttgtatttagaaaagtcttaagtattgtgctttgtaaag1320
aagatgattaaaatgaaattttgtgagaataaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1379
<210> 38
<211> 581
<212> DNA
<213> Homo Sapiens
<400> 38
gaaggatatggactattgcaatacatttcttccttcaaatcctgccactgttttgttggc 60
ccacaactaataggacctcaaaataagccatgctgctttgcacacacactagccttcttt 120
tgtacttttcattctggatgggcttggccaaaacaggctcaggccaaagacctcccaagc 180
tgtatgtacttccagtatcctgaaacagtgtttggtgacataatgccaagggtaaacaag 240
cctgatttaggcactgctttatccaggggcttcacccatgaaattaataaaacttatctg 300
agtcacttgaaacttggttcccagaaaacacatttctggtttataatctccttttatgct 360
cacctgacattaattatctatccttgatgatgtgtttaaactgagtagcagaaaacagag 420
gccacactttctgggaaattttaaaggaagaaaccatttttaatgagatgaaaatattta 480
acgaatttaaaaagctaatgacaattttgagaaaaggtttgggatgtatattgctatgta 540
atttaataaactgattttatggatataaaaaaaaaaaaaaa 581
<210> 39
<211> 1284
<212> DNA
<213> Homo Sapiens
<400>
39
cccacgcgtccgcggagacaggacactcttcctgaagcggggcgcagatcagaggcagaa 60
gaggaggaggccaggaccatcagagtgacacctgtcaggggccgagagaggctcaatgag 120
gaggagcctccaggtgggcaagacccttggaaattgctgaaggagcaagaggagcggaag 180
aagtgtgtcatctgccaggaccagagcaagacagtgttgctcctgccctgccggcatctg 240
tgcctgtgccaggcctgcactgaaatcctgatgcgccaccccgtctaccaccgcaattgc 300
ccgctctgccgccggggcatcctgcagaccctcaatgtctacctctgaagcctccttccc 360
tgcctgcccacccctccatgctccacgcaggcactcacgctaggacagcattaacacctc 420
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
atctccgggtcctggtctgaatcccctcctacccctgtggccatcctgccatacatccag480
gacattgagttggaagactatgatctgggtgggggcaggataacatggcttctctttacc540
cagtgggtcccttcgatgctgagggtggtgagtatgtcactatgcaagggccctgagact600
atttgctgtgggctctcctccagcctgcccagggcccacccagatgcctctggggttacc660
cctgtctgcttctggtttttctgttggagatctataggtccttttcctgcctccttcaca720
tttcctccccagcttttgcggccacaacacatcagtgtcatttgggtgttttggcaactc780
aggggccttcggatgatcttaaacctttgtgttcagccagagcccctgtgccctggtagg840
cgttggggttagtatctctcgggtgccctcagagccacctctgcctgtgatcg~ctgatg900
aggctccctcccaacctgatccaaaagccagtctcaggagtttacccctgggatggggga960
tgcatctgcacctgactttggggccacgtgccctgtggcaccccagctcactgggagtct1020
caggagggataaccggatttctgctctttcccctgtcactcccacatcacacagaaaaat1080
ggcattcctctctgtctctccctggcatggagagggcagactgtgcacatttcactaggg1140
tccaaatacagaagggcccagggcccaggggcttgcagcttcgtgaggggtctctggccc1200
agtttccaatgaataaagttctcttgacagctmaaaaaaaaaaaaaaaaaaaaaaaaaaa1260
aaaaaaaaaaaaaaaaaaactcga 1284
<210> 40
<211> 792
<212> DNA
<213> Homo Sapiens
<400>
40
cagacatggctgaaatccagtcccgcctggcctacgtgtcctgtgtgcggcagctagagg 60
ttgtcaagtccagctcctactgcgagtacctgcgcccgcccatcgactgcttcaagacca 120
tggactttgggaagttcgaccagatctatgatgtgggctaccagtacgggaaggcggtgt 180
ttggaggctggagccgtggcaacgtcattgagaaaatgctcacagaccggcggtctacag 240
accttaatgagagccgccgtgcagacgtgcttgccttcccaagctctggcttcactgact 300
tggcagagattgtgtcccggattgagccccccacgagctatgtctctgatggctgtgctg 360
acggagaggagtcagattgtctgacagagtatgaggaggacgccggacccgactgctcga 420
gggatgaaggggggtcccccgagggcgcaagccccagcactgcctccgagatggaggagg 480
agaagtcgattctccggcaacgacgctgtctgccccaggagccgcccggctcagccacag 540
atgcctgaggacctcgacaggggtcaccccctccctcccacccctggactgggctggggg 600
tggccccgtgggggtagctcactccccctcctgctgctatgcctgtgacccccgcggccc 660
acacactggactgacctgccctgagcggggatgcagtgttgcactgatgacttgaccagc 720
ccctcccccaataaactcgcctcttggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 780
aaaaaaaaaaas 792
<210> 41
<211> 1351
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (864)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (876)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (883)
<223> n equals a,t,g, or c
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
31
<400>
41
ctttgagcccctctcattccactttagcaatctttttggtaagaactcttaaagccaaaa 60
gtctgctgaaaagatttgctgattattagtttaaaaatcttgtaacactcagcagtgcta 120
ttttgagtcatcccagtttcctgaaagtaatgcccagtcttcctgaatcctccttaatag 180
cagaaccttggtgattttgttggctcatatgaatgcttgtcatggatatgttaacaattt 240
agtgtttgacattgcttcctctgccacaaagacaatactctggtgacacatgtctagacc 300
cagcacaggctgtaggcccaggagtgactcaaaggagtttttccctctttcttacggttc 360
aaaggtgaccctggtggtggccagagcagtaatgcttgtttgatgctcttcatggctcat 420
ctgcttctcagaacccacccgttgagtttgtgggtaaccagcaggcaggccaaagactgg 480
tgcttttcatttcatcctttagagggatgaaacagttatttccgtctgatgagcattcgg 540
tagaatttttgaagtgagattttatgaagtcaaaggggactttacacagatctcgacctg 600
ctttgaaacctagaggtggccctttgatttgtgcgtgtccttgccctctggacaacttaa 660
tatttcaagtaatcgaataccaacttccctgccagcccacctgccttccgccccgcttgt 720
gtaacagtcctgttttgttgagttgctgctattgcactgccagtgcagcccacaccaaat 780
cacaacccaagatactcagataggaagactccttcctctcccagtactttaccaaaggaa 840
cccccgccaggacccacatggggnccacgtgttggncagtggnaatcagcctgtgcaggc 900
tggggatctcaggctgatcagtaggggccagctttggagccagccaagctgaatcccaca 960
ctccaggtctgtgctcaagagaccagatggtgtatttccaaatggscctctctggtatgg 1020
gcaataggcaagctcctggggtctggttatgtggaagattcttagtggatgttccgcctg 1080
gttagctggttctcttcagagaatataaagtgaatgcctttaggggtagctctgaaagag 1140
aaacccaacaacttcattcctagccatgaaagtagcacgatcatattgtactgtattgtt 1200
attgtaaaatgaytatttgccatgtcatgagtaggtagatgttttgccacaaatatgaat 1260
gtgtttgttgttcctgactttaagcaatgaagattgagacaataaatagcactcagagaa 1320
tgaaaaaaaaaaaaaaaaaatgaccctcgag 1351
<210> 42
<211> 2812
<212> DNA
<213> Homo Sapiens
<400>
42
aaagttcatgttgttgatctaaaggcagaatctgtagctgctcctataactgtycgtgct 60
tacttaaatcagacagttacagaattcaaacaactgatttcaaaggccatccatttacct 120
gctgaaacaatgagaatagtgctggaacgctgctacaatgatttgcgtcttctcagtgtc 180
tccagtaaaaccctgaaagctgaaggattttttagaagtaacaaggtgtttgttgaaagc 240
tccgagactttggattaccagatggcctttgcagactctcatttatggaaactcctggat 300
cggcatgcaaatacaatcagattatttgttttgctacctgaacaatccccagtatcttat 360
tccaaaaggacagcataccagaaagctggaggcgattctggtaatgtggatgatgactgt 420
gaaagagtcaaaggacctgtaggaagcctaaagtctgtggaagctattctagaagaaagc 480
actgaaaaactcaaaagcttgtcactgcagcaacagcaggatggagataatggggacagc 540
agcaaaagtactgagacaagtgactttgaaaacatcgaatcacctctcaatgagagggac 600
tcttcagcatcagtggataatagagaacttgaacagcatattcagacttctgatccagaa 660
aattttcagtctgaagaacgatcagactcagatgtgaataatgacaggagtacaagttca 720
gtggacagtgatattcttagctccagtcatagcagtgatactttgtgcaatgcagacaat 780
gctcagatccctttggctaatggacttgactctcacagtatcacaagtagtagaagaacg 840
aaagcaaatgaagggaaaaaagaaacatgggatacagcagaagaagactctggaactgat 900
agtgaatatgatgwgagtggcaagagtaggggagaaatgcagtacatgtatttcaaagct 960
gaaccttatgctgcagatgaaggttctggggaaggacataaatggttgatggtgcatgtt 1020
gataaaagaattactctggcagctttcaaacaacatttagagccctttgttggagttttg 1080
tcctctcacttcaaggtctttcgagtgtatgccagcaatcaagagtttgagagcgtccgg 1140
ctgaatgagacactttcatcattttctgatgacaataagattacaattagactggggaga 1200
gcacttaaaaaaggagaatacagagttaaagtataccagcttttggtcaatgaacaagag 1260
ccatgcaagtttctgctagatgctgtgtttgctaaaggaatgactgtacggcaatcaaaa 1320
gaggaattaattcctcagctcagggagcaatgtggtttagagctcagtattgacaggttt 1380
cgtctaaggaaaaaaacatggaagaatcctggcactgtctttttggattatcatatttat 1440
gaagaagatattaatatttccagcaactgggaggttttccttgaagttcttgatggggta 1500
gagaagatgaagtccatgtcacagcttgcagttttgtcaagacggtggaagccttcagag 1560
atgaagttggatcccttccaggaggttgtattggaaagcagtagtgtggacgaattgcga 1620
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
gagaagcttagtgaaatcagtgggattcctttggatgatattgaatttgctaagggtaga1680
ggaacatttccctgtgatatttctgtccttgatattcatcaagatttagactggaatcck1740
aaagtttctaccctgaatgtctggcctctttatatctgtgatgatggtgcggtcatattt1800
tatagggataaaacagaagaattaatggaattgacagatgagcaaagaaatgaactgatg1860
aaaaaagaaagcagtcgactccagaagactggacatcgtgtaacatactcacctcgtaaa1920
gagaaagcactaaaaatatatctggatggagcaccaaataaagatctgactcaagactga1980
ctctgatagtgtagcattttccctgggggagttttggttttaattagatggttcactacc2040
actgggtagtgccattttggccggacatggttggggtaacccagtgacaccagcactgat2100
tggactgccctacaccaatcagaagctcagtgcccaatgggccactgttttgactcggaa2160
tcatgttgtgcactatagtcaaatgtactgtaaagtgaaaagggatgtgcaaaaaaawar2220
araaaaacaacaaraaragctaaccttctattasawaaggggacaggggaatgagtarac2280
ttcttttattgcggacaaatgtgcacatagccgctagtaaaactagcctcaaacaggatg2340
ctcatagcttaataataaaagctgtgcaaaggccatgaatgaatgaatttcctgtttatt2400
tcactgatgcacacattacctcattgaacaattcagaagtaaatccaacgtgtgttgact2460
cttggaaagcagcaaaarcaggagctgaagaaaagaaattcttggaaccagccgtaaccc2520
agtaaggaattgtgaagttgtgtttttattttgtttcattttttgcagagtattaagaac2580
attattctggaacatcagaacgtttcccttagaccgatcccagcaggtggcagctcagat2640
tgctgcagtgttgtaattataactgattgtacttaagttatggatgtagagaatatgttt2700
cattcatttattcagcatgtaaataaaattgatcctgttgagttatcataattgcagttc2760
aactatctgccmtggttattcttttcacgtatcattcattctgtacatttgg 2812
<210> 43
<211> 1145
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (251)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (901)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1142)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (1145)
<223> n equals or c
a,t,g,
<400> 43
gccgcgctcc tgcctcctgccccagcaggcaggaagaatgggggctgacctctacctcgg 60
tgctcaagag agaggccccagctggcagggacccagaagagcctggagatgttggtgctg 120
gagaccccaa ctctgatcagggactccctgtgctgatgactcagggaacagaggacctaa 180
agggcccagg acaaaggtgtgagaatgagccactgctggaccctgttggccctgagcctc 240
tggggcctga nagtcagtcagggaagggagacatggtggagatggccacacggtttgggt 300
ccaccctgca gctagacctggaaaagggaaggagagtctgttggagaagaggctggtggc 360
agaggaggaa gaggacgaagaggaggtggaagaggatggccccagcagctgctcggagga 420
cgattacagt gagctgctgcaggagatcacagacaacctgacgaagaaggagattcagat 480
agagaagatc catttggacacrtcctccttcrtggaggagctgcctggagagaaggacct 540
tgcccacgtg gtagagatctatgactttgaaccagcgctcaagacggaggacctgctggc 600
aacgttttct gagttccaagagaaggggttcaggattcagtgggtggatgatactcacgc 660
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
~3
actcggcatctttccctgcckggcctcagctgcggaagccctgacccgggagttctcggt 720
gctcaagatccggcccctcacrcagggaaccaagcagtcaaagctcaaagccttgcagag 780
gccaaaactcctgcgtctggtgaaggagaggccacagacaaatgcgactgtggcccggcg 840
gctggtggcccgggccctgggactccaacacaaaaagaaagagcggcctgctgtccgggg 900
ntccgctgccgccctgaggcctggagacccaactggcctggatctgcgtcccgacgtagc 960
tggcgcccccaacaccataagccttcacagacgccagagcagccccgcaccaccctcgag 1020
cttcaccatggggtgtggtgggctttagt:tagtcccagaaatggagaaaaaataaaaac 1080
tcacgttgttctaatgtgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggggg 1140
gnccn 1145
<210> 44
<211> 1509
<212> DNA
<213> Homo sapiens
<400>
44
ggcacgagtaactgcctactatccaatgtcagttaattggtgtcttcccccctcatttgc 60
tctcttccctaaaatgtgtcccagatgccttcatttgctgttttacttctatgttctgct 120
tttcctcctctctttgttcccttcctgtctatccattgagtttatgaaatggaagagtta 180
actgcatgcactagtgtttggagggtgttgtggtttgtctttctaattaggtgtatagcc 240
tattcactttcctagaataaatctcttaacctaaatttgagtagtctgcattttggcaac 300
tcctctagcagcttggtagcctagtacaggttgtttttttaaaaaaggaaaagcaggaag 360
gaggagtgaattttattaacatgtttgccaaatgtattgagatttggcctctgaagaaca 420
ctttttcagtgttaagtttctttaccttaagattcagaaatactttagaatattattaat 480
tttaagtcctgtctttacatccttttggaaaacttgtattaccatgagtttggaaaaagg 540
acaacgaaaggcttttcatgtaaagataagatctttagctatctctaaccctgtcctttt 600
ttcactgcattttttctagttttgcttcattgcttatcattaggatagggtaagtgaagt 660
ttgctatgctgctagcatcctaagatgatacctttgttgaaagaattgtgaatagcatga 720
ttcatttctagcagaggctgagtttaggacagcagcttccattgagaagtctttctgtgt 780
cgtgaatagcattttaatgacctcttggctcacataagcaaacaacatagggacgtatct 840
gctatgaaaatccacaaatttttcagatagtgccctaaaaacaattttatatgcctcact 900
ggttgttattcttaggttattcccacacttgactttatcattgtttactactagtaaaaa 960
gcagcattgccaaataatccctaattttccactaaaaatataatgaaatgatgttaagct 1020
ttttgaaaagtttaggttaaacctactgttgttagattaatgtatttgttgcttcccttt 1080
atctggaatgtggcattagcttttttattttaaccctctttaattcttattcaattccat 1140
gacttaaggttggagagctaaacactgggatttttggataacagactgacagttttgcat 1200
aattataatcggcattgtacatagaaaggatatggctaccttttgttaaatctgcacttt 1260
ctaaatatcaaaaaagggaaatgaagtataaatcaatttttgtataatctgtttgaaaca 1320
tgagttttatttgcttaatattagggctttgccccttttctgtaagtctcttgggatcct 1380
gtgtagaagctgttctcattaaacaccaaacagttaagtccattctctggtactagctac 1440
aaattcggtttcatattctacttaacaatttaaataaactgaaatatttctaaaaaaaaa 1500
aaaaaaaaa 1509
<210> 45
<211> 1857
<212> DNA
<213> Homo Sapiens
<400>
45
ggcacgagcaaaaaataattcttttaagaaaaaatgtaaaaatgtttattctaaaaagct 60
gcattaaagggacaacctataaaaagttttgctagctcatctttagaaggaagaaagaat 120
attagcttgggtgatgtttaatttgggtggcgatagtttctgtaggctaaacttgatgag 180
aaaagtgtacctactctataaaggtaataaatgtaaaacctcttgctgttattgaggaag 240
ctcttcaactaccctaaatttcacaaatgtaacttataacactatgaaaagattgaccaa 300
caatttacgtttgctgtgtgcttagtttttgtttaagcatattctttgctgaattctgtg 360
ttcatgagagttaggtgttttatgctctgaactaatttataacatatttaatatattacc 420
agttaagatataaaatcatttgtacatagcgaattgtaaagcagctattaaagtaggtga 480
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
2~1
aataaagtatatatttgccggttatccatatcttttagaagtcctgacagaacaaccagt 54C
ttatttgcacataggtagcttctgtttgaaggaaggtaaagttataaggaaactcaaata 600
ctataagatgtgtcaaggtatttctccagaattaattgcaaagctagtgctgaaggattt 660
taatcagcttctaaaattttcttctcaataaggcatatgttttgattacttagggaagat 720
tcctcatttttatttgccctttatgcatt~aatccacatgataggacattaaaaattaat 780
ataaagaaaaatcgtgctcatactgtaca~ctgtttctgtgcttggaactacttgttaat 840
agtttttatcgaagctgtcagcaataagggacataaaactgctgtattatacattgtgga 900
attgaataaacagcctaattttttttttc~agtatagggtacttaagcatttccactttt 960
ggaagaaaagtgtattagtattttatattgcatttcatttaaaaggacagtttttttttt 1020
tttttttgtaaatccattcattgaaatggtttctaaactgtataatgtaatttggagcct 1080
atttagtaatagaattaaatgtcctatgtagtgctacaatttttgaattagaaagtgatc 1140
aaatgtaagaaaaaaatttaaaaattcagcccagaaaacaaaatagtgtattaaattagt 1200
ttaatgtaaaaggaatttataagatttttttcctcaatatagatacctcacttgaaaaga 1260
aagcacagcatacttaaagtagttctagtaaacatgtcctagaaaacagttgctaaatgt 1320
aggacatcttttgaggaattagtttatgagaaataaaattttacttgtttttactatcct 1380
gttagaagtatttgtttatcctgataattttaagccaacatagtagtcttaaattacttt 1440
tgaattcctaatctgtgaaggcagtaaatgaaatatctgttctgcaactgttgaaacaaa 1500
taattggctacattgaccataattaaagtcaaaattttgccaatgatgtacagttttatg 1560
gttaaagttgctgtggttggttgcattacatgacacagaaaactgtcctctacctcacgt 1620
gaaataaatattttatatggttttactaaaaataagactcatgtatctggtcacctagtt 1680
tacaaattttgaattatatttattgaaacatgacatactgtgctctgagcttatacctca 1740
attgtattttgtgctgttttccattttcatgccttgtaaataacttgtatagattgtgga 1800
tcaaatactaaataaaaacttttaatgccaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1857
<210> 46
<211> 1810
<212> DNA
<213> Homo Sapiens
<400>
46
cggcacgagctgcttctcagccaggtgtcttgcaaatgtgtggttttatccctatgggga 60
accccatgggaacgatggacatatggcagactgaacagacacaccccatcactggagatc 120
ctgtgcagaaatcatgagctgtgctctcacacacacagttccctcgactaggtctgcttt 180
cttgttcttactgattttgtaagcttttcagagctgatgataaaatttgtgcttatacca 240
ggaaaacagaaaaggatgttgctcttaagtcactggcaacattctaataacatgcactta 300
actggcttgatctttttgttcttggtttctcatcctaatcaagacccttggctgtcttgt 360
cagaaagagagcagcagagaaaatagtgaccttgaatattaagacattgggcaagaaaaa 420
gatattcatccaggcttctcatataacaggatgtctagactaataccactttgttttatt 480
atgtccaatactaggaatctttgagggattaaaagagaaaagttcaccttggcttctttt 540
ttaaaaattcagaagattaaacataaaattttaaccagtgtttactgattccttttcaat 600
tttatattatgatcagttaaggtacaggagagaaatcctgagatccaagaggaatcaaga 660
gtagaaagagaagaaagaggcaagaggaaaggagaaatggtgctaagacagaggaggttg 720
ccctggctgagtgctgcctaaactcagcaactgctcctcatcccaccagtcatccccttc 780
agatctgctggactgacaggactcttcctcttgctgcacctgggccccaggtcctaattc 840
ggtttacctggttccctgccaaaaattcttaaagcagtgttcaggcctcctccagcatgt 900
gctccaggttaccaccccatcactgagaactgttgctccatgtgctcttagtggaagtcc 960
cacgtgcctttttttcttcccagtgtaaactttcctctctgcccccgagtgtcccttgtt 1020
catgttgtgttgtatttttgtgtgtggttatgagatgattaatgtttgcatcttcccacc 1080
tctgtccatcgttcccaggtcttcccttgagagaggagaagagtggttcccaaataccat 1140
ttaagcactagtgatagagacaaagttcttgtcagactatggcaaaatgagaaaaataaa 1200
ggggaacatagaatatggaaacacatactgcattgtttataagtgcatatgtgtatgaaa 1260
cctcattggtggaagcactctgaaaaaccatgaaatgttgtggaactgtgcattattgtt 1320
gttgttgtttttttttttttaatctccatttaaccttagagtcttgcagctgccccagct 1380
aaccactagaatgcaaggcctgtggctgccatcgcttcccttcctctccagcgcacaaac 1440
ttatttctgtcagcatttcctcctggggaagaattgcctcttgcctgggccacacctgtt 1500
cagaggtatagcccatcaaacttacagacttggcctctaagaggacccaaaagaagtatg 1560
attttaggaaatttatactactctgccttcttttatcttagtgcagtagtgttatctttc 1620
taagacttacaagcatttcttgtttcttgtttgggattgtttgaatggaaggcatgatcc 1680
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
2j
cttccttacg ttgcttacat tccaaacgtt ttatcgtcct tgtaagcaaa caagacaatt 1740
tatggcattg ccaaaaagtg tccgtgtaag ctttaaaatt acttacatgt tcaaaaaaaa 1800
aaaaaaaaaa 1810
<210> 47
<211> 1879
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (41)
<223> n equals a,t,g, or c
<400>
47
agacctttgataacataaccattagcagagaggctcagggngaggtccctgcctcggact 60
caaagaccgaatgcacggccttgtaggggacgccccagattgtcagggatkgggggatgg 120
tccttggagttttgcatgctctcctccctcccacttctgcaccctttcaccacctcgagg 180
agatttgctccccattagcgaatgaaattgatgcagtcctacctaactcgattccctttg 240
gcttggtgggtaggcctgcagggcacttttattccaacccctggtcaytcagtamtstkt 300
tactccaggaaggcacaggatggtacctaaagagaattagagaatgaacctggckrgacg 360
gatgtctaatcctgcrcctagctgggttggtcagtagaacctattttcagactcaaaaac 420
catcttcagaaagaaaaggcccagggaaggaatgtatgagaggctctcccagatgaggaa 480
gtgtactctctatgactatcaagctcaggcctctccctttttttaaaccaaagtctggca 540
accaagagcagcagctccatggcctccttgccccagatcagcctgggtcaggggacatag 600
tgtcattgtttggaaactgcagacacaaggtgtgggtctatcccacttcctagtgctccc 660
cacattccccatcagggcttcctcacgtggamaggtktgctartccaggcagttcacttg 720
cagtttccttgtcctcatgcytcggggatgggagccmcgmcygaactagagttcaggctg 780
gatacatgtgctcacctgctgctcttgtcttcctaagagacagagagtggggcagatgga 840
ggagaagaaagtgaggaatgagtagcatagcattctgccaaaagggccccagattcttaa 900
tttagcaaactaagaagcccaattcaaaagcattgtggctaaagtctaacgctcctctct 960
tggtcagataacaaaagccctccctgttggatcttttgaaataaaacgtgcaagttatcc 1020
aggctcgtagcctgcatgctgccaccttgaatcccagggagtatctgcacctggaatagc 1080
tctccacccctctctgcctccttactttctgtgcaagatgacttcctgggttaacttcct 1140
tctttccatccacccacccactggaatctctttccaaacatttttccattttcccacaga 1200
tgggctttgattagctgtcctctctccatgcctgcaaagctccagatttttggggaaagc 1260
tgtacccaactggactgcccagtgaactgggatcattgagtacagtcgagcacacgtgtg 1320
tgcatgggtcaaaggggtgtgttccttctcatcctagatgccttctctgtgccttccaca 1380
gcctcctgcctgattacaccactgcccccgccccaccctcagccatcccaattcttcctg 1440
gccagtgcgctccagccttatctaggaaaggargagtgggtgtagccgtgcagcaagatt 1500
ggggcctcccccatcccagcttctccaccatcccagcaagtcaggatatcagacartcct 1560
cccctgaccctcccccttgtagatatcaattcccaaacagagccaaatactctatatcta 1620
tagtcacagccctgtacagcatttttcataagttatatagtaaatggtctgcatgatttg 1680
tgcttctagtgctctcatttggaaatgaggcaggcttcttctatgaaatgtaaagaaaga 1740
aaccactttgtatattttgtaataccacctctgtggccatgcctgccccgcccactctgt 1800
atatatgtaagttaaacccgggyaggggctgtggccgtctttgtactctggtgattttta 1860
aaaattgaatctttgtact 1879
<210> 48
<211> 556
<212> DNA
<213> Homo Sapiens
<400> 48
tgacctgtct gggcccagca tgttgcagat gtgtatttat gcgcaatggt atgcatatct 60
ctgtgtgact gtcagtgttg caagctggct ggatccaacc atctcttctg aaataatgca 120
tccaaagggt tgatattctg ggggaggtca ctgcagaagg atggaactga cctttattcc 180
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
26
ccagtgggcagttactgagctttcctcctcagagccatgctggcagccctgggacagaga 240
acggtgtggctttggctgcctctgcatggaatcttgccccggactcctgaagactgcaca 300
aggaatgaggaagatcagggacaacctgggaactgaataactttcaaagccagtgctcag 360
cttctctgctccgtactagcgtttacaggtcttaattcaaaccagatgcctgtactagtt 420
tttagaccccaagtcaacctttctgagccacagcttcccgctgggaataatgatgcctgc 480
cctatctacctcacagacttgttatgaggataaagtgagattaaactgcctcaaagtgaa 540
aaaaaaaaaaaaaaaa 556
<210>
49
<211>
607
<212>
DNA
<213> Sapiens
Homo
<400>
49
ggcacgagtgggcctggaattcgccacaggacggatcttacagaggcaagtggtccctgg 60
acctctcttgcatccattctctagacggccgtgtcagaggctccaccctgttgtgaactt 120
ggtatggaggcaaaggcttagaggctggaccagcattcttgggcaaggactgactctcga 180
agggttttgttcttggctttggacacctgagaaccccctcctcccctcccccaatacaag 240
gtttttgacatgagtgtactcctgcttagttcctcttgtggggctgcatttgcggtgctt 300
tgccctccccactgtgagtgaggggccaagggatctcctcaatcctgtctccccagcggc 360
tctgtttcctccttctttccttggcctctgtcctttgctgacttcctcttccttacccag 420
cagaactcaccctggggtcggggcagtggggaggggcctatccactgctcttcctagtcc 480
ttggcagctggcctaggtgggcagactataggagggactggttaggagtctgcattgctt 540
tgacttccctctccttggttaataaacacaaatgcttgtttctcaaaaaaaaaaaaaaaa 600
aaaaaaa 607
<210> 50
<211> 596
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (564)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (569)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (571)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (578)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (580)
<223> n equals a,t,g, or c
<400> 50
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
27
cagctatgacccatgattacgccaagctcgaaattaaccctcactaaagggaacaaaagc 60
tggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaat 120
tcggcacgagaattggggaaaagggtattcaatatttattaagtaacaatgagaaatgca 180
acaaacttccttatttatgccttatgaataaaagagtgtggaatgtaaggtgaaatttta 240
tgtgcaagatcctgaaggaatgggtattcttgaaggcagggactgtcttttctatgtttg 300
ttctgtacagctctcagcacactcttggtgcttgataaatgttatgatgttatataatac 360
caataactaagcacatttttagatcttttttcaaatggcttgtgtaaaatttggttaatg 420
tacaatggttggtttgaagctatcatgtaaaattggcctctccaaatataatcagaaata 480
aaccgaaaaaaatataaaaaaaaaaaaaaaaaaaactcgagggggggcccggtacccaat 540
tcgccctatagtgagtcgtatacnctaantnctttttntncctatagtgagtctta 596
<210> 51
<211> 284
<212> DNA
<213> Homo sapiens
<400>
51
ggcacgagattttcttcaaatatataatggcaattttcagatatctcaccttaccatatc 60
tttccttattttcactgcatgcatttaatcactgtattacttaatgtttgatttgttatt 120
atgggcatttcaaataggcaagcattgaattgtaatgacaaaaaggctattttatattaa 180
ggatatatgcatttgtatttcacacaccagagatgatattaaacactgattattttatgc 240
tgctgtttattaaaaatgtttactataaaaaaaaaaaaaaaaaa 284
<210> 52
<211> 1615
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (591)
<223> n equals a,t,g, or c
<400>
52
ctttctacttttctttggcactcttactgcctgtaaggagtagaactgttagggcacact 60
gttgctatacagtttaactcccattttcatgttttgtctttcttttcccatttctggggc 120
ttacctcctgatacctgcttactttctggaagtagtgggcaagtaagatttggctcttgg 180
tttctaatttttaaatttctgaatactgccctagtctgaacttggcctttatagattaat 240
ctttgcttcacatttttagtgttgtatttaaactattttataatttaaaaatagattcta 300
atctgaagatacttttcaagaaatattattaactgatgtcatcctcatcccagcagctca 360
tctgttaggaatgaagttgagatgcttctattccatgtttttgtatttgggaaggattca 420
aagttgaaggtttattgtcgttgggtttttcagatggtgacatgtaaactcaggatagca 480
aaccctaatgttcacacagtgctctgcctctgcactcagttgggatagttgctccytttg 540
agtgktttaatcatcgtataactaatcgtagtgycaagaagtycataatgnkgtatgtag 600
ctaatgtcactgaaaaacagtcctaccatttaggtaagaccaaacagagtctctaaccca 660
aggacttgttacacctgacaacctatagtatatttgctttttctcacaaaatgaaaccaa 720
ttttgccgaaagctagctgggataataggatcatcacaagttgcagtttctataactaaa 780
attagattgaaatctcttctgacctagaacattttacttcaggcattcagcagatttcag 840
aaagaattaccttattttaagttagtttctttgttagtttactgtgtgtctcttattcaa 900
taaacaagcagaatttgtgtcctgccctatccatgtcttaaagatgagaagttggatcca 960
ctgagttagtttcattggggcgggggaaagaactgtaattaaacttgtttaatccttatt 1020
ttgtattgtagctattttttgtaaaagcaacttaaaatcttttaaaaattttatagtgac 1080
attagagacaatggtcatacaaattatcacataaacatggacttgaaaaattaggctttt 1140
cataaaacacatcacatgtcattgactgctttttagaaatacacttccaaggcagtacat 1200
ctgtattgctactgaaaagtgccawttcacagaacacagacttctttttgcyctttgaca 1260
tcttgaaaacatctgtttttcttttttaatacmaaactttgtgctcaagacmaatcttac 1320
atgaaactctcataaaccatgaaaatgtagctggccttcgggccttaggcatgaaataag 1380
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
h
catgaggaac atattcccct aacttctacc cccagcccag caagttatcc tttaagaaat 1440
ctcctaggaa ttctggagtt tgaaaacaat tgctctatgt tattcctgct tccagtctct 1500
aagtaacaag ggcatttaaa agcatagtct cttaaggtcc actatagtgg ttctttattt 1560
aaggaataac tcagctgggt gcagtggctc acgsctgtta atcccagcac tttta 1615
<210> 53
<211> 1961
<212> DNA
<213> Homo Sapiens
<400>
53
ggggaaaatgaaagaaaagagatatgatgccagcaaagggatggaaagtctagaggacac 60
ctttgtatctcaagctaatgctctacaaaggaaaggccgagcaggcgttgttgcatctgg 120
ggtctgcttccatttattcactagccatcactacaatcaccagcttttaaaacaacagct 180
accagaaatacaaagagtgccattggaacagctgtgtctaagaattaaaattttagagat 240
gtttagtgctcataatctccagtctgtgttctctcggctcattgaacctccacacaccga 300
ttctcttcgtgcctcaaaaatacgattacgagacttaggagcattaactccagatgaaag 360
attgacccctcttgggtatcatttggcctctctgcccgtggatgtgagaattggcaaact 420
aatgttgtttgggtctatcttccgctgtttggatcctgctctcaccattgctgccagttt 480
ggcttttaagtckccgtttgtatctccctgggataaaaaagaagaagctaaccagaaaaa 540
gctggaatttgcattcgcaaacagtgattatctggcccttctacaagcgtataagggatg 600
gcagctaagtacaaaagaaggcgtgcgtgcaagttataattactgcagacaaaacttctt 660
gtctggaagagttctgcaggaaatggccagcctcaaacgacaattcacggaactgttatc 720
ggatatagggtttgcaagggaagggctcagagcaagggaaattgagaaaagggcccaagg 780
aggagatggtgtcttagatgccacaggagaagaggcaaactcaaatgctgagaaccccaa 840
gctgatatcagcaatgctgtgtgctgctttgtatccaaatgtagtgcaggtgaaaagccc 900
agaaggaaaatttcagaagaccagtactggagctgtcagaatgcaaccaaaatcagctga 960
gttgaagtttgtcaccaagaacgatggatatgtacacattcacccttcatcagtgaacta 1020
tcaggtgagacactttgacagcccctacctgttgtaccacgagaagataaaaactagtcg 1080
agtattcatccgagactgcagcatggtgtctgtgtacccgctggtcttgtttggaggagg 1140
ccaagtgaatgtgcagcttcaaagaggagagttcgttgtctccctggatgatggttggat 1200
ccgttttgtagctgcttcccatcaggtggctgaactggtaaaggagcttcgttgcgaact 1260
tgatcagcttctccaggataaaattaaaamcccaagcattgatctgtgtacgtgtcctcg 1320
aggatcccggatcatcagcacaattgtgaaacttgtcaccacacaatrraaagcagtctt 1380
aragagtgcttgctactcacctgcttctagctcacctgggaaataacagcagaacctcta 1440
cctcgaactaaagacctattggggctggccctggtggaggagcccagggcatgaagccca 1500
aggcagctgaggcagtgtatatacccttagggccatttctaacaaagccttggccactcc 1560
cagcacaatttggagtgtcaagggtgagagcctaaaacccagcttgcctgtctttgtctc 1620
tgtgattgttctggagtgaattaagttcacctgataactcaaaagtgaatgtataataca 1680
attctgttttaatctgtgtattctttttctcctactttttactggggtgagaggggcatg 1740
aagagaaatacgcctttttttttttcttttcctgtcgccaaggctcgactgagagaagtc 1800
agaacagagaaggggaaaaaaaacccaaaattatgtgaacaagcaaaattaaaatttcat 1860
tttaggctattggctactgagtaaacttgacttgtgaggggtttttatttttactcatta 1920
aaagtcaacttaaaaaaaaaaaaaaaaaaaaaaaactcgag 1961
<210> 54
<211> 1357
<212> DNA
<213> Homo Sapiens
<400> 54
ggcacgagtgacttaaatcatggctgtattatttcatttttaaagaaaataaagtataaa 60
ataatacatttaaaatacctgtcaaagttttaaaatataagatagaaccttctttttttg 120
ccttcttacacatgaatgtacttaaagtattgttatgtgatcttgggatacttatttatt 180
tttgaaatttttgtatatatagcatttctgaacttagcaaataatatgtgtaaaggaatt 240
aaccaaaaatgaaagaactggcattttcatatgggatacatatttgcttaaactaaataa 300
aataacttagatttatttaaatcaggaatatttacgttgttgatcatttttaaagtatat 360
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
ttgttcaaatattcctaatttgtttttcagcacacttgtttaagttcttgcctttcaggt420
atactacagcgttatatgaacataatgttttaaaaaaatcttggttgtagtttctaattt480
tcactgcataacaaatttgaaaccaaatgttgaatttctctgtgagggtacttattttgc540
ctacagtttcaaatatattttcaaattcatctctttcttactagactgtgagctccttgt600
ggacaaggatgttattttagttataatgacaactttaatatctagcaaagtgccaggcat660
agagtattcctttattgaaatgaattgatagatattgattattaaaatgctactacagta720
ttctacgatgcaggctgaatgtatattacagtaattctctggctaattttaaaagtaaga780
catagaaaacaaaacacctgtagcattttctttatttaaaattgaaactctgttttgaat840
cctttttattttgtcaaacattctatgcaaatattgaaatatgcaacagctaaactttat900
ggtacatattaattagttttattttccttttcaaaatcagaaatgctgtatttaagcttc960
ctggaaatgtcgacaatcattttaatgaccaaaggtgctacttatttttcaacattgacc1020
ttgatcataagtgcttctatctgctgagctttatttattgtttttggacagaaagtttgg1080
tgggaaggttgcaataaaatcagaatctctcttgtctgaattatgcagtttaaccctgtc1140
catgttcctctgtactctattcttactgtattttagttattgtttcttacttatcactaa1200
tttttactgcaggtctgtgctgtttcttagaaagtacatgcatatatatttctataatat1260
gtaagaaaaaacctgtattgcttattaaattaaaattaaaataaaaaaattcatgttaaa1320
tttttgaaatgaaaaaaaaaaaaaaaaaaaaaaaaaa 1357
<210> 55
<211> 785
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (9)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (22)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (39)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (44)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (774)
<223> n equals or c
a,t,g,
<400> 55
caattgganc gcaaccggcaanttaaatggtggagttanc ccantcaattaggcacccca 60
ggctttacac tttatgcttcccggctcgtatgttgtgtgg aattgtgagcggataacaat 120
ttcaccacag gaaacagctatgaccatgattacgccaagc tcgaaattaaccctcactaa 180
agggaacaaa agctggagctccaccgcggtggcggccgct ctagaactagtggatccccc 240
gggctgcagg aattcggcacgagtgtgagctaggtcttca agatttatagaatgttactt 300
atgaacaaaa tataattatttatggtacaattcttgtact ttagcaaatctggagttagt 360
tcatagtcaa agtcagttaatatttcttagaggaaagttt tgctttttgtggcaacattt 420
ttatagcttg tgtgagttcttttttatttaatgatttgaa agcatttttgacagtcgtg 480
c
accgtgtgtg gtggcgtcactgtaaccaaagtatatgcac cagcccttgtgcatttattg 540
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
tttctcctgattttgtggatttaaatgtccaaatgcaaacctttgtgacttcctttggag 600
gacttggcagcacagcatgcccccgtgacctgcctgctgtggtatgagctatgaccaaga 660
gcaggcttcctgctccatggagtcctgag~tgctctggggcaggggattacgttatgaaa 720
actaaccatgtgtaacaataaatctaccttagcagaaaaaaaaaaaaaaaaaanaaaaac 780
tcgag 785
<210> 56
<211> 2440
<212> DNA
<213> Homo Sapiens
<400>
56
tacggctgcgagacgacagaaggggccttctagcagaaatggcggctgcggcggctcgag 60
tggtgttgtcatccgcggcgcgcggcggc~ctggggtttcagcgagagtcttctaatccg 120
aggcgctgcgggacggtcattatattttggagagaacagattaagaagtacacaggctgc 180
tacccaagttgttctgaatgttcctgaaacaagagtaacatgtttagaaagtggactcag 240
agtagcttcggaagactctgggctctcaacatgcacagttggactctggattgatgctgg 300
aagtagatacgaaaatgagaagaacaatggaacagcacactttctggagcatatggcttt 360
caagggcaccaagaagagatcccagttagatctggaacttgagattgaaaatatgggtgc 420
tcatctcaatgcctatacctccagagagcagactgtatactatgccaaagcattctctaa 480
agacttgccaagagctgtagaaattcttgctgatataatacaaaacagcacattgggaga 540
agcagagattgaacgtgagcgtggagtaatccttagagagatgcaggaagttgaaaccaa 600
tttacaagaagttgtttttgattatcttcatgccacagcttatcaaaatactgcacttgg 660
acggacaattttgggaccaactgaaaatatcaaatctataagtcgtaaggacttagtgga 720
ttatataaccacacattataaggggccaagaatagtgcttgctgctgctggaggtgtttc 780
ccatgatgaattgcttgacttagcaaagtttcatttcggtgactctttatgcacacacaa 840
aggagaaataccagctctgcctccctgcaaattcacaggaagtgagattcgtgtgaggga 900
tgacaagatgcctttggcgcaccttgcaatagctgttgaagctgttggttgggcacatcc 960
agatacaatctgtctcatggttgcaaacacgctgattggcaactgggatcgctcttttgg 1020
gggaggaatgaatttatctagcaagctggcccagctcacttgtcatggcaatctttgcca 1080
tagctttcagtctttcaacacttcctacacagatacaggattatggggactgtatatggt 1140
ttgtgaatcatccactgttgcagacatgctacatgttgttcaaaaagaatggatgcgact 1200
ctgtacaagtgtcacagaaagtgaggttgcacgagccagaaatcttctgaaaacaaacat 1260
gttgttgcagcttgatggttcaactccaatttgtgaagatattggtaggcaaatgttatg 1320
ctataatagaaggattcccatccctgagcttgaagcaagaattgatgctgtgaatgctga 1380
gmcaattcgagaagtatgtaccaaatacatttataataggagtccagctattgctgctgt 1440
tggattctttcttcttaggatataatcacagaagtgaacttcatgaatggaaatggaaca 1500
agttattttccaaaaggcaaactatttcatactcctacgtgaacatccttttaccacagc 1560
ctcagccacagtacgtctaattatttaaaatttgtgattatcgctggtggtcaacaattt 1620
ttttgtttttcatcctttaactggaaaaaggaggggctgtctcagtttttcttctgactc 1680
tgtgtgtcacttacaattaataatgctagctgttaacatctacatagcagttgacatgtg 1740
ccaggcctgtttaccagttaatcttctccatgatcctatgaggaaagtgctattgctgtc 1800
tccatttcatagatgaggaaaatgaggcacagaggagacgttatgtagccactaccactg 1860
caacttgctcaaacttccggccaagtcggctctagtccagacagcctgactcctcagcct 1920
gcgtctgtatgctgcctctcctaattcattatgtttattaataatttttccccgttttgt 1980
taacacttatgtttcaaaaacagtcatctttatttacattgactgatcatttcttttgta 2040
atttcttctagtacttacaatagttctagtttcattaacatttcctatagactgaaaaaa 2100
ttttttaatctttttggaattttaatatagtcaagaaagtatctaaacttctcatttttc 2160
aaaatcttactgtcatttaataatccttcccttttccactgawwwgtgacactataatag 2220
taaattttacaagggcttgttttggttacttattcttgccctggggttatctttaaactg 2280
accacagytttctaatatatttgatcgtaacttccaaaaactgtgtaatttttgcttatt 2340
tgtcttgcatctagtcacttaactctcttttgcttttctaggcagatcctatattatcta 2400
caaataatatatttatttccttctttccaaaaaaaaaaaa 2440
e210> 57
<211> 810
<212> DNA
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
31
<213> Homo Sapiens
<220>
<221> SITE
<222> (5)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (22)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (26)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (43)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (54)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (59)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (88)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (806)
<223> n equals or c
a,t,g,
<400> 57
ggaangagcg ccccaattacgnaaanccggctttttcccc ggnggggttg gccnggatnc60
attaatgcca gctggcacggacaggttncccggactggaa aagccgggcc agtgagcgca120
acgcaattaa tggggggttggctcacttcattaggcaccc caggctttac actttatgct180
tccggctcgt atgttgtgtggaattgtgagcggataacaa tttcacacag gaaacagcta240
tgaccatgat tacgccaagctcgaaattaaccctcactaa agggaacaaa agctggagct300
ccaccgcggt ggcggccgctctagaactagtggatccccc gggctgcagg aattcggcac360
gagccgagaa ggcccttaactcaaagtagcttatttatcc aaaatgttct ggatgcatca420
tctccaacca aggaccccttatttatcatgcctttgttct cttttccctc agatgtatat480
ttctttaaaa ataattttcctaataacaaaacttatttct aaaacagctt aaaaattcaa540
agaaaaaccc caaacactgacattacctacacttccacta cccaaagaca aaatgtgccc600
actgtgtgct tttgagtgtattttcttttagtttgttttt tgttgggtgc atatttatga660
taataacaat gatggacttcaattgtactcactgttctat tgttggtttt aattagcagc720
aagttgtgat cactttcccaggtgaataaatcatttcaaa gcaaaaaaaa aaaaaaaaaa780
aaaaaaaaaa aaaaaaaaaaaaaaantcga 810
attttaggaaatttatactactctgccttcttttatcttagtgcagtagtgttatctttc 1620
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
32
<210> 58
<211> 484
<212> DNA
<213> Homo Sapiens
<400>
58
ggcacgaggggagacaataatgagggactttcagttttactttacataattttcttttaa 60
gtattggaatttaggtgatttttcctttggggttttctgtattttccaatcacaataaat 120
aaaataagttataaatatttgttgcatgaatgaaatgtataaacccatttatgtatgtat 180
ttttttaaaattagtatattattaagtctatacaatattagtatattgttatgtatgtat 240
aagctttttaacatgaagtttgcagaatatagtacttcttccaaactctatgacatgggg 300
ggaactgaagtatggggatatcttgtaccagtgtaagaattcaagaagagaccgtgtgtg 360
gtggctcatgcctatagtcccagcactttgggatgccaaagcaggatgatctcttggagc 420
taggcgttcaagaccagcctgggcaacatatcaagaccccatctctaaaaaaaaaaaaaa 480
aaaa 484
<210> 59
<211> 431
<212> DNA
<213> Homo Sapiens
<400>
59
ggacgctgggttcattaattacctttaaaccgttccttattttttttaagattttaaatt 60
gtattttggcttttgcctccagtatcctttctggttgctctggtttgaattaagttccta 120
ttatgctgcagcacatatcaaccttccctaagtaaccatttcctggaatgtgaagcatcg 180
gtgccattagcagaccatatgcagaaatgtcgtgtacttgcatttcttttttgtgcactc 240
tataaggctggttgtgactcagatcagcttaactttttatattatgttatttcactaact 300
gctacagtcaaaatgatcaaatctttgtacaatagaaaattatttaaattttatttttct 360
actgacatttctaattctagtgtaaatgtttatcaataaaaaattactttcaaaaaaaaa 420
aaaaaaaaaaa 431
<210> 60
<211> 38
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (38)
<223> Xaa equals stop translation
<400> 60
Met Ile Leu Lys Gln Gly Leu Ile Cys Thr Trp Gln Val Leu Leu Leu
1 5 10 15
Ala Ser Ala Leu Glu Met Leu Val Phe Ile Cys Ala Met Glu Cys Leu
20 25 30
Thr Gln Phe Gln Val Xaa
<210> 61
<211> 6
<212> PRT
<213> Homo Sapiens
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
3
<220>
<221> SITE
<222> (6)
<223> Xaa equals stop translation
<400> 61
Met His Ile I1e Gln Xaa
1 5
<210> 62
<211> 45
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (45)
<223> Xaa equals stop translation
<400> 62
Met Asp Thr Trp Ile Phe Leu Leu Val Thr Lys Ile Phe Lys Leu Phe
1 5 10 15
Val Tyr Thr His Thr His Thr His Thr His Thr His Val Ser Val Pro
20 25 30
Arg Asn Val Tyr Arg Gly Gly Gln Phe Ser Glu Asp Xaa
35 40 45
<210> 63
<211> 39
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (39)
<223> Xaa equals stop translation
<400> 63
Val Leu Leu Ile Leu Lys Leu Leu Leu Leu Lys Gly Ala Arg Ser Ile
1 5 10 15
Gln Ile Phe Met Phe Arg Cys Leu Ile Ala Phe Ala Leu Ile Thr Lys
20 25 30
Leu Gln Asn Tyr Met Asp Xaa
<210> 64
<211> 39
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
CA 02361277 2001-08-28
WO 00/55177 PCTNS00/06058
34
<222> (39)
<223> Xaa equals stop translatio:~
<400> 64
Met Arg Thr G''~u Val Gly Ala Glr_ Arg Ala Cys Ala Val Arg Cys Val
1 5 10 15
Leu Ala Leu Gly Ala Val Val Ser Leu Leu Val Pro Pro Thr Pro Ala
20 25 30
Leu Ser Ser Ile Pro Ser Xaa
<210> 65
<211> 53
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (53)
<223> Xaa equals stop translation
<400> 65
Met Thr Ala Ser Pro Asp Tyr Leu Val Val Leu Phe Gly Ile Thr Ala
1 5 10 15
Gly Ala Thr Gly Ala Lys Leu Gly Ser Asp Glu Lys Glu Leu Ile Leu
20 25 30
Leu Phe Trp Lys Val Val Asp Leu Ala Asn Lys Lys Val Gly Gln Leu
35 40 45
His Glu Ser Ser Xaa
<210> 66
<211> 19
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (19)
<223> Xaa equals stop translation
<400> 66
Val Leu Cys Ser Leu Ser Cys Met Leu Lys Leu Gly Val Cys Trp Arg
1 5 10 15
Ala Ser Xaa
<210> 67
<211> 39
<212> PRT
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
3
<213> Homo sapiens
<220>
<221> SITE
<222> (39)
<223> Xaa equals stop translation
<400> 67
Met Arg Thr Ala Leu Phe Pro Thr Glu Cys Cys Leu Pro Met Cys Val
1 5 10 15
Val Leu Ala Val Phe Tyr Leu Pro Ile Val Phe Ser Arg Ile Ile Glu
20 25 30
Ser Ala Asp Ser Phe Asp Xaa
<210> 68
<211> 57
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (57)
<223> Xaa equals stop translation
<400> 68
Gly Trp Phe His Leu Phe Trp Gln Glu Trp Glu Gln Glu Pro Gly Gln
1 5 10 15
Asn Lys Leu Leu Glu Ala Leu Val Leu Gly Thr Ala Ala Gly Arg Val
20 25 30
Gly Thr Arg Gln Asn Cys Leu Gln Asp Glu Ser Gln Glu Arg Thr Leu
35 40 45
Ser Pro Val Ser Gly Val Trp Leu Xaa
50 55
<210> 69
<211> 34
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (34)
<223> Xaa equals stop translation
<400> 69
Met Val Pro Arg Ser Val Ala Phe Val Lys Thr Leu Ala Leu Leu Glu
1 5 10 15
Leu Gly Phe Ala Leu Ala Val Met Gln Gly Cys Ala Glu Pro Ile Ser
20 25 30
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
36
Met Xaa
<210> 70
<211> 98
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (98)
<223> Xaa equals stop translation.
<400> 70
Met Glu Val Val Val Thr Val Thr Pro Lys Thr Cys Pro Leu Se= Ser
1 5 10 1 5
Leu Leu Leu Phe Leu Leu Tyr Phe Leu Val Ile Gly Ser Val Ile His
20 25 30
Leu Thr Ala Gly Phe Arg Ile Leu Val Leu Gly Leu Val Phe Lea Phe
35 40 45
Phe Pro Tyr Pro Pro Tyr Pro Asn Cys His Gln Val Leu Leu His Ala
50 55 60
Leu Met Ile Ser His Leu Ser Tyr Pro Ser Ser Phe Gln Ile Gly Pro
65 70 75 80
Ser Asp Phe Asn Leu Gly His Ser His Tyr Leu Leu Tyr Tyr Gly Lys
85 90 95
Ile Xaa
<210> 71
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
<400> 71
Met Val Lys Leu Leu Val Lys Leu Thr Phe Ile Ile Ser Pro Leu Ile
1 5 10 15
Lys Ser Ser Asp Ser Gly Ile Thr Ser Leu Ser Cys Ser Tyr Gln Arg
20 25 30
Ala Ile Phe Xaa
<210> 72
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
37
<211> 18
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (18)
<223> Xaa equals stop translation
<400> 72
Met Gln Lys Leu Lys Gly Gly Ile Ser Val Phe Leu Ala Phe Leu Leu
1 5 10 15
Met Xaa
<210> 73
<211> 32
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (32)
<223> Xaa equals stop translation
<400> 73
Met Thr Glu Ser Leu Leu Tyr Leu Gln Leu Ile Leu Leu Trp Gly Ile
1 5 10 15
Ser Glu Ile Pro Ser Ser Asn Thr Glu Met Tyr Arg Lys Cys Pro Xaa
20 25 30
<210> 74
<211> 17
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (17)
<223> Xaa equals stop translation
<400> 74
Met Gln Lys Gly Arg Ala Val Cys Leu Ser Pro Asp Leu Ala His Gly
1 5 10 15
Xaa
<210> 75
<211> 12
<212> PRT
ttatgctgcagcacatatcaaccttccctaagtaaccatt
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
38
<213> Homo Sapiens
<220>
<221> SITE
<222> (12)
<223> Xaa equals stop translatio~.
<400> 75
Met Phe Val Cys Leu Phe Leu Ile Asn Asn Ile Xaa
1 5 10
<210> 76
<211> 35
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (28)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (35)
<223> Xaa equals stop translation
<400> 76
Met Ile Ile Trp Phe Leu Pro Phe Thr Leu Leu Val Trp Phe Ile Thr
1 5 10 15
Phe Ile Asp Leu Phe Met Leu Asn His Pro Cys Xaa Pro Gly Ile Asn
20 25 30
Leu Thr Xaa
<210> 77
<211> 6
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (6)
<223> Xaa equals stop translation
<400> 77
His Ala Cys Phe Leu Xaa
1 5
<210> 78
<211> 33
<212> PRT
<213> Homo sapiens
<220>
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
3 ~)
<221> SITE
<222> (33)
<223> Xaa equals stop translation
<400> 78
Met Phe Cys Gly Ala Cys Gln Ile Gly Trp Asn Leu Trp Gly Leu Leu
1 5 10 15
Trp Glu Met Pro Arg Glu His Arg Phe Arg Arg Trp Glu Gln Leu Val
20 25 30
Xaa
<210> 79
<211> 25
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (25)
<223> Xaa equals stop translation
<400> 79
Val Met Glu Val Leu Met Thr Met Val Ala Ile Ile Ile Thr Ala Met
1 5 10 15
Gly Met Met Ala Leu Met Thr Glu Xaa
20 25
<210> 80
<211> 40
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (19)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (40)
<223> Xaa equals stop translation
<400> 80
Met Leu Asn Asn Phe Leu Gly Ile Asn Leu Gln Ala Trp Thr Ser Arg
1 5 10 15
Leu Ser Xaa Gln Phe Leu Leu Thr Phe Ala Thr Tyr Cys Tyr Ala Asn
20 25 30
Phe Gln Lys Asn Cys Thr Gln Xaa
35 40
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
<210> 81
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
<400> 81
Met Lys Thr Gln Asn His Cys Ile Phe Cys Ile Val Ile Leu Phe Ser
1 5 10 15
Thr Ser Val Pro Pro Leu Ile Trp Ser Trp Gln Cys Val Ser Vai His
20 25 30
Ser Leu Phe Xaa
<210> 82
<211> 6
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (6)
<223> Xaa equals stop translation
<400> 82
Met Asn Gly Leu Leu Xaa
1 5
<210> 83
<211> 40
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (40)
<223> Xaa equals stop translation
<400> 83
Met Arg Ser Ser Gly Ala Trp Gln Ala Met Val Gly Val Trp Ile Leu
1 5 10 15
Phe Leu Ser Ala Val Glu Ser Gln Gly Arg Val Leu Ala Glu Gln Arg
20 25 30
Cys Asn Leu Ala Trp Ala Leu Xaa
35 40
<210> 84
<211> 27
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
41
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (27)
<223> Xaa equals stop translation
<400> 84
Met Tyr Cys Ile Ser Trp Glu Val Tyr Phe Met Ser Phe Leu Ala Phe
1 5 10 15
Phe Phe Pro Thr Ala Thr Ala Asn Glu Gly Xaa
20 25
<210> 85
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
<400> 85
Met Ile Lys Leu Val His Gln Ile Val Ile Leu Cys Val Met Arg Ile
1 5 10 15
Val Ala Gly Val Ile Leu Lys Cys Trp Tyr Leu Asp Arg Thr Ala Ser
20 25 30
Pro Gly Phe Xaa
<210> 86
<211> 73
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (73)
<223> Xaa equals stop translation
<400> 86
Met Leu Asp Val Phe Leu Lys Ser Cys Phe Val Ser Phe Leu Ser Leu
1 5 10 15
Ile Val Lys Leu Leu Asn Ile Asn Arg Phe Ala Gln Pro Gln Arg Met
20 25 30
Arg Val Asp Asn Thr Glu Glu Val Met Gln Lys Gln Lys Ile Thr Leu
35 40 45
Leu Ile Ile Asp Ser Ile Thr Asn Lys Cys Leu Phe Leu Ser Leu Pro
50 55 60
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
4?
Pro Phe Leu Pro Leu Pro Ser Ser laa
65 70
<210> 87
<211> 38
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (38)
<223> Xaa equals stop translatio_~_
<400> 87
Met Leu Leu Cys Thr His Thr Ser Leu Leu Leu Tyr Phe Ser Phe Trp
1 5 10 15
Met Gly Leu Ala Lys Thr Gly Ser Gly Gln Arg Pro Pro Lys Leu Tyr
20 25 30
Val Leu Pro Val Ser Xaa
<210> 88
<211> 18
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (18)
<223> Xaa equals stop translation
<400> 88
Met Pro Leu Gly Leu Pro Leu Ser Ala Ser Gly Phe Ser Val Gly Asp
1 5 10 15
Leu Xaa
<210> 89
<211> 46
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (46)
<223> Xaa equals stop translation
<400> 89
Met Arg Ala Ala Val Gln Thr Cys Leu Pro Ser Gln Ala Leu Ala Ser
1 5 10 15
Leu Thr Trp Gln Arg Leu Cys Pro Gly Leu Ser Pro Pro Arg Ala Met
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
4;
20 25 30
Ser Leu Met Ala Val Leu Thr Glu =rg Ser Gln Ile Val Xaa
35 40 45
<210> 90
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
<400> 90
Met Leu Phe Met Ala His Leu Leu '~eu Arg Thr His Pro Leu Ser Leu
1 5 10 15
Trp Val Thr Ser Arg Gln Ala Lys Asp Trp Cys Phe Ser Phe His Pro
20 25 30
Leu Glu Gly Xaa
<210> 91
<211> 103
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (72)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (103)
<223> Xaa equals stop translation
<400> 91
Met Thr Gly Val Gln Val Gln Trp Thr Val Ile Phe Leu Ala Pro Val
1 5 10 15
Ile Ala Val Ile Leu Cys Ala Met Gln Thr Met Leu Arg Ser Leu Trp
20 25 30
Leu Met Asp Leu Thr Leu Thr Val Ser Gln Val Val Glu Glu Arg Lys
35 40 45
Gln Met Lys Gly Lys Lys Lys His Gly Ile Gln Gln Lys Lys Thr Leu
50 55 60
Glu Leu Ile Val Asn Met Met Xaa Val Ala Arg Val Gly Glu Lys Cys
65 70 75 80
Ser Thr Cys Ile Ser Lys Leu Asn Leu Met Leu Gln Met Lys Val Leu
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
44
85 90 95
Gly Lys Asp Ile Asn Gly Xaa
1C0
<210> 92
<211> 37
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (16)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (37)
<223> Xaa equals stop translation
<400> 92
Met Ser His Cys Trp Thr Leu Leu Ala Leu Ser Leu Trp Gly Leu Xaa
1 5 10 15
Val Ser Gln Gly Arg Glu Thr Trp Trp Arg Trp Pro His Gly Leu Gly
20 25 30
Pro Pro Cys Ser Xaa
<210> 93
<211> 45
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (45)
<223> Xaa equals stop translation
<400> 93
Leu Ile Gly Val Phe Pro Pro His Leu Leu Ser Ser Leu Lys Cys Val
1 5 10 15
Pro Asp Ala Phe Ile Cys Cys Phe Thr Ser Met Phe Cys Phe Ser Ser
20 25 30
Ser Leu Cys Ser Leu Pro Val Tyr Pro Leu Ser Leu Xaa
35 40 45
<210> 94
<211> 17
<212> PRT
<213> Homo Sapiens
<220>
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
=) i
<221> SITE
<222> (17)
<223> Xaa equa-~s stop translation
<400> 94
Glu Lys Ile Asp Gln Gln Phe Thr Phe Ala Val Cys Leu Val Phe Val
1 5 10 15
Xaa
<210> 95
<211> 13
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (13)
<223> Xaa equals stop translation
<400> 95
Asn Trp Leu Asp Leu Phe Val Leu Gly Phe Ser Ser Xaa
1 5 10
<210> 96
<211> 54
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (54)
<223> Xaa equals stop translation
<400> 96
Met Gly Gln Arg Gly Val Phe Leu Leu Ile Leu Asp Ala Phe Ser Val
1 5 10 15
Pro Ser Thr Ala Ser Cys Leu Ile Thr Pro Leu Pro Pro Pro His Pro
20 25 30
Gln Pro Ser Gln Phe Phe Leu Ala Ser Ala Leu Gln Pro Tyr Leu Gly
35 40 45
Lys Glu Glu Trp Val Xaa
<210> 97
<211> 38
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (38)
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
46
<223> Xaa equals stop translatic~
<400> 97
Met Leu Gln N.et Cys Ile Tyr Ala Gln Trp Tyr Ala Tyr Leu Cys Val
1 5 10 15
Thr Val Ser Val Ala Ser Trp Leu asp Pro Thr Ile Ser Ser Glu Ile
20 25 30
Met His Pro Lys Gly Xaa
<210> 98
<211> 24
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (24)
<223> Xaa equals stop translation
<400> 98
Met Ser Val Leu Leu Leu Ser Ser Ser Cys Gly Ala Ala Phe Ala Val
1 5 10 15
Leu Cys Pro Pro His Cys Glu Xaa
<210> 99
<211> 4
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (4)
<223> Xaa equals stop translation
<400> 99
Gly Tyr Ser Xaa
1
<210> 100
<211> 11
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (11)
<223> Xaa equals stop translation
<400> 100
Met His Leu Ile Thr Val Leu Leu Asn Val Xaa
1 5 10
WO.00/55177 cA 02361277 2001-08-28 pCT/US00/06058
47
<210> 101
<211> 26
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (26)
<223> Xaa equals stop translation
<400> 101
Met Phe Cys Leu Ser Phe Pro Ile Ser Gly Ala Tyr Leu Leu Ile Pro
1 5 10 15
Ala Tyr Phe Leu Glu Val Val Gly Lys Xaa
20 25
<210> 102
<211> 335
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (290)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (316)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (321)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (335)
<223> Xaa equals stop translation
<400> 102
Met Leu Phe Gly Ser Ile Phe Arg Cys Leu Asp Pro Ala Leu Thr Ile
1 5 10 15
Ala Ala Ser Leu Ala Phe Lys Ser Pro Phe Val Ser Pro Trp Asp Lys
20 25 30
Lys Glu Glu Ala Asn Gln Lys Lys Leu Glu Phe Ala Phe Ala Asn Ser
35 40 45
Asp Tyr Leu Ala Leu Leu Gln Ala Tyr Lys Gly Trp Gln Leu Ser Thr
50 55 60
Lys Glu Gly Val Arg Ala Ser Tyr Asn Tyr Cys Arg Gln Asn Phe Leu
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
1 ~;
65 70 75 80
Ser Gly Arg Val Leu Gln Glu Met Ala Ser Leu Lys Arg Gln Phe Thr
85 90 95
Glu Leu Leu Ser ~.sp Ile Gly Phe Ala Arg Glu Gly Leu Arg Ala Arg
100 105 110
Glu Ile Glu Lys Arg Ala Gln Gly Gly Asp Gly Val Leu Asp Ala Thr
115 120 125
Gly Glu Glu Ala Asn Ser Asn Ala Glu Asn Pro Lys Leu Ile Ser Ala
130 135 140
Met Leu Cys Ala Ala Leu Tyr Pro Asn Val Val Gln Val Lys Ser Pro
145 150 155 160
Glu Gly Lys Phe Gln Lys Thr Ser Thr Gly Ala Val Arg Met Gln Pro
165 170 175
Lys Ser Ala Glu Leu Lys Phe Val Thr Lys Asn Asp Gly Tyr Val His
180 185 190
Ile His Pro Ser Ser Val Asn Tyr Gln Val Arg His Phe Asp Ser Pro
195 200 205
Tyr Leu Leu Tyr His Glu Lys Ile Lys Thr Ser Arg Val Phe Ile Arg
210 215 220
Asp Cys Ser Met Val Ser Val Tyr Pro Leu Val Leu Phe Gly Gly Gly
225 230 235 240
Gln Val Asn Val Gln Leu Gln Arg Gly Glu Phe Val Val Ser Leu Asp
245 250 255
Asp Gly Trp Ile Arg Phe Val Ala Ala Ser His Gln Val Ala Glu Leu
260 265 270
Val Lys Glu Leu Arg Cys Glu Leu Asp Gln Leu Leu Gln Asp Lys Ile
275 280 285
Lys Xaa Pro Ser Ile Asp Leu Cys Thr Cys Pro Arg Gly Ser Arg Ile
290 295 300
Ile Ser Thr Ile Val Lys Leu Val Thr Thr Gln Xaa Lys Ala Val Leu
305 310 315 320
Xaa Ser Ala Cys Tyr Ser Pro Ala Ser Ser Ser Pro Gly Lys Xaa
325 330 335
<210> 103
<211> 39
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (39)
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
49
<223> Xaa equals stop translation
<400> 103
Met Tyr Leu Lys Tyr Cys Tyr Val Ile Leu Gly Tyr Leu Phe Ile Phe
1 5 10 15
Glu Ile Phe Val Tyr Ile Ala Phe Leu Asn Leu Ala Asn Asn Met Cys
20 25 30
Lys Gly Ile Asn Gln Lys Xaa
<210> 104
<211> 12
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (12)
<223> Xaa equals stop translation
<400> 104
Met His Gln Pro Leu Cys Ile Tyr Cys Phe Ser Xaa
1 5 10
<210> 105
<211> 11
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (11)
<223> Xaa equals stop translation
<400> 105
Met Thr Arg Cys Leu Trp Arg Thr Leu Gln Xaa
1 5 10
<210> 106
<211> 6
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (6)
<223> Xaa equals stop translation
<400> 106
Ile Val Gly Phe Asn Xaa
1 5
<210> 107
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
<211> 54
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (54)
<223> Xaa equals stop translatio~:
<400> 107
Met Tyr Phe Phe Lys Ile Ser Ile Leu Leu Ser Leu Tyr Asn Ile Ser
1 5 10 15
Ile Leu Leu Cys Met Tyr Lys Leu Phe Asn Met Lys Phe Ala Glu Tyr
20 25 30
Ser Thr Ser Ser Lys Leu Tyr Asp ?~Iet Gly Gly Thr Glu Val Trp Gly
35 40 45
Tyr Leu Val Pro Val Xaa
<210> 108
<211> 67
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (67)
<223> Xaa equals stop translation
<400> 108
Met Gln Lys Cys Arg Val Leu Ala Phe Leu Phe Cys Ala Leu Tyr Lys
1 5 10 15
Ala Gly Cys Asp Ser Asp Gln Leu Asn Phe Leu Tyr Tyr Val Ile Ser
20 25 30
Leu Thr Ala Thr Val Lys Met Ile Lys Ser Leu Tyr Asn Arg Lys Leu
35 40 45
Phe Lys Phe Tyr Phe Ser Thr Asp Ile Ser Asn Ser Ser Val Asn Val
50 55 60
Tyr Gln Xaa
<210> 109
<211> 70
<212> PRT
<213> Homo sapiens
<400> 109
Phe Phe Phe Phe Phe Glu Thr Glu Ser Cys Ser Val Ala Glu Ala Gly
1 5 10 15
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
51
Val Gln Trp Cys Asp Leu Gly Ser Leu Lys Ser Pro Pro Pro Gly Ser
20 25 30
Ser Asp Ser Pro Ala Ser Ala Ser Arg Val Ala Gly Ile Thr Gly Met
35 40 45
His His His Thr Gln Leu Ile Phe Val Phe Leu Val Glu Thr Gly Ser
50 55 60
His Met Gln Leu Ser Asp
65 70
<210> 110
<211> 71
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (44)
<223> Xaa equals stop translation
<400> 110
Phe Phe Phe Phe Phe Glu Thr Glu Ser His Pro Val Ala Gln Ala Gly
1 5 10 15
Val Gln Trp His Asp Leu Gly Ser Leu Gln Ser Leu Pro Pro Arg Phe
20 25 30
Lys Arg Phe Pro Asn Leu Ser Leu Pro Ser Ser Xaa Gln Tyr Lys Cys
35 40 45
Ala His His Asn Arg Val Ile Phe Val Phe Leu Val Glu Thr Gly Phe
50 55 60
Ser His Val Gly Gln Ala Asp
65 70
<210> 111
<211> 95
<212> PRT
<213> Homo Sapiens
<400> 111
Thr Gln Thr Ser Pro Val Ala Asp Ala Pro Thr Gly Val Gln Trp His
1 5 10 15
Asp Phe Gly Ser Leu Gln Pro Leu Pro Pro Gly Phe Lys Arg Phe Ser
20 25 30
Cys Leu Ser Leu Pro Arg Ser Trp Asp Tyr Arg His Pro Pro Pro Arg
35 40 45
Pro Ala Asn Phe Glu Phe Leu Val Glu Thr Gly Phe Leu His Val Gly
50 55 60
Gln Ala Gly Leu Glu Leu Leu Thr Ser Gly Asp Leu Pro Ala Ser Ala
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
52
65 70 75 80
Ser Gln Ser Ala Arg Ile Thr Gly Val Ser His Arg Ala Arg Pro
85 90 95
<210> 112
<211> 93
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (73)
<223> Xaa equals stop translation
<400> 112
Thr Glu Ser Cys Ser Val Ala Gln Ala Gly Val Gln Trp His Asp Leu
1 5 10 15
Ser Ser Leu Gln Ala Pro Pro Pro Gly Phe Met Pro Phe Ser Cys Leu
20 25 30
Ser Leu Pro Ser Ser Trp Asp Tyr Arg Leu Pro Leu Pro Tyr Leu Ala
35 40 45
Asn Phe Phe Val Val Leu Val Glu Met Gly Phe His His Ile Ser Gln
50 55 60
Asp Gly Leu Asn Leu Leu Thr Ser Xaa Ser Ala Arg Leu Ala Ser Gln
65 70 75 80
Ser Ala Gly Ile Thr Gly Val Ser His Leu Ala Arg Pro
85 90
<210> 113
<211> 80
<212> PRT
<213> Homo sapiens
<400> 113
His Leu Arg Ser Gly Val Gln Asp Tyr Pro Gly Gln His Gly Lys Ile
1 5 10 15
Pro Ser Leu Leu Lys Ile Gln Glu Leu Ala Gly His Gly Gly Arg Cys
20 25 30
Leu Gln Ser Gln Leu Leu Arg Arg Leu Arg Gln Glu Asn His Leu Asn
35 40 45
Ser Gly Gly Arg Gly Cys Ser Glu Pro Lys Ser His Leu Cys Ile Pro
50 55 60
Ala Trp Val Thr Glu Gly Asp Ser Val Ser Lys Gln Asn Lys Thr Lys
65 70 75 80
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
>3
<210> 114
<211> 80
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (15)
<223> Xaa equals stop translation
<220>
<221> SITE
<222> (34)
<223> Xaa equals stop translation
<220>
<221> SITE
<222> (70)
<223> Xaa equals stop translation
<220>
<221> SITE
<222> (76)
<223> Xaa equals stop translation
<400> 114
His Leu Arg Ser Gly Val Gln Asp Gln Leu Gly Gln His Gly Xaa Ser
1 5 10 15
Pro Ser Leu Leu Lys Val Gln Lys Leu Ala Trp His Gly Gly Met His
20 25 30
Leu Xaa Phe Gln Leu Leu Gly Arg Leu Arg Gln Glu Asn Cys Leu Ser
35 40 45
Pro Gly Gly Glu Gly Cys Ser Glu Pro Arg Ser His Arg Cys Thr Pro
50 55 60
Ala Trp Ala Thr Glu Xaa Asp Ser Val Thr Lys Xaa Asn Ile Asn Lys
65 70 75 gp
<210> 115
<211> 393
<212> PRT
<213> Homo Sapiens
<400> 115
Ala Tyr Cys Lys Gln His Ala Asp Arg Leu Asp Arg Lys Trp Lys Arg
1 5 10 15
Lys Asn Tyr Leu Ala Leu Gln Ser Tyr Cys Lys Met Ser Leu Gln Glu
20 25 30
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
5-i
Arg Glu Lys Gln Leu Ser Pro Glu ria G1n Ala Arg Ile Asn Ala Arg
35 40 45
Leu Gln Gln Tyr Arg Ala Lys Ala Glu Leu Ala Arg Ser Thr Arg Pro
50 55 60
Gln Ala Trp Val Pro Arg Glu Lys Leu Pro Arg Pro Leu Thr Ser Ser
65 70 75 80
Ala Ser Ala Ile Arg Lys Leu Met Arg Lys Ala Glu Leu Met Gly Ile
85 90 95
Ser Thr Asp Ile Phe Pro Val Asp Asn Ser Asp Thr Ser Ser Ser Val
100 105 110
Asp Gly Arg Arg Lys His Lys Gln Pro Ala Leu Thr Ala Asp Phe Val
115 120 125
Asn Tyr Tyr Phe Glu Arg Asn Met Arg Met Ile Gln Ile Gln Glu Asn
130 135 140
Met Ala Glu Gln Lys Asn Ile Lys Asp Lys Leu Glu Asn Glu Gln Glu
145 150 155 160
Lys Leu His Val Glu Tyr Asn Lys Leu Cys Glu Ser Leu Glu Glu Leu
165 170 175
Gln Asn Leu Asn Gly Lys Leu Arg Ser Glu Gly Gln Gly Ile Trp Ala
180 185 190
Leu Leu Gly Arg Ile Thr Gly Gln Lys Leu Asn Ile Pro Ala Ile Leu
195 200 205
Arg Ala Pro Lys Glu Arg Lys Pro Ser Lys Lys Glu Gly Gly Thr Gln
210 215 220
Lys Thr Ser Thr Leu Pro Ala Val Leu Tyr Ser Cys Gly Ile Cys Lys
225 230 235 240
Lys Asn His Asp Gln His Leu Leu Leu Leu Cys Asp Thr Cys Lys Leu
245 250 255
His Tyr His Leu Gly Cys Leu Asp Pro Pro Leu Thr Arg Met Pro Arg
260 265 270
Lys Thr Lys Asn Ser Tyr Trp Gln Cys Ser Glu Cys Asp Gln Ala Gly
275 280 285
Ser Ser Asp Met Glu Ala Asp Met Ala Met Glu Thr Leu Pro Asp Gly
290 295 300
Thr Lys Arg Ser Arg Arg Gln Ile Lys Glu Pro Val Lys Phe Val Pro
305 310 315 320
Gln Asp Val Pro Pro Glu Pro Lys Lys Ile Pro Ile Arg Asn Thr Arg
325 330 335
Thr Arg Gly Arg Lys Arg Ser Phe Val Pro Glu Glu Glu Lys His Glu
340 345 350
CA 02361277 2001-08-28
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SS
Glu Arg Val Pro Arg Glu Arg Arg Gln Arg Gln Ser Val Leu Gln Lys
355 360 365
Lys Pro Lys Ala Glu Asp Leu Arg =hr Glu Cys Ala Thr Cys Lys Gly
370 375 380
Thr Gly Asp Asn Glu Asn Leu Val Arg
385 390
<210> 116
<211> 393
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (345)
<223> Xaa equals any of the natvurally occurring L-amino acids
<220>
<221> SITE
<222> (346)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (347)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (348)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (349)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (350)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (351)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (352)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (353)
WO 00/55177 CA 02361277 2001-08-28
PCT/US00/06058
56
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (354)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (355)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (356)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (357)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (358)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (359)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (360)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (361)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (362)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (363)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 116
Ala Tyr Cys Lys Gln His Ala Asp Arg Leu Asp Arg Lys Trp Lys Arg
1 5 10 15
Lys Asn Tyr Leu Ala Leu Gln Ser Tyr Cys Lys Met Ser Leu Gln Glu
20 25 30
CA 02361277 2001-08-28
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57
Arg Glu Lys Gln Leu Ser Pro Glu Ala Gln Ala Arg Ile Asn Ala Arg
35 40 45
Leu Gln Gln Tyr Arg Ala Lys Ala Glu Leu Ala Arg Ser Thr Arg Pro
50 55 50
Gln Ala Trp Val Pro Arg Glu Lys Leu Pro Arg Pro Leu Thr Ser Ser
65 70 75 80
Ala Ser Ala Ile Arg Lys Leu Met Arg Lys Ala Glu Leu Met Gly Ile
85 90 95
Ser Thr Asp Ile Phe Pro Val Asp Asn Ser Asp Thr Ser Ser Ser Val
100 105 110
Asp Gly Arg Arg Lys His Lys Gln Pro Ala Leu Thr Ala Asp Phe Val
115 12C 125
Asn Tyr Tyr Phe Glu Arg Asn Met Arg Met Ile Gln Ile Gln Glu Asn
130 135 140
Met Ala Glu Gln Lys Asn Ile Lys Asp Lys Leu Glu Asn Glu Gln Glu
145 150 155 160
Lys Leu His Val Glu Tyr Asn Lys Leu Cys Glu Ser Leu Glu Glu Leu
165 170 175
Gln Asn Leu Asn Gly Lys Leu Arg Ser Glu Gly Gln Gly Ile Trp Ala
180 185 190
Leu Leu Gly Arg Ile Thr Gly Gln Lys Leu Asn Ile Pro Ala Ile Leu
195 200 205
Arg Ala Pro Lys Glu Arg Lys Pro Ser Lys Lys Glu Gly Gly Thr Gln
210 215 220
Lys Thr Ser Thr Leu Pro Ala Val Leu Tyr Ser Cys Gly Ile Cys Lys
225 230 235 240
Lys Asn His Asp Gln His Leu Leu Leu Leu Cys Asp Thr Cys Lys Leu
245 250 255
His Tyr His Leu Gly Cys Leu Asp Pro Pro Leu Thr Arg Met Pro Arg
260 265 270
Lys Thr Lys Asn Ser Tyr Trp Gln Cys Ser Glu Cys Asp Gln Ala Gly
275 280 285
Ser Ser Asp Met Glu Ala Asp Met Ala Met Glu Thr Leu Pro Asp Gly
290 295 300
Thr Lys Arg Ser Arg Arg Gln Ile Lys Glu Pro Val Lys Phe Val Pro
305 310 315 320
Gln Asp Val Pro Pro Glu Pro Lys Lys Ile Pro Ile Arg Asn Thr Arg
325 330 335
Thr Arg Gly Arg Lys Arg Ser Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
340 345 350
CA 02361277 2001-08-28 pCT/US00/06058
WO 00/55177
ti
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Val Leu Gln Lys
355 360 365
Lys Pro Lys Ala Glu Asp Leu Arg Thr Glu Cys Ala Thr Cys Lys Gly
370 375 380
Thr Gly Asn GluAsnLeu VaiArg
Asp
385 390
<210>
117
<211>
114
<212>
PRT
<213> Sapiens
Homo
<400>
117
Lys Cys Val SerProSer CysGluLeuCys ProAsnGln AspGly
Gly
1 5 10 15
Ile Phe Glu ThrAspAla GlyArgTrpVal HisIleVal CysAla
Lys
20 25 30
Leu Tyr Pro GlyValAla PheGlyAspIle AspLysLeu ArgPro
Val
35 4C 45
Val Thr Thr GluMetAsn TyrSerLysTyr GlyAlaLys GluCys
Leu
50 55 60
Ser Phe Glu AspProArg PheAlaArgThr GlyValCys IleSer
Cys
65 70 75 80
Cys Asp Gly MetCysArg AlaTyrPheHis ValThrCys AlaGln
Ala
85 90 95
Lys Glu Leu LeuSerGlu AlaAlaAlaGlu GluAspIle AlaAsp
Gly
100 105 110
Pro Phe
<210>
118
<211>
114
<212>
PRT
<213> Sapiens
Homo
<400>
118
Lys Cys Ser SerCys GluLeuCys ProAsnGlnAsp Gly
Gly Val Pro
1 5 10 15
Ile Phe Glu Thr AlaGly ArgTrpVal HisIleValCys Ala
Lys Asp
20 25 30
Leu Tyr Pro Gly AlaPhe GlyAspIle AspLysLeuArg Pro
Val Val
35 40 45
Val Thr Thr Glu AsnTyr SerLysTyr GlyAlaLysGlu Cys
Leu Met
50 55 60
CA 02361277 2001-08-28
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59
Ser Phe Cys Glu Asp Pro Arg Phe Ala Arg Thr Gly Val Cys Ile Ser
65 70 75 80
Cys Asp Ala Gly Met Cys Arg Ala Tyr Phe His Val Thr Cys Ala Gln
85 90 95
Lys Glu Gly Leu Leu Ser Glu Ala Ala Ala Glu Glu Asp I1e Ala Asp
100 105 110
Pro Phe
<210>
119
<211>
79
<212>
PRT
<213> sapiens
Homo
<400>
119
Cys Gly Cys LysLysAsnHis AspGlnHis LeuLeuLeu LeuCys
Ile
1 5 10 15
Asp Thr Lys LeuHisTyrHis LeuGlyCys LeuAspPro ProLeu
Cys
20 25 30
Thr Arg Pro ArgLysThrLys AsnSerTyr TrpGlnCys SerGlu
Met
35 40 45
Cys Asp Ala GlySerSerAsp MetGluAla AspMetAla MetGlu
Gln
50 55 60
Thr Leu Asp GlyThrLysArg SerArgArg GlnIleLys Glu
Pro
65 70 75
<210>
120
<211>
78
<212>
PRT
<213> sapiens
Homo
<400>
120
Cys Ala Cys LysGlyThrGly AspAsnGlu AsnLeuVal ArgCys
Thr
5 10 15
Asp Glu Arg LeuCysTyrHis PheGlyCys LeuAspPro ProLeu
Cys
20 25 30
Lys Lys Pro LysGlnThrGly TyrGlyTrp IleCysGln GluCys
Ser
35 40 45
Asp Ser Ser SerLysGluAsp GluAsnGlu AlaGluArg LysAsn
Ser
50 55 60
Tle Ser Glu LeuAsnMetGlu GlnLysAsn ProLysLys
Gln
65 70 75
<210> 121
CA 02361277 2001-08-28
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<211> 26
<212> PRT
<213> Homo Sapiens
<400> 121
Glu Thr Glu Ser Cys Ser Val Ala Glu Ala Gly Val Gln Trp Cys Asp
1 5 10 15
Leu Gly Ser Leu Lys Ser Pro Pro Pro Gly
20 25
<210> 122
<211> 26
<212> PRT
<213> Homo Sapiens
<400> 122
Glu Thr Glu Ser His Ser Val Val Gln Ala Gly Val Gln Trp Arg Asn
1 5 10 15
Leu Gly Ser Leu Gln Pro Pro Pro Pro Gly
20 25
<210> 123
<211> 17
<212> PRT
<213> Homo sapiens
<400> 123
Gly Ser Ser Asp Ser Pro Ala Ser Ala Ser Arg Val Ala Gly Ile Thr
1 5 10 15
Gly
<210> 124
<211> 17
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (9)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (11)
<223> Xaa equals stop translation
<400> 124
Gly Ser Ser Asp Ser Pro Ala Ser Xaa Phe Xaa Val Ala Gly Val Thr
1 5 10 15
Gly
CA 02361277 2001-08-28
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61
<210> 125
<211> 26
<212> PRT
<213> Homo Sapiens
<400> 125
Asp Ser Pro A'~a Ser Ala Ser Arg Val Ala Gly Ile Thr Gly Met His
1 5 10 15
His His Thr G,_n Leu Ile Phe Val Phe Leu
20 25
<210> 126
<211> 26
<212> PRT
<213> Homo Sapiens
<400> 126
Asp Leu Pro Thr Ser Ala Ser Pro Ser Ala Gly Ile Thr Gly Val Ser
1 5 10 15
His Cys Ala Arg Leu Thr Val Ser Phe Leu
20 25
<210> 127
<211> 16
<212> PRT
<213> Homo Sapiens
<400> 127
His His Thr Gln Leu Ile Phe Val Phe Leu Val Glu Thr Gly Ser His
1 5 10 15
<210> 128
<211> 16
<212> PRT
<213> Homo Sapiens
<400> 128
His His Thr Arg Leu Ile Phe Val Phe Leu Val Gly Met Arg Phe His
1 5 10 15
<210> 129
<211> 48
<212> PRT
<213> Homo Sapiens
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
6?
<400> 129
Lys Lys Leu Ile His His Asp Gln ',lal Gly Phe Ile Pro Gly Met Gln
1 5 10 15
Gly Trp Phe Asn Ile Arg Lys Ser Ile f-an Val Ile Gln His Ile Asn
20 25 30
Arg Ala Lys Asp Lys Asn His Met Ile Ile Ser Ile Asp Ala Glu Lys
35 40 45
<210> 130
<211> 48
<212> PRT
<213> Homo sapiens
<220>
<221>
SITE
<222>
(15)
<223> thenat urally L-amino acids
Xaa equals occurring
any of
<400>
130
Lys Arg IleHis AspGln ValArg Phe ProGlyXaa
Ser His Ile Gln
1 5 10 15
Gly Trp AsnIle LysSer IleAsn Val AsnHisThr
Phe Asn Ile Ser
20 25 30
Lys Val GlyLys HisMet IleIle Thr AspAlaLys
Lys Asn Thr Lys
35 40 45
<210> 131
<211> 23
<212> PRT
<213> Homo sapiens
<400> 131
Leu Pro Asn Ser Phe Tyr Glu Ala Ser Ile Ile Leu Ile Pro Lys Pro
1 5 10 15
Gly Arg Asp Thr Thr Lys Lys
<210> 132
<211> 23
<212> PRT
<213> Homo sapiens
<400> 132
Leu Pro Asn Ser Phe Tyr Asp Val Gly Ile Thr Leu Ile Pro Lys Arg
1 5 10 15
CA 02361277 2001-08-28
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63
Asp Lys Asp Thr Thr Arg Lys
<210> 133
<211> 26
<212> PRT
<213> Homo sapiens
<400> 133
Thr Lys Lys Glu Asn Phe Arg Pro Ile Ser Leu Met Asn Ile Asb Ala
1 5 10 15
Lys Ile Leu Asn Lys Ile Leu Ala Asn Arg
20 25
<210> 134
<211> 26
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (14)
<223> Xaa equals any of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (20)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 134
Thr Leu Gln Glu Asn Tyr Arg Ser Ile Ser Leu Met Asn Xaa Tyr Ala
1 5 10 15
Lys Ile Leu Xaa Lys Ile Leu Thr Asn Gln
20 25
<210>
135
<211>
113
<212>
PRT
<213> Sapiens
Homo
<400>
135
Glu Val LysSer AsnAsnVal GluMetAsp TrpValLeuLys His
Phe
1 5 10 15
Thr Gly AsnSer ProAspThr AlaAsnAsp GlyPheValArg Leu
Pro
20 25 30
Arg Gly ProPhe GlyCysSer LysGluGlu IleValGlnPhe Phe
Leu
35 40 45
Ser Gly GluIle ValProAsn GlyIleThr LeuProValAsp Phe
Leu
50 55 60
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
6-1
Gln Gly Arg Ser Thr Gly Glu Ala Phe Val Gln Phe Ala Ser Gln Glu
65 70 75 g0
Ile Ala Glu Lys Ala Leu Lys Lys His Lys Glu Arg Ile Gly His Arg
85 90 95
Tyr Ile Glu Ile Phe Lys Ser Ser Arg Ala Glu Val Arg Thr His Tyr
100 105 110
Asp
<210>
136
<211>
110
<212>
PRT
<213> sapiens
Homo
<400>
136
Gln Ala Lys SerAsnGly IleGluMet AspTrpValMet LysHis
Phe
1 5 10 15
Asn Gly Asn AspAlaSer AspGlyThr ValArgLeuArg GlyLeu
Pro
20 25 30
Pro Phe Cys SerLysGlu GluIleVal GlnPhePheGln GlyLeu
Gly
35 40 45
Glu Ile Pro AsnGlyIle ThrLeuThr MetAspTyrGln GlyArg
Val
50 55 60
Ser Thr Glu AlaPheVal GlnPheAla SerLysGluIle AlaGlu
Gly
65 70 75 80
Asn Ala Gly LysHisLys GluArgIle GlyHisArgTyr IleGl.u
Leu
85 90 95
Ile Phe Ser SerArgSer GluIleLys GlyPheTyrAsp
Arg
100 105 110
<210> 137
<211> 203
<212> PRT
<213> Homo Sapiens
<400> 137
Asn Gln Lys Thr Phe Arg Pro Lys Lys Ser Ala Pro Ser Gly Thr Lys
1 5 10 15
Gly Ala Glu Leu Arg Lys His Ile Asp Ala Thr Leu Gly Ser Gly Asn
20 25 30
Leu Arg Glu Ala Val Lys Leu Pro Pro Gly Glu Asp Leu Asn Glu Trp
35 40 45
Leu Ala Val Asn Thr Val Asp Phe Phe Asn Gln Val Asn Leu Leu Phe
50 55 60
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
6s
GlyThrLeuT:~_rGluPheCys ThrProGlu AsnCysSerThr MetThr
65 70 75 80
AlaGlyProLys TyrGluTyr ArgTrpAla AspGlyValGln IleLys
85 90 95
LysProileG~_uValSerAla ProLysTyr ValGluTyrLeu MetAsp
100 105 110
TrpIleGluThr GlnLeuAsp AspGluThr IlePheProGln LysLeu
115 120 125
GlyAlaAlaPhe ProProAsn PheLysGlu ValValLysThr IlePhe
130 135 140
LysArgLeuPhe ArgValTyr AlaHisIle TyrHisSerHis PheGln
145 150 155 160
LysIleValSer LeuLysGlu GluAlaHis LeuAsnThrCys PheLys
165 170 175
HisPheIleLeu PheThrHis GluPheVal LeuIleAspLys LysGlu
180 185 190
LeuAlaProLeu GlnGluLeu IleGluSer Ile
195 200
<210> 138
<211> 203
<212> PRT
<213> Homo sapiens
<400> 138
Ser Ser Lys Thr Phe Lys Pro Lys Lys Asn Ile Pro Glu Gly Ser His
1 5 10 15
Gln Tyr Glu Leu Leu Lys His Ala Glu Ala Thr Leu Gly Ser Gly Asn
20 25 30
Leu Arg Gln Ala Val Met Leu Pro Glu Gly Glu Asp Leu Asn Glu Trp
35 40 45
Ile Ala Val Asn Thr Val Asp Phe Phe Asn Gln Ile Asn Met Leu Tyr
50 55 60
Gly Thr Ile Thr Glu Phe Cys Thr Glu Ala Ser Cys Pro Val Met Ser
65 70 75 80
Ala Gly Pro Arg Tyr Glu Tyr His Trp Ala Asp Gly Thr Asn Ile Lys
85 90 95
Lys Pro Ile Lys Cys Ser Ala Pro Lys Tyr Ile Asp Tyr Leu Met Thr
100 105 110
Trp Val Gln Asp Gln Leu Asp Asp Glu Thr Leu Phe Pro Ser Lys Ile
115 120 125
Gly Val Pro Phe Pro Lys Asn Phe Met Ser Val Ala Lys Thr Ile Leu
CA 02361277 2001-08-28
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66
130 135 140
Lys Arg Leu Phe Arg Val Tyr Ala His Ile Tyr His Gln His Phe Asp
145 150 155 160
Ser Val Met Gln Leu Gln Glu Glu Ala His Leu Asn Thr Ser Phe Lys
165 170 175
His Phe Ile Phe Phe Val Gln Glu Phe Asn Leu Ile Asp Arg Arg Glu
180 185 190
Leu Ala Pro Leu Gln Glu Leu Ile Glu Lys Leu
195 200
<210> 139
<211> 74
<212> PRT
<213> HomoSapiens
<400> 139
Asp Arg LeuLeuCysHis ProGlyTrp SerAlaValVal GlnSer
Val
1 5 10 15
Leu Phe ValAlaSerThr PheLeuVal LysGlnSerSer CysLeu
Thr
20 25 30
Gly Leu SerSerTrpAsp TyrArgArg IleProProHis LeuAla
Pro
35 40 45
Asn Phe PhePheCysArg AsnLysSer LeuThrMetLeu ProArg
Ser
50 55 60
Leu Ile AsnSerTrpPro GlnValIle
Leu
65 70
<210> 140
<211> 73
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (54)
<223> Xaa equals stop translation
<220>
<221> SITE
<222> (61)
<223> Xaa equals stop translation
<220>
<221> SITE
<222> (69)
<223> Xaa equals stop translation
<400> 140
Asp Arg Val Trp Leu Cys His Pro Gly Trp Ser Ala Val Val Gln Ser
CA 02361277 2001-08-28
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67
1 5 10 15
Trp Leu Thr Ala Ala Ser Thr Ser Trp Gly Gln Ala Ile Leu Leu Ser
20 25 30
Leu Pro Pro Ser Ser Trp Asp Tyr Arg His Met Pro Pro Gln Ser Thr
35 40 45
Thr Phe Phe Tyr Leu Xaa Arg Gly Gly Phe Ala Met Xaa Pro Arg Leu
50 55 60
Val Ser Ser Ser Xaa Ala Gln Ala Ile
65 70
<210> 141
<211> 181
<212> PRT
<213> Homo Sapiens
<400> 141
Asp Met Ala Glu Ile Gln Ser Arg Leu Ala Tyr Val Ser Cys Val Arg
1 5 10 15
Gln Leu Glu Val Val Lys Ser Ser Ser Tyr Cys Glu Tyr Leu Arg Pro
20 25 30
Pro Ile Asp Cys Phe Lys Thr Met Asp Phe Gly Lys Phe Asp Gln Ile
35 40 45
Tyr Asp Val Gly Tyr Gln Tyr Gly Lys Ala Val Phe Gly Gly Trp Ser
50 55 60
Arg Gly Asn Val Ile Glu Lys Met Leu Thr Asp Arg Arg Ser Thr Asp
65 70 75 80
Leu Asn Glu Ser Arg Arg Ala Asp Val Leu Ala Phe Pro Ser Ser Gly
85 90 95
Phe Thr Asp Leu Ala Glu Ile Val Ser Arg Ile Glu Pro Pro Thr Ser
100 105 110
Tyr Val Ser Asp Gly Cys Ala Asp Gly Glu Glu Ser Asp Cys Leu Thr
115 120 125
Glu Tyr Glu Glu Asp Ala Gly Pro Asp Cys Ser Arg Asp Glu Gly Gly
130 135 140
Ser Pro Glu Gly Ala Ser Pro Ser Thr Ala Ser Glu Met Glu Glu Glu
145 150 155 160
Lys Ser Ile Leu Arg Gln Arg Arg Cys Leu Pro Gln Glu Pro Pro Gly
165 170 175
Ser Ala Thr Asp Ala
180
<210> 142
CA 02361277 2001-08-28
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68
<211>
181
<212>
PRT
<213> sapiens
Homo
<400>
142
Asp Met Glu IleGlnSer ArgLeuAlaTyr ValSerCys ValArg
Ala
1 5 10 15
Gln Leu Val ValLysSer SerSerTyrCys GluTyrLeu ArgPro
Glu
20 25 30
Pro Ile Cys PheLysThr MetAspPheGly LysPheAsp GlnIle
Asp
35 40 45
Tyr Asp Gly TyrGlnTyr GlyLysAlaVal PheGlyGly TrpSer
Val
50 55 60
Arg Gly Val IleGluLys MetLeuThrAsp ArgArgSer ThrAsp
Asn
65 70 75 80
LeuAsnGluSer ArgArgAlaAsp ValLeuAla PheProSer SerGly
85 90 95
PheThrAspLeu AlaGluIleVal SerArgIle GluProPro ThrSer
100 105 110
TyrValSerAsp GlyCysAlaAsp GlyGluGlu SerAspCys LeuThr
115 120 125
GluTyrGluGlu AspAlaGlyPro AspCysSer ArgAspGlu GlyGly
130 135 140
SerProGluGly AlaSerProSer ThrAlaSer GluMetGlu GluGlu
145 150 155
160
LysSerIleLeu ArgGlnArgArg CysLeuPro GlnGluPro ProGly
165 170 175
SerAlaThrAsp Ala
180
<210>
143
<211>
414
<212>
PRT
<213> sapiens
Homo
<400>
143
Gly Val ArgGlu MetArgTyr AsnSerArgArg HisLeuSer Arg
His
1 5 10 15
Leu Thr ThrPhe ValSerLys AlaAsnAlaLys GlnArgSer Gly
Asp
20 25 30
Arg Ala ArgVal GlnGluGly IleCysTyrHis LeuPheSer Lys
Gly
35 40 45
Phe Lys AspThr GlnPheLeu SerTyrGlnThr ProGluIle Leu
His
50 55 60
CA 02361277 2001-08-28
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69
Arg Leu Asn Leu Gln Glu Val Val Leu Arg Val Lys Met Cys Gln Met
65 70 75 g0
Gly Asp Val Gln Asp Val Leu Gly Lys Ala Leu Asp Pro Pro Ser Ser
85 90 95
Thr Asn Ile Ile Arg Ala Leu Glu Lys Leu His Gln Val Gly Ala Leu
100 105 110
Ser Glu Asn Glu Lys Leu Thr Lys Leu Gly Lys Phe Leu Ser Gln Leu
115 120 125
Pro Val Asp Ala Asn Leu Gly Lys Ile Leu Val Leu Gly Cys Phe Tyr
130 135 140
Lys Cys Val Asp Ala Ala Ser Ser Ile Val Ala Met Leu Thr Ile Gly
145 150 155 160
Ser Pro Phe Arg Lys Ser Val Asp Asn Glu Phe Ser Ala Asn Lys Ala
165 170 175
Arg Leu Ser Phe Ala Lys Glu Asn Thr Arg Ser Asp Leu Val Leu Met
180 185 190
Tyr Tyr Ala Tyr Cys Ala Trp Arg Glu Ile Cys Leu Ser Pro Leu Gly
195 200 205
Pro Asp Glu Asp Ser Phe Ala Lys Glu Lys Tyr Leu Asn Leu Glu Ala
210 215 220
Leu Ser Met Thr Glu Ser Leu Lys Ile Gln Leu Leu Ser Glu Leu Lys
225 230 235 240
Asp Met Lys Leu Leu Gly Ala Ser Asp Val Asp Thr Cys Lys Ser Leu
245 250 255
Lys Arg Ser Ile Cys Arg Arg Phe Ala Val Ile Pro Lys Glu His Asp
260 265 270
Ile Asn Ser Gly Asn Ala Glu Ile Leu Cys Gly Val Ile Ala Ala Ser
275 280 285
Leu Tyr Pro Asn Ile Leu Arg Tyr Asp Tyr Glu Lys Arg Gln Trp Ser
290 295 300
Thr Leu Ser Thr Asn Lys Arg Val Arg Ile Leu Asp Val Ser Val Asn
305 310 315 320
Asn Arg Ser Glu Leu Pro Asn Met Pro Ser Lys Phe Val Ala Tyr Thr
325 330 335
Asn Met Met Ser Ser Thr Arg Ala Ser Glu Tyr Val Asn Glu Thr Thr
340 345 350
Met Val Thr Leu Arg Gln Leu Leu Met Met Cys Gly Leu Lys Val Glu
355 360 365
Asn Arg Val Ser Val Gly Gln Ala Lys Leu Asp Asn Phe Thr Val Tyr
CA 02361277 2001-08-28
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370 375 380
Phe Glu Asn Val Tyr Val Ser Ala Ser Leu Ser Ile Leu Arg Arg Phe
385 390 395 400
Ile Glu Thr Ser Leu Asn Glu Phe Phe Ala Glu Pro Asp Lys
405 410
<210> 144
<211> 429
<212> PRT
<213> Homo Sapiens
<400> 144
Gly Lys Met Lys Glu Lys Arg Tyr Asp Ala Ser Lys Gly Met Glu Ser
1 5 10 15
Leu Glu Asp Thr Phe Val Ser Gln rla Asn Ala Leu Gln Arg Lys Gly
2~ 25 30
Arg Ala Gly Val Val Ala Ser Gly Val Cys Phe His Leu Phe Thr Ser
35 40 45
His His Tyr Asn His Gln Leu Leu Lys Gln Gln Leu Pro Glu Ile Gln
50 55 60
Arg Val Pro Leu Glu Gln Leu Cys Leu Arg Ile Lys Ile Leu Glu Met
65 70 75 80
Phe Ser Ala His Asn Leu Gln Ser Val Phe Ser Arg Leu Ile Glu Pro
85 90 95
Pro His Thr Asp Ser Leu Arg Ala Ser Lys Ile Arg Leu Arg Asp Leu
100 105 110
Gly Ala Leu Thr Pro Asp Glu Arg Leu Thr Pro Leu Gly Tyr His Leu
115 120 125
Ala Ser Leu Pro Val Asp Val Arg Ile Gly Lys Leu Met Leu Phe Gly
130 135 140
Ser Ile Phe Arg Cys Leu Asp Pro Ala Leu Thr Ile Ala Ala Ser Leu
145 150 155 160
Ala Phe Lys Ser Pro Phe Val Ser Pro Trp Asp Lys Lys Glu Glu Ala
165 170 175
Asn Gln Lys Lys Leu Glu Phe Ala Phe Ala Asn Ser Asp Tyr Leu Ala
180 185 190
Leu Leu Gln Ala Tyr Lys Gly Trp Gln Leu Ser Thr Lys Glu Gly Val
195 200 205
Arg Ala Ser Tyr Asn Tyr Cys Arg Gln Asn Phe Leu Ser Gly Arg Val
210 215 220
Leu Gln Glu Met Ala Ser Leu Lys Arg Gln Phe Thr Glu Leu Leu Ser
225 230 235 240
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Asp Ile Gly Phe Ala Arg Glu Gly Leu Arg Ala Arg Glu Ile Glu Lys
245 250 255
Arg Ala Gln G1y Gly Asp Gly Val Leu Asp Ala Thr Gly Glu Glu Ala
260 265 270
Asn Ser Asn Ala Glu Asn Pro Lys Leu Ile Ser Ala Met Leu Cys Ala
275 280 285
Ala Leu Tyr Pro Asn Val Val Gln Val Lys Ser Pro Glu Gly Lys Phe
290 295 300
Gln Lys Thr Ser Thr Gly Ala Val Arg Met Gln Pro Lys Ser Ala Glu
305 310 315 320
Leu Lys Phe Val Thr Lys Asn Asp Gly Tyr Val His Ile His Pro Ser
325 330 335
Ser Val Asn Tyr Gln Val Arg His Phe Asp Ser Pro Tyr Leu Leu Tyr
340 345 350
His Glu Lys Ile Lys Thr Ser Arg Val Phe Ile Arg Asp Cys Ser Met
355 360 365
Val Ser Val Tyr Pro Leu Val Leu Phe Gly Gly Gly Gln Val Asn Val
370 375 380
Gln Leu Gln Arg Gly Glu Phe Val Val Ser Leu Asp Asp Gly Trp Ile
385 390 395 400
Arg Phe Val Ala Ala Ser His Gln Val Ala Glu Leu Val Lys Glu Leu
405 410 415
Arg Cys Glu Leu Asp Gln Leu Leu Gln Asp Lys Ile Lys
420 425
<210>
145
<211>
462
<212>
PRT
<213> sapiens
Homo
<400>
145
Val Val Ser SerAlaAla ArgArgArg LeuTrpGlyPhe SerGlu
Leu
1 5 10 15
Ser Leu Ile ArgGlyAla AlaGlyArg SerLeuTyrPhe GlyGlu
Leu
20 25 30
Asn Arg Arg SerThrGln AlaAlaThr GlnValValLeu AsnVal
Leu
35 40 45
Pro Glu Arg ValThrCys LeuGluSer GlyLeuArgVal AlaSer
Thr
50 55 60
Glu Asp Gly LeuSerThr CysThrVal GlyLeuTrpIle AspAla
Ser
65 70 75 80
CA 02361277 2001-08-28
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7?
Gly Ser Arg Tyr Glu Asn Glu Lys Asn Asn Gly Thr Ala His Phe Leu
85 90 95
Glu His Met Ala Phe Lys Gly Thr Lys Lys Arg Ser Gln Leu Asp Leu
100 105 110
Glu Leu Glu Ile Glu Asn Met Gly Ala His Leu Asn Ala Tyr Thr Ser
115 120 125
Arg Glu Gln Thr Val Tyr Tyr Ala Lys Ala Phe Ser Lys Asp Leu Pro
130 135 140
Arg Ala Val Glu Ile Leu Ala Asp Ile Ile Gln Asn Ser Thr Leu Gly
145 150 155 160
Glu Ala Glu Iie Glu Arg Glu Arg Gly Val Ile Leu Arg Glu Met Gln
165 170 175
Glu Val Glu Thr Asn Leu Gln Glu Val Val Phe Asp Tyr Leu His Ala
180 185 190
Thr Ala Tyr Gln Asn Thr Ala Leu Gly Arg Thr Ile Leu Gly Pro Thr
195 200 205
Glu Asn Ile Lys Ser Ile Ser Arg Lys Asp Leu Val Asp Tyr Ile Thr
210 215 220
Thr His Tyr Lys Gly Pro Arg Ile Val Leu Ala Ala Ala Gly Gly Val
225 230 235 240
Ser His Asp Glu Leu Leu Asp Leu Ala Lys Phe His Phe Gly Asp Ser
245 250 255
Leu Cys Thr His Lys Gly Glu Ile Pro Ala Leu Pro Pro Cys Lys Phe
260 265 270
Thr Gly Ser Glu Ile Arg Val Arg Asp Asp Lys Met Pro Leu Ala His
275 280 285
Leu Ala Ile Ala Val Glu Ala Val Gly Trp Ala His Pro Asp Thr Ile
290 295 300
Cys Leu Met Val Ala Asn Thr Leu Ile Gly Asn Trp Asp Arg Ser Phe
305 310 315 320
Gly Gly Gly Met Asn Leu Ser Ser Lys Leu Ala Gln Leu Thr Cys His
325 330 335
Gly Asn Leu Cys His Ser Phe Gln Ser Phe Asn Thr Ser Tyr Thr Asp
340 345 350
Thr Gly Leu Trp Gly Leu Tyr Met Val Cys Glu Ser Ser Thr Val Ala
355 360 365
Asp Met Leu His Val Val Gln Lys Glu Trp Met Arg Leu Cys Thr Ser
370 375 380
Val Thr Glu Ser Glu Val Ala Arg Ala Arg Asn Leu Leu Lys Thr Asn
385 390 395 400
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Met Leu Leu Gln Leu Asp Gly Ser Thr Pro Ile Cys Glu Asp Ile Gly
405 410 415
Arg Gln Met Leu Cys Tyr Asn Arg Arg Ile Pro Ile Pro Glu Leu Glu
420 425 430
Ala Arg Ile Asp Ala Val Asn Ala Glu Thr Ile Arg Glu Val Cys Thr
435 440 445
Lys Tyr Ile Tyr Asn Arg Ser Pro Ala Ile Ala Ala Val Gly
450 455 460
<210> 146
<211> 460
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (440)
<223> Xaa equals any of the naturally occurring L-amino acids
<400> 146
Val Val Ile Arg Gly Ala Arg Arg Leu Trp Gly Phe Ser Glu Ser Leu
1 5 10 15
Leu Ile Arg Gly Ala Ala Gly Arg Ser Leu Tyr Phe Gly Glu Asn Arg
20 25 30
Leu Arg Ser Thr Gln Ala Ala Thr Gln Val Val Leu Asn Val Pro Glu
35 40 45
Thr Arg Val Thr Cys Leu Glu Ser Gly Leu Arg Val Ala Ser Glu Asp
50 55 60
Ser Gly Leu Ser Thr Cys Thr Val Gly Leu Trp Ile Asp Ala Gly Ser
65 70 75 80
Arg Tyr Glu Asn Glu Lys Asn Asn Gly Thr Ala His Phe Leu Glu His
85 90 95
Met Ala Phe Lys Gly Thr Lys Lys Arg Ser Gln Leu Asp Leu Glu Leu
100 105 110
Glu Ile Glu Asn Met Gly Ala His Leu Asn Ala Tyr Thr Ser Arg Glu
115 120 125
Gln Thr Val Tyr Tyr Ala Lys Ala Phe Ser Lys Asp Leu Pro Arg Ala
130 135 140
Val Glu Ile Leu Ala Asp Ile Ile Gln Asn Ser Thr Leu Gly Glu Ala
145 150 155 160
Glu Ile Glu Arg Glu Arg Gly Val Ile Leu Arg Glu Met Gln Glu Val
165 170 175
Glu Thr Asn Leu Gln Glu Val Val Phe Asp Tyr Leu His Ala Thr Ala
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180 185 190
Tyr Gln Asn Thr Ala Leu Gly Arg Thr Ile Leu Gly Pro Thr Glu Asn
195 200 205
Ile Lys Ser Ile Ser Arg Lys Asp Leu Val Asp Tyr Ile Thr Thr His
210 215 220
Tyr Lys Gly Pro Arg Ile Val Leu Ala Ala Ala Gly Gly Val Ser His
225 230 235 240
Asp Glu Leu Leu Asp Leu Ala Lys Phe His Phe Gly Asp Ser Leu Cys
245 250 255
Thr His Lys Gly Glu Ile Pro Ala Leu Pro Pro Cys Lys Phe Thr Gly
260 265 270
Ser Glu Ile Arg Val Arg Asp Asp Lys Met Pro Leu Ala His Leu Ala
275 280 285
Ile Ala Val Glu Ala Val Gly Trp Ala His Pro Asp Thr Ile Cys Leu
290 295 300
Met Val Ala Asn Thr Leu Ile Gly Asn Trp Asp Arg Ser Phe Gly Gly
305 310 315 320
Gly Met Asn Leu Ser Ser Lys Leu Ala Gln Leu Thr Cys His Gly Asn
325 330 335
Leu Cys His Ser Phe Gln Ser Phe Asn Thr Ser Tyr Thr Asp Thr Gly
340 345 350
Leu Trp Gly Leu Tyr Met Val Cys Glu Ser Ser Thr Val Ala Asp Met
355 360 365
Leu His Val Val Gln Lys Glu Trp Met Arg Leu Cys Thr Ser Val Thr
370 375 380
Glu Ser Glu Val Ala Arg Ala Arg Asn Leu Leu Lys Thr Asn Met Leu
385 390 395 400
Leu Gln Leu Asp Gly Ser Thr Pro Ile Cys Glu Asp Ile Gly Arg Gln
405 410 415
Met Leu Cys Tyr Asn Arg Arg Ile Pro Ile Pro Glu Leu Glu Ala Arg
420 425 430
Ile Asp Ala Val Asn Ala Glu Xaa Ile Arg Glu Val Cys Thr Lys Tyr
435 440 445
Ile Tyr Asn Arg Ser Pro Ala Ile Ala Ala Val Gly
450 455 460
<210> 147
<211> 53
<212> PRT
<213> Homo sapiens
CA 02361277 2001-08-28
WO 00/55177 PCT/US00/06058
<400> 147
Phe Phe Ile Phe Phe Phe Arg Asp Arg Val Leu Leu Cys His Pro Gly
1 5 10 15
Trp Ser Ala Val Val Gln Ser Leu Phe Thr Val Ala Ser Thr Phe Leu
20 25 30
Val Lys Gln Ser Ser Cys Leu Gly Leu Pro Ser Ser Trp Asp Tyr Arg
35 40 45
Arg Ile Pro Pro His
<210> 148
<211> 43
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (19)
<223> Xaa equals stop translation
<400> 148
Phe Phe Phe Phe Phe Phe Arg Asp Gly Val Leu Ile Cys Cys Pro Gly
1 5 10 15
Trp Ser Xaa Thr Pro Ser Ser Lys Arg Ser Ser Cys Phe Gly Ile Pro
20 25 30
Lys Cys Trp Asp Tyr Arg His Glu Pro Pro His
35 40
