U.S. patent application number 10/213535 was filed with the patent office on 2006-04-06 for secreted and transmembrane polypeptides and nucleic acids encoding the same.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to David Botstein, Luc Desnoyers, Napoleone Ferrara, Sherman Fong, Wei-Qiang Gao, Audrey Goddard, Austin L. Gurney, James Pan, Margaret Ann Roy, Timothy A. Stewart, Daniel Tumas, Colin K. Watanabe, William I. Wood.
Application Number | 20060073546 10/213535 |
Document ID | / |
Family ID | 29550507 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073546 |
Kind Code |
A1 |
Botstein; David ; et
al. |
April 6, 2006 |
Secreted and transmembrane polypeptides and nucleic acids encoding
the same
Abstract
The present invention is directed to novel polypeptides and to
nucleic acid molecules encoding those polypeptides. Also provided
herein are vectors and host cells comprising those nucleic acid
sequences, chimeric polypeptide molecules comprising the
polypeptides of the present invention fused to heterologous
polypeptide sequences, antibodies which bind to the polypeptides of
the present invention and to methods for producing the polypeptides
of the present invention.
Inventors: |
Botstein; David; (Belmont,
CA) ; Desnoyers; Luc; (San Francisco, CA) ;
Ferrara; Napoleone; (San Francisco, CA) ; Fong;
Sherman; (Alameda, CA) ; Gao; Wei-Qiang; (Palo
Alto, CA) ; Goddard; Audrey; (San Francisco, CA)
; Gurney; Austin L.; (Belmont, CA) ; Pan;
James; (Zitobicoke, CA) ; Roy; Margaret Ann;
(San Francisco, CA) ; Stewart; Timothy A.; (San
Francisco, CA) ; Tumas; Daniel; (Orinda, CA) ;
Watanabe; Colin K.; (Moraga, CA) ; Wood; William
I.; (Hillsborough, CA) |
Correspondence
Address: |
Ginger R. Dreger;Knobbe Martens Olson & Bear
Suite 1150
201 California Street
San Francisco
CA
94111-3335
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
29550507 |
Appl. No.: |
10/213535 |
Filed: |
August 7, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09866034 |
May 25, 2001 |
|
|
|
10213535 |
Aug 7, 2002 |
|
|
|
PCT/US99/28634 |
Dec 1, 1999 |
|
|
|
09866034 |
May 25, 2001 |
|
|
|
60112851 |
Dec 16, 1998 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/183; 435/252.33; 435/254.2; 435/320.1; 435/358; 530/350;
530/388.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/47 20130101 |
Class at
Publication: |
435/069.1 ;
435/183; 435/358; 435/320.1; 435/252.33; 435/254.2; 530/350;
530/388.1; 536/023.2 |
International
Class: |
C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C12N 9/00 20060101
C12N009/00; C12N 5/06 20060101 C12N005/06; C12N 1/18 20060101
C12N001/18 |
Claims
1. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence that encodes an amino acid
sequence selected from the group consisting of the amino acid
sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG.
6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO: 11), FIG. 10 (SEQ ID NO:16),
FIG. 12 (SEQ ID NO: 18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID
NO:22), and FIG. 18 (SEQ ID NO:24).
2. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence selected from the group
consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:
1), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8), FIG. 7 (SEQ ID NO:
10), FIG. 9 (SEQ ID NO:15), FIG. 11 (SEQ ID NO:17), FIG. 13 (SEQ ID
NO:19), FIG. 15 (SEQ ID NO:21) and FIG. 17 (SEQ ID NO:23).
3. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence selected from the group
consisting of the full-length coding sequence of the nucleotide
sequence shown in FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:6), FIG.
5 (SEQ ID NO:8), FIG. 7 (SEQ ID NO: 10), FIG. 9 (SEQ ID NO: 15),
FIG. 11 (SEQ ID NO: 17), FIG. 13 (SEQ ID NO: 19), FIG. 15 (SEQ ID
NO:21) and FIG. 17 (SEQ ID NO:23).
4. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to the full-length coding sequence of the DNA deposited
under ATCC accession number 203538, 203661, 203583, 203657, 203576,
203573, 203553, 203651 and 203537.
5. A vector comprising the nucleic acid of any one of claims 1 to
4.
6. The vector of claim 5 operably linked to control sequences
recognized by a host cell transformed with the vector.
7. A host cell comprising the vector of claim 5.
8. The host cell of claim 7, wherein said cell is a CHO cell.
9. The host cell of claim 7, wherein said cell is an E. coli.
10. The host cell of claim 7, wherein said cell is a yeast
cell.
11. A process for producing a PRO polypeptides comprising culturing
the host cell of claim 7 under conditions suitable for expression
of said PRO polypeptide and recovering said PRO polypeptide from
the cell culture.
12. An isolated polypeptide having at least 80% amino acid sequence
identity to an amino acid sequence selected from the group
consisting of the amino acid sequence shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID
NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO: 18), FIG. 14
(SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), and FIG. 18 (SEQ ID
NO:24).
13. An isolated polypeptide scoring at least 80% positives when
compared to an amino acid sequence selected from the group
consisting of the amino acid sequence shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID
NO:11), FIG. 10 (SEQ ID NO:16), FIG. 12 (SEQ ID NO: 18), FIG. 14
(SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), and FIG. 18 (SEQ ID
NO:24).
14. An isolated polypeptide having at least 80% amino acid sequence
identity to an amino acid sequence encoded by the full-length
coding sequence of the DNA deposited under ATCC accession number
203538, 203661, 203583, 203657, 203576, 203573, 203553, 203651 and
203537.
15. A chimeric molecule comprising a polypeptide according to any
one of claims 12 to 14 fused to a heterologous amino acid
sequence.
16. The chimeric molecule of claim 15, wherein said heterologous
amino acid sequence is an epitope tag sequence.
17. The chimeric molecule of claim 15, wherein said heterologous
amino acid sequence is a Fc region of an immunoglobulin.
18. An antibody which specifically binds to a polypeptide according
to any one of claims 12 to 14.
19. The antibody of claim 18, wherein said antibody is a monoclonal
antibody, a humanized antibody or a single-chain antibody.
20. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to: (a) a nucleotide sequence encoding the polypeptide
shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID
NO:9), FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO: 16), FIG. 12 (SEQ
ID NO: 18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), or FIG.
18 (SEQ ID NO:24), lacking its associated signal peptide; (b) a
nucleotide sequence encoding an extracellular domain of the
polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7),
FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO: 11), FIG. 10 (SEQ ID
NO:16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16
(SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24), with its associated
signal peptide; or (c) a nucleotide sequence encoding an
extracellular domain of the polypeptide shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID
NO: 11), FIG. 10 (SEQ ID NO: 1 6), FIG. 12 (SEQ ID NO: 18), FIG. 14
(SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24),
lacking its associated signal peptide.
21. An isolated polypeptide having at least 80% amino acid sequence
identity to: (a) the polypeptide shown in FIG. 2 (SEQ ID NO:2),
FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO: 1l),
FIG. 10 (SEQ ID NO: 16), FIG. 12 (SEQ ID NO: 18), FIG. 14 (SEQ ID
NO:20), FIG. 16 (SEQ ID NO:22), or FIG. 18 (SEQ ID NO:24), lacking
its associated signal peptide; (b) an extracellular domain of the
polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7),
FIG. 6 (SEQ ID NO:9), FIG. 8 (SEQ ID NO: I1), FIG. 10 (SEQ ID NO:
16), FIG. 12 (SEQ ID NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ
ID NO:22), or FIG. 18 (SEQ ID NO:24), with its associated signal
peptide; or (c) an extracellular domain of the polypeptide shown in
FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:7), FIG. 6 (SEQ ID NO:9),
FIG. 8 (SEQ ID NO:11), FIG. 10 (SEQ ID NO: 16), FIG. 12 (SEQ ID
NO:18), FIG. 14 (SEQ ID NO:20), FIG. 16 (SEQ ID NO:22), or FIG. 18
(SEQ ID NO:24), lacking its associated signal peptide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the
identification and isolation of novel DNA and to the recombinant
production of novel polypeptides.
BACKGROUND OF THE INVENTION
[0002] Extracellular proteins play important roles in, among other
things, the formation, differentiation and maintenance of
multicellular organisms. The fate of many individual cells, e.g.,
proliferation, migration, differentiation, or interaction with
other cells, is typically governed by information received from
other cells and/or the immediate environment. This information is
often transmitted by secreted polypeptides (for instance, mitogenic
factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones) which are, in turn, received
and interpreted by diverse cell receptors or membrane-bound
proteins. These secreted polypeptides or signaling molecules
normally pass through the cellular secretory pathway to reach their
site of action in the extracellular environment.
[0003] Secreted proteins have various industrial applications,
including as pharmaceuticals, diagnostics, biosensors and
bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons, interleukins, erythropoietins,
colony stimulating factors, and various other cytokines, are
secretory proteins. Their receptors, which are membrane proteins,
also have potential as therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new,
native secreted proteins. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted proteins. Examples of screening
methods and techniques are described in the literature [see, for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);
U.S. Pat. No. 5,536,637)].
[0004] Membrane-bound proteins and receptors can play important
roles in, among other things, the formation, differentiation and
maintenance of multicellular organisms. The fate of many individual
cells, e.g., proliferation, migration, differentiation, or
interaction with other cells, is typically governed by information
received from other cells and/or the immediate environment. This
information is often transmitted by secreted polypeptides (for
instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and hormones) which are, in
turn, received and interpreted by diverse cell receptors or
membrane-bound proteins. Such membrane-bound proteins and cell
receptors include, but are not limited to, cytokine receptors,
receptor kinases, receptor phosphatases, receptors involved in cell
cell interactions, and cellular adhesin molecules like selectins
and integrins. For instance, transduction of signals that regulate
cell growth and differentiation is regulated in part by
phosphorylation of various cellular proteins. Protein tyrosine
kinases, enzymes that catalyze that process, can also act as growth
factor receptors. Examples include fibroblast growth factor
receptor and nerve growth factor receptor.
[0005] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interactions. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0006] Efforts are being undertaken by both industry and academia
to identify new, native receptor or membrane-bound proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor or
membrane-bound proteins.
1. PRO1800
[0007] Hep27 protein is synthesized and accumulated in the nucleus
of human hepatoblastoma cells (HepG2 cells) following growth arrest
induced by butyrate treatment (Gabrielli et al., Eur. J. Biochem.
232:473-477 (1995)). The synthesis of Hep27 is inhibited in cells
that, released from the butyrate block, have resumed DNA synthesis.
The Hep27 protein sequence shows significant homology to the known
short-chain alcohol dehydrogenase (SCAD) family of proteins and it
has been suggested that Hep27 is a new member of the SCAD family of
proteins. In agreement with its nuclear localization, Hep27 has a
region similar to the bipartite nuclear-targeting sequence and
Hep27 mRNA expression and protein synthesis suggests the existence
of a regulation at the post-transcriptional level.
[0008] We herein describe the identification and characterization
of novel polypeptides having homology to Hep27 protein, designated
herein as PRO1800 polypeptides.
2. PRO539
[0009] Development of multicellular organisms depends, at least in
part, on mechanisms which specify, direct or maintain positional
information to pattern cells, tissues, or organs. Various secreted
signaling molecules, such as members of the transforming growth
factor-beta (TGF-.beta.), Wnt, fibroblast growth factors and
hedgehog families have been associated with patterning activity of
different cells and structures in Drosophila as well as in
vertebrates. Perrimon, Cell 80:517-520(1995).
[0010] Costal-2 is a novel kinesin-related protein in the Hedgehog
signaling pathway. Hedgehog (Hh) was first identified as a
segment-polarity gene by a genetic screen in Drosophila
melanogaster, Nusslein-Volhard et al., Roux. Arch. Dev. Biol. 193:
267-282 (1984), that plays a wide variety of developmental
functions. Perrimon, supra. Although only one Drosophila Hh gene
has been identified, three mammalian Hh homologues have been
isolated: Sonic Hh (SHh), Desert Hh (DHh) and Indian Hh (IHh),
Echelard et al., Cell 75: 1417-30 (1993); Riddle et al, Cell 75:
1401 -16 (1993). SHh is expressed at high level in the notochord
and floor plate of developing vertebrate embryos. In vitro explant
assays as well as ectopic expression of SHh in transgenic animals
show that SHh plays a key role in neuronal tube patterning,
Echelard et al., supra., Krauss et al., Cell 75, 1432-44 (1993),
Riddle et al., Cell 75: 1401-16(1993), Roelinketal, Cell 81: 445-55
(1995). In vitro explant assays as well as ectopic expression of
SHh in transgenic animals show that SHh plays a key role in neural
tube patterning, Echelard et al. (1993), supra.; Ericson et al.,
Cell 81: 747-56(1995); Marti et al., Nature 375:322-5(1995);
Roelink et al. (1995), supra; Hynes et al., Neuron 19: 15-26
(1997). Hh also plays a role in the development of limbs (Krauss et
al., Cell 75: 1431-44(1993); Laufer et al., Cell 79, 993-1003
(1994)), somites (Fan and Tessier-Lavigne, Cell 79, 1175-86(1994);
Johnson et al., Cell 79: 1165-73(1994)), lungs (Bellusciet al.,
Develop. 124: 53-63 (1997) and skin (Oro et al., Science 276:
817-21 (1997). Likewise, IHh and DHh are involved in bone, gut and
germinal cell development, Apelqvist et al., Curr. Biol. 7:
801-4(1997); Bellusci et al., Dev. Suppl. 124: 53-63 (1997);
Bitgood et al., Curr. Biol. 6: 298-304 (1996); Roberts et al.,
Development 121: 3163-74 (1995). SHh knockout mice further
strengthened the notion that SHh is critical to many aspect of
vertebrate development, Chiang et al., Nature 383: 407-13 (1996).
These mice show defects in midline structures such as the notochord
and the floor plate, absence of ventral cell types in neural tube,
absence of distal limb structures, cyclopia, and absence of the
spinal column and most of the ribs.
[0011] At the cell surface, the Hh signals is thought to be relayed
by the 12 transmembrane domain protein Patched (Ptch) [Hooper and
Scott, Cell 59: 751-65 (1989); Nakano et al., Nature 341: 508-13
(1989)] and the G-protein coupled like receptor Smoothened (Smo)
[Alcedo et al., Cell 86: 221-232 (1996); van den Heuvel and Ingham,
Nature 382: 547-551 (1996)]. Both genetic and biochemical evidence
support a receptor model where Ptch and Smo are part of a
multicomponent receptor complex, Chen and Struhl, Cell 87: 553-63
(1996); Marigo et al, Nature 384: 176-9 (1996); Stone et al.,
Nature 384: 129-34 (1996). Upon binding of Hh to Ptch, the normal
inhibitory effect of Ptch on Smo is relieved, allowing Smo to
transduce the Hh signal across the plasma membrane. Loss of
function mutations in the Ptch gene have been identified in
patients with the basal cell nevus syndrome (BCNS), a hereditary
disease characterized by multiple basal cell carcinomas (BCCs).
Disfunctional Ptch gene mutations have also been associated with a
large percentage of sporadic basal cell carcinoma tumors,
Chidambaram et al., Cancer Research 56: 4599-601 (1996); Gailani et
al., Nature Genet. 14: 78-81 (1996); Hahn et al., Cell 85: 841-51
(1996); Johnson et al., Science 272: 1668-71 (1996); Unden et al.,
Cancer Res. 56: 4562-5; Wicking et al., Am. J. Hum. Genet. 60: 21-6
(1997). Loss of Ptch function is thought to cause an uncontrolled
Smo signaling in basal cell carcinoma. Similarly, activating Smo
mutations have been identified in sporatic BCC tumors (Xie et al.,
Nature 391: 90-2 (1998)), emphasizing the role of Smo as the
signaling subunit in the receptor complex for SHh. However, the
exact mechanism by which Ptch controls Smo activity still has yet
to be clarified and the signaling mechanisms by which the Hh signal
is transmitted from the receptor to downstream targets also remain
to be elucidated. Genetic epistatic analysis in Drosophila has
identified several segment-polarity genes which appear to function
as components of the Hh signal transduction pathway, Ingham, Curr.
Opin. Genet. Dev. 5: 492-8 (1995); Perrimon, supra. These include a
kinesin-like molecule, Costal-2 (Cos-2) [Robbins et al., Cell 90:
225-34 (1997); Sisson et al., Cell 90: 235-45 (1997)], a protein
designated fused [Preat et al., Genetics 135: 1047-62 (1990);
Therond et al., Proc. Natl Acad Sci. USA 93: 4224-8 (1996)], a
novel molecule with unknown function designated Suppressor of fused
[Pham et al., Genetics 140: 587-98 (1995); Preat, Genetics 132:
725-36 (1992)] and a zinc finger protein Ci. [Alexandre et al.,
Genes Dev. 10: 2003-13 (1996); Dominguez et al, Science 272:1621-5
(1996); Orenic et al, Genes Dev. 4: 1053-67 (1990)]. Additional
elements implicated in Hh signaling include the transcription
factor CBP [Akimaru et al., Nature 386: 735-738 (1997)], the
negative regulator slimb [Jiang and Struhl, Nature 391: 493-496
(1998)] and the SHh response element COUP-TFII [rishnan et al.,
Science 278: 1947-1950 (1997)].
[0012] Mutants in Cos-2 are embryonicly lethal and display a
phenotype similar to Hh over expression, including duplications of
the central component of each segment and expansion domain of Hh
responsive genes. In contrast, mutant embryos for fused and Ci show
a phenotype similar to Hh loss of function including deletion of
the posterior part of each segment and replacement of a mirror-like
image duplication of the anterior part or each segment and
replacement of a mirror-like duplication of the anterior part,
Busson et al., Roux. Arch. Dev. Biol. 197: 221-230 (1988).
Molecular characterizations of Ci suggested that it is a
transcription factor which directly activates Hh responsive genes
such as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez
et al., (1996) supra. Likewise, molecular analysis of fused reveals
that it is structurally related to serine threonine kinases and
that both intact N-terminal kinase domain and a C-terminal
regulatory region are required for its proper function, Preat et
al., Nature 347: 87-9 (1990); Robbins et al., (1997), supra;
Therond et al., Proc. Natl. Acad Sci. USA 93: 4224-8 (1996).
Consistent with the putative opposing functions of Cos-2 and fused,
fused mutations are suppressed by Cos-2 mutants and also by
Suppressor of fused mutants, Preat et al., Genetics 135: 1047-62
(1993). However, where as fused null mutations and N-terminal
kinase domain mutations can be fully suppressed by Suppressor of
fused mutations, C-terminus mutations of fused display a strong
Cos-2 phenotype in a Suppressor of fused background. This suggests
that the fused kinase domain can act as a constitutive activator of
SHh signaling when Suppressor of Fused is not present. Recent
studies have shown that the 92 kDa Drosophila fused, Cos-2 and Ci
are present in a microtubule associated multiprotein complex and
that Hh signaling leads to dissociation of this complex from
microtubules, Robbins et al, Cell 90: 225-34 (1997); Sisson et al.,
Cell 90: 235-45 (1997). Both fused and Cos-2 become phosphorylated
in response to Hh treatment, Robbins et al., supra; Therond et al.,
Genetics 142: 1181-98 (1996), but the kinase(s) responsible for
this activity(ies) remain to be characterized. To date, the only
known vertebrate homologues for these components are members of the
Gli protein family (e.g., Gli-1, Gli-2 and Gli-3). These are zinc
finger putative transcription factors that are structurally related
to Ci. Among these, Gli-1 was shown to be a candidate mediator of
the SHh signal [Hynes et al., Neuron 15: 35-44 (1995), Lee et al.,
Development 124: 2537-52 (1997); Alexandre et al., Genes Dev. 10:
2003-13 (1996)] suggesting that the mechanism of gene activation in
response to Hh may be conserved between fly and vertebrates. To
determine whether other signaling components in the Hh cascade are
evolutionarily conserved and to examine the function of fused in
the Hh signaling cascade on the biochemical level, Applicants have
isolated and characterized the human fused cDNA. Tissue
distribution on the mouse indicates that fused is expressed in SHh
responsive tissues. Biochemical studies demonstrate that fused is a
functional kinase. Functional studies provide evidence that fused
is an activator of Gli and that a dominant negative form of fused
is capable of blocking SHh signaling in Xenopus embryos. Together
this data demonstrated that both Cos-2 and fused are directly
involved in Hh signaling.
[0013] For additional references related to the Costal-2 protein,
see Simpson et al., Dev. Biol. 122:201-209 (1987), Grau et al.,
Dev. Biol. 122:186-200 (1987), Preat et al., Genetics 135:1047-1062
(1993), Sisson et al., Cell 90:235-245 (1997) and Robbins et al.,
Cell 90:225-234 (1997).
[0014] Applicants have herein identified and describe a cDNA
encoding a human Costal-2 homolog polypeptide, designated herein as
PRO539.
3. PRO982
[0015] Efforts are being undertaken by both industry and academia
to identify new, native secreted proteins. Many efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel secreted proteins. We herein
describe the identification and characterization of novel secreted
polypeptides, designated herein as PRO982 polypeptides.
4. PRO1434
[0016] The nel gene has been described to encode a protein that is
expressed in the neural tissues of chicken (Watanabeet al.,
Genomics 38(3):273-276 (1996)). Recently, two novel human cDNAs
(designated NELL1 and NELL2) have been isolated and characterized
which encode polypeptides having homology to that encoded by the
chicken nel gene, wherein those human polypeptides contain six
EGF-like repeats (Watanabe et al., supra). Given the
neural-specific expression of these genes, it is suggested that
they may play a role in neural development. There is, therefore,
significant interest in identifying and characterizing novel
polypeptides having homology to nel, NELL1 and NELL2.
[0017] We herein describe the identification and characterization
of novel polypeptides having homology to the nel protein,
designated herein as PRO1434 polypeptides.
5. PRO1863
[0018] Efforts are being undertaken by both industry and academia
to identify new, native transmembrane proteins. Many efforts are
focused on the screening of mammalian recombinant DNA libraries to
identify the coding sequences for novel transmembrane proteins. We
herein describe the identification and characterization of novel
transmembrane polypeptides, designated herein as PRO1863
polypeptides.
6. PRO1917
[0019] The characterization of inositol phosphatases is of interest
because it is fundamental to the understanding of signaling
activities that stimulate the release of Ca.sup.2+ from the
endoplasmic reticulum. Molecular cloning allowed the identification
of a multiple inositol polyphosphate phosphatase which is highly
expressed in kidney and liver (Craxton et al. (1997) Biochem J.
328:75-81).
7. PRO1868
[0020] The inflammatory response is complex and is mediated by a
variety of signaling molecules produced locally by mast cells,
nerve endings, platelets, leucocytes and complement activation.
Certain of these signaling molecules cause the endothelial cell
lining to become more porous and/or even to express selectins which
act as cell surface molecules which recognize and attract
leucocytes through specific carbohydrate recognition. Stronger
leucocyte binding is mediated by integrins, which mediate leukocyte
movement through the endothelium. Additional signaling molecules
act as chemoattractants, causing the bound leucocytes to crawl
towards the source of the attractant. Other signaling molecules
produced in the course of an inflammatory response escape into the
blood and stimulate the bone marrow to produce more leucocytes and
release them into the blood stream.
[0021] Inflammation is typically initiated by an antigen, which can
be virtually any molecule capable of initiating an immune response.
Under normal physiological conditions these are foreign molecules,
but molecules generated by the organism itself can serve as the
catalyst as is known to occur in various disease states.
[0022] T-cell proliferation is a mixed lymphocyte culture or mixed
lymphocyte reaction (MLR) is an established indication of the
ability of a compound to stimulate the immune system. In an
inflammatory response, the responding leucocytes can be
neutrophilic, eosinophilic, monocytic or lymphocytic. Histological
examination of the affected tissues provides evidence of an immune
stimulating or inhibiting response. See Current Protocols in
Immunology, ed. John E. Coligan, 1994, John Wiley and Sons,
Inc.
[0023] Inflammatory bowel disease (IBD) is a term used to
collectively describe gut disorders including both ulcerative
colitis (UC) and Crohn's disease, both of which are classified as
distinct disorders, but share common features and likely share
pathology. The commonality of the diagnostic criteria can make it
difficult to precisely determine which of the two disorders a
patient has; however the type and location of the lesion in each
are typically different. UC lesions are characteristically a
superficial ulcer of the mucosa and appear in the colon, proximal
to the rectum. CD lesions are characteristically extensive linear
fissures, and can appear anywhere in the bowel, occasionally
involving the stomach, esophagus and duodenum.
[0024] Conventional treatments for IBD usually involve the
administration of antiinflammatory or immunosuppressive agents,
such as sulfasalazine, corticosteriods,
6-mercaptopurine/azathoprine, or cyclospoine all of which only
bring partial relief to the afflicted patient. However, when
antiinflammatory/immunosuppressive therapies fail, colectomies are
the last line of defense. Surgery is required for about 30% of CD
patients within the first year after diagnosis, with the likelihood
for operative procedure increasing about 5% annually thereafter.
Unfortunately, CD also has a high rate of reoccurrence as about 5%
of patients require subsequent surgery after the initial year. UC
patients further have a substantially increased risk of developing
colorectal cancer. Presumably, this is due to the recurrent cycles
of injury to the epithelium, followed by regrowth, which
continually increases the risk of neoplastic transformation.
[0025] A recently discovered member of the immunoglobulin
superfamily known as Junctional Adhesion Molecule (JAM) has been
identified to be selectively concentrated at intercellular
junctions of endothelial and epithelial cells of different origins.
Martin-Padura, I. et al., J. Cell Biol. 142(1): 117-27 (1998). JAM
is a type I integral membrane protein with two extracellular,
intrachain disulfide loops of the V-type. JAM bears substantial
homology to A33 antigen (FIG. 1 or FIG. 18). A monoclonal antibody
directed to JAM was found to inhibit spontaneous and
chemokine-induced monocyte transmigration through an endothelial
cell monolayer in vitro. Martin-Padura, supra.
[0026] It has been recently discovered that JAM expression is
increased in the colon of CRF2-4-/- mice with colitis. CRF
2-4-/-(IL-10R subunit knockout mice) develop a spontaneous colitis
mediated by lymphocytes, monocytes and neutrophils. Several of the
animals also developed colon adenocarcinoma. As a result, it is
foreseeable likely that the compounds of the invention are
expressed in elevated levels in or otherwise associated with human
diseases such as inflammatory bowel disease, other inflammatory
diseases of the gut as well as colorectal carcinoma.
[0027] The compounds of the invention also bear significant
homology to A33 antigen, a known colorectal cancer-associated
marker. The A33 antigen is expressed in more than 90% of primary or
metastatic colon cancers as well as normal colon epithelium. In
carcinomas originating from the colonic mucosa, the A33 antigen is
expressed homogeneously in more than 95% of all cases. The A33
antigen, however, has not been detected in a wide range of other
normal issues, i.e., its expression appears to be organ specific.
Therefore, the A33 antigen appears to play an important role in the
induction of colorectal cancer.
[0028] Since colon cancer is a widespread disease, early diagnosis
and treatment is an important medical goal. Diagnosis and treatment
of colon cancer can be implemented using monoclonal antibodies
(mAbs) specific therefore having fluorescent, nuclear magnetic or
radioactive tags. Radioactive gene, toxins and/or drug tagged mAbs
can be used for treatment in situ with minimal patient description.
mAbs can also be used to diagnose during the diagnosis and
treatment of colon cancers. For example, when the serum levels of
the A33 antigen are elevated in a patient, a drop of the levels
after surgery would indicate the tumor resection was successful. On
the other hand, a subsequent rise in serum A33 antigen levels after
surgery would indicate that metastases of the original tumor may
have formed or that new primary tumors may have appeared.
[0029] Such monoclonal antibodies can be used in lieu of, or in
conjunction with surgery and/or other chemotherapies. For example,
preclinical analysis and localization studies in patients infected
with colorectal carcinoma with amAb to A33 are described in Welt et
al., J. Clin. Oncol. 8: 1894-1906 (1990) and Welt et al., J. Clin.
Oncol. 12:1561-1571 (1994), while U.S. Pat. No. 4,579,827 and U.S.
Ser. No. 424,991 (E.P. 199,141) are directed to the therapeutic
administration of monoclonal antibodies, the latter of which
relates to the application of anti-A33 mAb.
[0030] We herein describe the identification and characterization
of novel polypeptides having homology to A33 antigen protein,
designated herein as PRO1868 polypeptides.
8. PRO3434
[0031] Efforts are being undertaken by both industry and academia
to identify new, native secreted proteins. Many efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel secreted proteins. We herein
describe the identification and characterization of novel secreted
polypeptides, designated herein as PRO3434 polypeptides.
9. PRO1927
[0032] Proteins are glycosylated by a complex set of reactions
which are mediated by membrane bound glycosyltransferases. There is
a large number of different glycosyltransferases that account for
the array of carbohydrate structures synthesized.
N-acetylglucosaminyltransferase proteins comprise a family of
glycosyltransferases that provide for a variety of important
biological functions in the mammalian organism. As an example,
UDP-N-acetylglucosamine: alpha-3-D-mannoside
beta-1,2-N-acetylglucosaminyltransferase 1 is an
glycosyltransferase that catalyzes an essential first step in the
conversion of high-mannose N-glycans to hybrid and complex
N-glycans (Sarkar et al., Proc. Natl. Acad. Sci. USA. 88:234-238
(1991). UPD-N-acetylglucosamine:alpha1,3-D-mannoside bet al,
4-N-acetylglucosaminyltransferase is an essential enzyme in the
production of tri- and tetra-antennary asparagine-linked
sugarchains, and has been recently been purified from bovine small
intestine using cDNA cloning (Minowa et al., J. Biol. Chem. (1998)
273(19): 11556-62). There is interest in the identification and
characterization of additional members of the
N-acetylglucosaminyltransferase protein family, and more generally,
the identification of novel glycosyltransferases.
SUMMARY OF THE INVENTION
1. PRO1800
[0033] A cDNA clone (DNA 35672-2508) has been identified, having
homology to nucleic acid encoding Hep27 protein, that encodes a
novel polypeptide, designated in the present application as
"PRO1800".
[0034] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1800
polypeptide.
[0035] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1800 polypeptide
having the sequence of amino acid residues from about 1 or about 16
to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or (b) the
complement of the DNA molecule of (a).
[0036] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1800 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
nucleotides 36 or about 81 and about 869, inclusive, of FIG. 1 (SEQ
ID NO:1). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0037] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203538 (DNA35672-2508) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 203538 (DNA35672-2508).
[0038] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 or about
16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or (b) the
complement of the DNA of (a).
[0039] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 230 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO 1800 polypeptide having the
sequence of amino acid residues from 1 or about 16 to about 278,
inclusive of FIG. 2 (SEQ ID NO:2), or (b) the complement of the DNA
molecule of (a), and, if the DNA molecule has at least about an 80%
sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0040] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1800
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, or is complementary to such encoding
nucleic acid molecule. The signal peptide has been tentatively
identified as extending from about amino acid position 1 to about
amino acid position 15 in the sequence of FIG. 2 (SEQ ID NO:2).
[0041] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 16 to about 278,
inclusive of FIG. 2 (SEQ ID NO:2), or (b) the complement of the DNA
of (a).
[0042] Another embodiment is directed to fragments of a PRO 1800
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments may be from about 20 to about
80 nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length and most preferably from about 20 to about 40
nucleotides in length and may be derived from the nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1).
[0043] In another embodiment, the invention provides isolated
PRO1800 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified.
[0044] In a specific aspect, the invention provides isolated native
sequence PRO1800 polypeptide, which in certain embodiments,
includes an amino acid sequence comprising residues 1 or about 16
to about 278 of FIG. 2 (SEQ ID NO:2).
[0045] In another aspect, the invention concerns an isolated
PRO1800 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 16 to about 278,
inclusive of FIG. 2 (SEQ ID NO:2).
[0046] In a further aspect, the invention concerns an isolated
PRO1800 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 16 to about 278, inclusive of FIG.
2 (SEQ ID NO:2).
[0047] In yet another aspect, the invention concerns an isolated
PRO1800 polypeptide, comprising the sequence of amino acid residues
1 or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO:2), or a
fragment thereof sufficient to provide a binding site for an
anti-PRO1800 antibody. Preferably, the PRO1800 fragment retains a
qualitative biological activity of a native PRO1800
polypeptide.
[0048] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1800
polypeptide having the sequence of amino acid residues from about 1
or about 16 to about 278, inclusive of FIG. 2 (SEQ ID NO: 3), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0049] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1800 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1800
antibody.
[0050] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1800 polypeptide
by contacting the native PRO1800 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide.
[0051] In a still further embodiment, the invention concerns a
composition comprising a PRO1800 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
2. PRO539
[0052] A cDNA clone (DNA47465-1561) has been identified, having
homology to nucleic acid encoding Costal-2 protein, that encodes a
novel polypeptide, designated in the present application as
"PRO539".
[0053] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO539
polypeptide.
[0054] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO539 polypeptide having
the sequence of amino acid residues from about 1 to about 830,
inclusive of FIG. 4 (SEQ ID NO:7), or (b) the complement of the DNA
molecule of (a).
[0055] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO539 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
nucleotides 186 and about 2675, inclusive, of FIG. 3 (SEQ ID NO:6).
Preferably, hybridization occurs under stringent hybridization and
wash conditions.
[0056] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203661 (DNA47465-1561) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 203661 (DNA47465-1561).
[0057] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 to about
830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) the complement of
the DNA of (a).
[0058] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 100 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO539 polypeptide having the
sequence of amino acid residues from 1 to about 830, inclusive of
FIG. 4 (SEQ ID NO:7), or (b) the complement of the DNA molecule of
(a), and, if the DNA molecule has at least about an 80% sequence
identity, preferably at least about an 85% sequence identity, more
preferably at least about a 90% sequence identity, most preferably
at least about a 95% sequence identity to (a) or (b), isolating the
test DNA molecule.
[0059] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO539 polypeptide,
with or without the initiating methionine, or is complementary to
such encoding nucleic acid molecule.
[0060] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 to about 830, inclusive of FIG. 4
(SEQ ID NO:7), or (b) the complement of the DNA of (a).
[0061] Another embodiment is directed to fragments of a PRO539
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length and most preferably from about 20 to about 40
nucleotides in length and may be derived from the nucleotide
sequence shown in FIG. 3 (SEQ ID NO:6).
[0062] In another embodiment, the invention provides isolated
PRO539 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified.
[0063] In a specific aspect, the invention provides isolated native
sequence PRO539 polypeptide, which in certain embodiments, includes
an amino acid sequence comprising residues 1 to about 830 of FIG. 4
(SEQ ID NO:7).
[0064] In another aspect, the invention concerns an isolated PRO539
polypeptide, comprising an amino acid sequence having at least
about 80% sequence identity, preferably at least about 85% sequence
identity, more preferably at least about 90% sequence identity,
most preferably at least about 95% sequence identity to the
sequence of amino acid residues 1 to about 830, inclusive of FIG. 4
(SEQ ID NO:7).
[0065] In a further aspect, the invention concerns an isolated
PRO539 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 to about 830, inclusive of FIG. 4 (SEQ ID
NO:7).
[0066] In yet another aspect, the invention concerns an isolated
PRO539 polypeptide, comprising the sequence of amino acid residues
1 to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or a fragment
thereof sufficient to provide a binding site for an anti-PRO539
antibody. Preferably, the PRO539 fragment retains a qualitative
biological activity of a native PRO539 polypeptide.
[0067] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO539
polypeptide having the sequence of amino acid residues from about 1
to about 830, inclusive of FIG. 4 (SEQ ID NO:7), or (b) the
complement of the DNA molecule of (a), and if the test DNA molecule
has at least about an 80% sequence identity, preferably at least
about an 85% sequence identity, more preferably at least about a
90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0068] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO539 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO539
antibody.
[0069] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO539 polypeptide
by contacting the native PRO539 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide. In a preferred embodiment, the biological activity is
either binding to microtubiles or the ability to complex with fused
and cubitus interruptus.
[0070] In a still further embodiment, the invention concerns a
composition comprising a PRO539 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
[0071] In yet another embodiment, the invention provides for
compounds and methods for developing antagonists against and
agonist promoting PRO539 modulation of Hedgehog signaling. In
particular, an antagonist of vertebrate PRO539 which blocks,
prevents, inhibits and/or neutralized the normal functioning of
PRO539 in SH signaling pathway, including both small bioorganic
molecules and antisense nucleotides.
[0072] In yet another embodiment, the invention provides for
alternatively spliced variants of human PRO539.
[0073] In still yet a further embodiment, the invention provides a
method of screening or assaying for identifying molecules that
alter the PRO539 modulation of hedgehog signaling. Preferably, the
molecules either prevent interaction of PRO539 with its associative
complexing proteins (such as fused or cubitus interruptus) or
prevent or inhibit dissociation of complexes. The assay comprises
the incubation of a mixture comprising PRO539 and a substrate with
a candidate molecule and detection of the ability of the candidate
molecule to modulate PRO539 hedgehog signaling. The screened
molecules preferably are small molecule drug candidates.
[0074] In yet another embodiment, the method relates to a technique
of diagnosing to determine whether a particular disorder is
modulated by hedgehog signaling, comprising: [0075] (a) culturing
test cells or tissues; [0076] (b) administering a compound which
can inhibit PRO539 modulated hedgehog signaling; and [0077] (c)
determining whether hedgehog signaling is modulated. 3. PRO982
[0078] A cDNA clone (DNA57700-1408) has been identified that
encodes a novel polypeptide, designated in the present application
as "PRO982."
[0079] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO982
polypeptide.
[0080] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO982 polypeptide having
the sequence of amino acid residues from 1 or about 22 to about
125, inclusive of FIG. 6 (SEQ ID NO:9), or (b) the complement of
the DNA molecule of (a).
[0081] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO982 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
residues 89 and about 400, inclusive, of FIG. 5 (SEQ ID NO:8).
Preferably, hybridization occurs under stringent hybridization and
wash conditions.
[0082] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203583 (DNA57700-1408), or (b) the
complement of the DNA molecule of (a). In a preferred embodiment,
the nucleic acid comprises a DNA encoding the same mature
polypeptide encoded by the human protein cDNA in ATCC Deposit No.
203583 (DNA57700-1408).
[0083] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues from 1 or
about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or the
complement of the DNA of (a).
[0084] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least about 50 nucleotides, and
preferably at least about 100 nucleotides and produced by
hybridizing a test DNA molecule under stringent conditions with (a)
a DNA molecule encoding a PRO982 polypeptide having the sequence of
amino acid residues from 1 or about 22 to about 125, inclusive of
FIG. 6 (SEQ ID NO:9), or (b) the complement of the DNA molecule of
(a), and, if the DNA molecule has at least about an 80% sequence
identity, preferably at least about an 85% sequence identity, more
preferably at least about a 90% sequence identity, most preferably
at least about a 95% sequence identity to (a) or (b), isolating the
test DNA molecule.
[0085] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO982 polypeptide,
with or without the N-terminal signal sequence and/or the
initiating methionine, or is complementary to such encoding nucleic
acid molecule. The signal peptide has been tentatively identified
as extending from amino acid position 1 through about amino acid
position 21 in the sequence of FIG. 6 (SEQ ID NO:9).
[0086] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 22 to about 125,
inclusive of FIG. 6 (SEQ ID NO:9), or (b) the complement of the DNA
of (a).
[0087] Another embodiment is directed to fragments of a PRO982
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length, and most preferably from about 20 to about
40 nucleotides in length.
[0088] In another embodiment, the invention provides isolated
PRO982 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove defined.
[0089] In a specific aspect, the invention provides isolated native
sequence PRO982 polypeptide, which in one embodiment, includes an
amino acid sequence comprising residues 1 or about 22 to 125 of
FIG. 6 (SEQ ID NO:9).
[0090] In another aspect, the invention concerns an isolated PRO982
polypeptide, comprising an amino acid sequence having at least
about 80% sequence identity, preferably at least about 85% sequence
identity, more preferably at least about 90% sequence identity,
most preferably at least about 95% sequence identity to the
sequence of amino acid residues 1 or about 22 to about 125,
inclusive of FIG. 6 (SEQ ID NO:9).
[0091] In a further aspect, the invention concerns an isolated
PRO982 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 22 to 125 of FIG. 6 (SEQ ID
NO:9).
[0092] In yet another aspect, the invention concerns an isolated
PRO982 polypeptide, comprising the sequence of amino acid residues
1 or about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or a
fragment thereof sufficient to provide a binding site for an
anti-PRO982 antibody. Preferably, the PRO982 fragment retains a
qualitative biological activity of a native PRO982 polypeptide.
[0093] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO982
polypeptide having the sequence of amino acid residues from 1 or
about 22 to about 125, inclusive of FIG. 6 (SEQ ID NO:9), or (b)
the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
4. PRO1434
[0094] A cDNA clone (DNA68818-2536) has been identified, having
homology to nucleic acid encoding nel protein, that encodes a novel
polypeptide, designated in the present application as
"PRO1434".
[0095] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1434
polypeptide.
[0096] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1434 polypeptide
having the sequence of amino acid residues from about 1 or about 28
to about 325, inclusive of FIG. 8 (SEQ ID NO: 11), or (b) the
complement of the DNA molecule of (a).
[0097] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1434 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
nucleotides 581 or about 662 and about 1555, inclusive, of FIG. 7
(SEQ ID NO: 10). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0098] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203657 (DNA68818-2536) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 203657 (DNA68818-2536).
[0099] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 or about
28 to about 325, inclusive of FIG. 8 (SEQ ID NO: 11), or (b) the
complement of the DNA of (a).
[0100] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 65 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO1434 polypeptide having the
sequence of amino acid residues from 1 or about 28 to about 325,
inclusive of FIG. 8 (SEQ ID NO:11), or (b) the complement of the
DNA molecule of (a), and, if the DNA molecule has at least about an
80% sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0101] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1434
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, and its soluble, i.e., transmembrane
domain deleted or inactivated variants, or is complementary to such
encoding nucleic acid molecule. The signal peptide has been
tentatively identified as extending from about amino acid position
1 to about amino acid position 27 in the sequence of FIG. 8 (SEQ ID
NO: 11). The transmembrane domain has been tentatively identified
as extending from about amino acid position 11 to about amino acid
position 30 in the PRO1434 amino acid sequence (FIG. 8, SEQ ID NO:
11).
[0102] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 28 to about 325,
inclusive of FIG. 8 (SEQ ID NO: 11), or (b) the complement of the
DNA of (a).
[0103] Another embodiment is directed to fragments of a PRO1434
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length and most preferably from about 20 to about 40
nucleotides in length and may be derived from the nucleotide
sequence shown in FIG. 7 (SEQ ID NO: 10).
[0104] In another embodiment, the invention provides isolated
PRO1434 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified.
[0105] In a specific aspect, the invention provides isolated native
sequence PRO1434 polypeptide, which in certain embodiments,
includes an amino acid sequence comprising residues 1 or about 28
to about 325 of FIG. 8 (SEQ ID NO: 11).
[0106] In another aspect, the invention concerns an isolated
PRO1434 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 28 to about 325,
inclusive of FIG. 8 (SEQ ID NO: 11).
[0107] In a further aspect, the invention concerns an isolated
PRO1434 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 28 to about 325, inclusive of FIG.
8 (SEQ ID NO: 11).
[0108] In yet another aspect, the invention concerns an isolated
PRO1434 polypeptide, comprising the sequence of amino acid residues
1 or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO: 11), or
a fragment thereof sufficient to provide a binding site for an
anti-PRO1434 antibody. Preferably, the PRO1434 fragment retains a
qualitative biological activity of a native PRO1434
polypeptide.
[0109] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1434
polypeptide having the sequence of amino acid residues from about 1
or about 28 to about 325, inclusive of FIG. 8 (SEQ ID NO: 11), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0110] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1434 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1434
antibody.
[0111] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1434 polypeptide
by contacting the native PRO1434 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide.
[0112] In a still further embodiment, the invention concerns a
composition comprising a PRO1434 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
5. PRO1863
[0113] A cDNA clone (DNA59847-2510) has been identified that
encodes a novel transmembrane polypeptide, designated in the
present application as "PRO1863".
[0114] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1863
polypeptide.
[0115] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1863 polypeptide
having the sequence of amino acid residues from about 1 or about 16
to about 437, inclusive of FIG. 10 (SEQ ID NO: 16), or (b) the
complement of the DNA molecule of (a).
[0116] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1863 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
nucleotides 17 or about 62 and about 1327, inclusive, of FIG. 9
(SEQ ID NO: 15). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0117] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203576 (DNA59847-2510) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 203576 (DNA59847-2510).
[0118] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 or about
16 to about 437, inclusive of FIG. 10 (SEQ ID NO: 16), or (b) the
complement of the DNA of (a).
[0119] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 345 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO1863 polypeptide having the
sequence of amino acid residues from 1 or about 16 to about 437,
inclusive of FIG. 10 (SEQ ID NO: 16), or (b) the complement of the
DNA molecule of (a), and, if the DNA molecule has at least about an
80% sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0120] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1863
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, and its soluble, i.e., transmembrane
domain deleted or inactivated variants, or is complementary to such
encoding nucleic acid molecule. The signal peptide has been
tentatively identified as extending from about amino acid position
1 to about amino acid position 17 in the sequence of FIG. 10 (SEQ
ID NO: 16). The transmembrane domain has been tentatively
identified as extending from about amino acid position 243 to about
amino acid position 260 in the PRO1863 amino acid sequence (FIG.
10, SEQ ID NO: 16).
[0121] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 16 to about 437,
inclusive of FIG. 10 (SEQ ID NO: 16), or (b) the complement of the
DNA of (a).
[0122] Another embodiment is directed to fragments of a PRO1863
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length and most preferably from about 20 to about 40
nucleotides in length and may be derived from the nucleotide
sequence shown in FIG. 9 (SEQ ID NO: 15).
[0123] In another embodiment, the invention provides isolated
PRO1863 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified.
[0124] In a specific aspect, the invention provides isolated native
sequence PRO1863 polypeptide, which in certain embodiments,
includes an amino acid sequence comprising residues 1 or about 16
to about 437 of FIG. 10 (SEQ ID NO:16).
[0125] In another aspect, the invention concerns an isolated
PRO1863 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 16 to about 437,
inclusive of FIG. 10 (SEQ ID NO: 16).
[0126] In a further aspect, the invention concerns an isolated
PRO1863 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 16 to about 437, inclusive of FIG.
10 (SEQ ID NO: 16).
[0127] In yet another aspect, the invention concerns an isolated
PRO1863 polypeptide, comprising the sequence of amino acid residues
1 or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO: 16),
or a fragment thereof sufficient to provide a binding site for an
anti-PRO1863 antibody. Preferably, the PRO1863 fragment retains a
qualitative biological activity of a native PRO1863
polypeptide.
[0128] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1863
polypeptide having the sequence of amino acid residues from about 1
or about 16 to about 437, inclusive of FIG. 10 (SEQ ID NO: 16), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0129] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1863 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1863
antibody.
[0130] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1863 polypeptide
by contacting the native PRO1863 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide.
[0131] In a still further embodiment, the invention concerns a
composition comprising a PRO1863 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
6. PRO1917
[0132] A cDNA clone (DNA76400-2528) has been identified that
encodes a novel polypeptide having homology to inositol phosphatase
and designated in the present application as "PRO1917".
[0133] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1917
polypeptide.
[0134] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1917 polypeptide
having the sequence of amino acid residues from 1 or about 31 to
about 487, inclusive of FIG. 12 (SEQ ID NO: 18), or (b) the
complement of the DNA molecule of (a).
[0135] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1917 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
residues 96 and about 1466, inclusive, of FIG. 11 (SEQ ID NO: 17).
Preferably, hybridization occurs under stringent hybridization and
wash conditions.
[0136] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203573 (DNA76400-2528), or (b) the
complement of the DNA molecule of (a). In a preferred embodiment,
the nucleic acid comprises a DNA encoding the same mature
polypeptide encoded by the human protein cDNA in ATCC Deposit No.
203573 (DNA76400-2528).
[0137] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues from 1 or
about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO: 18), or the
complement of the DNA of (a).
[0138] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least about 50 nucleotides, and
preferably at least about 100 nucleotides and produced by
hybridizing a test DNA molecule under stringent conditions with (a)
a DNA molecule encoding a PRO1917 polypeptide having the sequence
of amino acid residues from 1 or about 31 to about 487, inclusive
of FIG. 12 (SEQ ID NO:18), or (b) the complement of the DNA
molecule of (a), and, if the DNA molecule has at least about an 80%
sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0139] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1917
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, or is complementary to such encoding
nucleic acid molecule. The signal peptide has been tentatively
identified as extending from amino acid position 1 through about
amino acid position 30 in the sequence of FIG. 12 (SEQ ID
NO:18).
[0140] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 31 to about 487,
inclusive of FIG. 12 (SEQ ID NO: 18), or (b) the complement of the
DNA of (a).
[0141] Another embodiment is directed to fragments of a PRO1917
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length, and most preferably from about 20 to about
40 nucleotides in length.
[0142] In another embodiment, the invention provides isolated
PRO1917 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove defined.
[0143] In a specific aspect, the invention provides isolated native
sequence PRO1917 polypeptide, which in one embodiment, includes an
amino acid sequence comprising residues 1 or about 31 to 487 of
FIG. 12 (SEQ ID NO:18).
[0144] In another aspect, the invention concerns an isolated
PRO1917 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 31 to about 487,
inclusive of FIG. 12 (SEQ ID NO:18).
[0145] In a further aspect, the invention concerns an isolated
PRO1917 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 31 to 487 of FIG. 12 (SEQ ID NO:
18).
[0146] In yet another aspect, the invention concerns an isolated
PRO1917 polypeptide, comprising the sequence of amino acid residues
1 or about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO: 18),
or a fragment thereof sufficient to provide a binding site for an
anti-PRO1917 antibody. Preferably, the PRO1917 fragment retains a
qualitative biological activity of a native PRO1917
polypeptide.
[0147] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1917
polypeptide having the sequence of amino acid residues from 1 or
about 31 to about 487, inclusive of FIG. 12 (SEQ ID NO: 18), or (b)
the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0148] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1917 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1917
antibody.
[0149] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1917
polypeptide, by contacting the native PRO1917 polypeptide with a
candidate molecule and monitoring a biological activity mediated by
said polypeptide.
[0150] In a still further embodiment, the invention concerns a
composition comprising a PRO1917 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier
7. PRO1868
[0151] The present invention concerns compositions and methods for
the diagnosis and treatment of inflammatory diseases in mammals,
including humans. The present invention is based on the
identification of proteins (including agonist and antagonist
antibodies) which either stimulate or inhibit the immune response
in mammals. Inflammatory diseases can be treated by suppressing the
inflammatory response. Molecules that enhance an inflammatory
response stimulate or potentiate the immune response to an antigen.
Molecules which stimulate an inflammatory response can be inhibited
where suppression of the inflammatory response would be beneficial.
Molecules which stimulate the inflammatory response can be used
therapeutically where enhancement of the inflammatory response
would be beneficial. Such stimulatory molecules can also be
inhibited where suppression of the inflammatory response would be
of value. Neutralizing antibodies are examples of molecules that
inhibit molecules having immune stimulatory activity and which
would be beneficial in the treatment of inflammatory diseases.
Molecules which inhibit the inflammatory response can also be
utilized (proteins directly or via the use of antibody agonists) to
inhibit the inflammatory response and thus ameliorate inflammatory
diseases.
[0152] Accordingly, the proteins of the invention are useful for
the diagnosis and/or treatment (including prevention) of immune
related diseases. Antibodies which bind to stimulatory proteins are
useful to suppress the inflammatory response. Antibodies which bind
to inhibitory proteins are useful to stimulate inflammatory
response and the immune system. The proteins and antibodies of the
invention are also useful to prepare medicines and medicaments for
the treatment of inflammatory and immune related diseases.
[0153] In one embodiment, the invention concerns antagonists and
agonists of a PRO1868 polypeptide that inhibits one or more of the
functions or activities of a PRO1868 polypeptide.
[0154] In another embodiment, the invention concerns a method for
determining the presence of a PRO1868 polypeptide comprising
exposing a cell suspected of containing the polypeptide to an
anti-PRO1868 antibody and determining binding of the antibody to
the cell.
[0155] In yet another embodiment, the present invention relates to
a method of diagnosing an inflammatory related disease in a mammal,
comprising detecting the level of expression of a gene encoding a
PRO1868 polypeptide (a) in a test sample of tissue cells obtained
from the mammal, and (b) in a control sample of known normal tissue
cells of the same cell type, wherein a higher expression level in
the test sample indicates the presence of an inflammatory disease
in the mammal.
[0156] In another embodiment, the present invention relates to
method of diagnosing an inflammatory disease in a mammal,
comprising (a) contacting an anti-PRO1868 antibody with a test
sample of tissue culture cells obtained from the mammal, and (b)
detecting the formation of a complex between the antibody and the
PRO1868 polypeptide. The detection may be qualitative or
quantitative, and may be performed in comparison with monitoring
the complex formation in a control sample of known normal tissue
cells of the same cell type. A larger quantity of complexes formed
in the test sample indicates the presence of tumor in the mammal
from which the test tissue cells were obtained. The antibody
preferably carries a detectable label. Complex formation can be
monitored, for example, by light microscopy, flow cytometry,
fluorimetry, or other techniques known in the art. The test sample
is usually obtained from an individual suspected of having a
deficiency or abnormality relating to the inflammatory
response.
[0157] In another embodiment, the present invention relates to a
diagnostic kit, containing an anti-PRO1868 antibody and a carrier
(e.g., a buffer) in suitable packaging. The kit preferably contains
instructions for using the antibody to detect the PRO1868
polypeptide.
[0158] In a further embodiment, the invention concerns an article
of manufacture, comprising:
[0159] a container;
[0160] a label on the container; and
[0161] a composition comprising an active agent contained within
the container; wherein the composition is effective for stimulating
or inhibiting an inflammatory response in a mammal, the label on
the container indicates that the composition can be used to treat
an inflammatory disease, and the active agent in the composition is
an agent stimulating or inhibiting the expression and/or activity
of the PRO1868 polypeptide. In a preferred aspect, the active agent
is a PRO1868 polypeptide or an anti-PRO1868 antibody.
[0162] A further embodiment is a method for identifying a compound
capable of inhibiting the expression and/or activity of a PRO1868
polypeptide by contacting a candidate compound with a PRO1868
polypeptide under conditions and for time sufficient to allow these
two compounds to interact. In a specific aspect, either the
candidate compound or the PRO1868 polypeptide is immobilized on a
solid support. In another aspect, the non-immobilized component
carries a detectable label.
[0163] In yet a further aspect, the invention relates to a method
of treating an inflammatory disease, by administration of an
effective therapeutic amount of a PRO1868 antagonist to a patient
in need thereof for the treatment of a disease selected from:
inflammatory bowel disease, systemic lupus erythematosis,
rheumatoid arthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's
syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria),
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia), thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis), demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic polyneuropathy, hepatobiliary
diseases such as infectious hepatitis (hepatitis A, B, C, D, E and
other nonhepatotropic viruses), autoimmune chronic active
hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and
sclerosing cholangitis, inflammatory and fibrotic lung diseases
(e.g., cystic fibrosis, eosinophilic pneumonias, idiopathic
pulmonary fibrosis and hypersensitivity pneumonitis),
gluten-sensitive enteropathy, Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis, allergic
diseases of the lung such as eosinophilic pneumonias, idiopathic
pulmonary fibrosis and hypersensitivity pneumonitis,
transplantation associated diseases including graft rejection and
graft-verus host disease.
[0164] In a further embodiment, the present invention provides a
method of diagnosing tumor in a mammal, comprising detecting the
level of expression of a gene encoding a PRO1868 polypeptide (a) in
a test sample of tissue cells obtained from the mammal, and (b) in
a control sample of known normal tissue cells of the same cell
type, wherein a higher expression level in the test sample
indicates the presence of tumor in the mammal from which the test
tissue cells were obtained.
[0165] In another embodiment, the present invention provides a
method of diagnosing tumor in a mammal, comprising (a) contacting
an anti-PRO1868 antibody with a test sample of the tissue cells
obtained from the mammal, and (b) detecting the formation of a
complex between the anti PRO1868 and the PRO1868 polypeptide in the
test sample. The detection may be qualitative or quantitative, and
may be performed in comparison with monitoring the complex
formation in a control sample of known normal tissue cells of the
same cell type. A larger quantity of complexes formed in the test
sample indicates the presence of tumor in the mammal from which the
test tissue cells were obtained. The antibody preferably carries a
detectable label. Complex formation can be monitored, for example,
by light microscopy, flow cytometry, fluorimetry, or other
techniques known in the art. Preferably, the test sample is
obtained from an individual mammal suspected to have neoplastic
cell growth or proliferation (e.g., cancerous cells).
[0166] In another embodiment, the present invention provides a
cancer diagnostic kit, comprising an anti-PRO1868 antibody and a
carrier (e.g. a buffer) in suitable packaging. The kit preferably
contains instructions for using the antibody to detect the PRO1868
polypeptide.
[0167] In yet another embodiment, the invention provides a method
for inhibiting the growth of tumor cells comprising exposing a cell
which overexpresses a PRO1868 polypeptide to an effective amount of
an agent inhibiting the expression and/or activity of the PRO1868
polypeptide. The agent preferably is an anti-PRO1868 polypeptide, a
small organic and inorganic peptide, phosphopeptide, antisense or
ribozyme molecule, or a triple helix molecule. In a specific
aspect, the agent, e.g., anti-PRO1868 antibody induces cell death.
In a further aspect, the tumor cells are further exposed to
radiation treatment and/or a cytotoxic or chemotherapeutic
agent.
[0168] In a further embodiment, the invention concerns an article
of manufacture, comprising:
a container;
a label on the container, and
[0169] a composition comprising an active agent contained within
the container; wherein the composition is effective for inhibiting
the growth of tumor cells, the label on the container indicates
that the composition can be used for treating conditions
characterized by overexpression of a PRO1868 polypeptide, and the
active agent in the composition is an agent inhibiting the
expression and/or activity of the PRO1868 polypeptide. In a
preferred aspect, the active agent is an anti-PRO1868 antibody.
[0170] A cDNA clone (DNA77624-2515) has been identified, having
homology to nucleic acid encoding A33 antigen, that encodes a novel
polypeptide, designated in the present application as
"PRO1868".
[0171] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1868
polypeptide.
[0172] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1868 polypeptide
having the sequence of amino acid residues from about 1 or about 31
to about 310, inclusive of FIG. 14 (SEQ ID NO:20), or (b) the
complement of the DNA molecule of (a).
[0173] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1868 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
nucleotides 51 or about 141 and about 980, inclusive, of FIG. 13
(SEQ ID NO:19). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0174] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203553 (DNA77624-2515) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 203553 (DNA77624-2515).
[0175] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 or about
31 to about 310 inclusive of FIG. 14 (SEQ ID NO:20), or (b) the
complement of the DNA of (a).
[0176] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 390 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO1868 polypeptide having the
sequence of amino acid residues from 1 or about 31 to to about 310,
inclusive of FIG. 14 (SEQ ID NO:20), or (b) the complement of the
DNA molecule of (a), and, if the DNA molecule has at least about an
80% sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0177] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1868
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, and its soluble, i.e., transmembrane
domain deleted or inactivated variants, or is complementary to such
encoding nucleic acid molecule. The signal peptide has been
tentatively identified as extending from about amino acid position
1 to about amino acid position 30 in the sequence of FIG. 14 (SEQ
ID NO:20). The transmembrane domain has been tentatively identified
as extending from about amino acid position 243 to about amino acid
position 263 in the PRO1868 amino acid sequence (FIG. 14, SEQ ID
NO:20).
[0178] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 31 to about 310
inclusive of FIG. 14 (SEQ ID NO:20), or (b) the complement of the
DNA of (a).
[0179] Another embodiment is directed to fragments of a PRO1868
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length and most preferably from about 20 to about 40
nucleotides in length and may be derived from the nucleotide
sequence shown in FIG. 13 (SEQ ID NO: 19).
[0180] In another embodiment, the invention provides isolated
PRO1868 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified.
[0181] In a specific aspect, the invention provides isolated native
sequence PRO1868 polypeptide, which in certain embodiments,
includes an amino acid sequence comprising residues 1 or about 31
to about 310 of FIG. 14 (SEQ ID NO:20).
[0182] In another aspect, the invention concerns an isolated
PRO1868 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 31 to about 310,
inclusive of FIG. 14 (SEQ ID NO:20).
[0183] In a further aspect, the invention concerns an isolated
PRO1868 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 31 to about 310, inclusive of FIG.
14 (SEQ ID NO:20).
[0184] In yet another aspect, the invention concerns an isolated
PRO1868 polypeptide, comprising the sequence of amino acid residues
1 or about 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20), or
a fragment thereof sufficient to provide a binding site for an
anti-PRO1868 antibody. Preferably, the PRO1868 fragment retains a
qualitative biological activity of a native PRO1868
polypeptide.
[0185] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1868
polypeptide having the sequence of amino acid residues from about 1
or about 31 to about 310, inclusive of FIG. 14 (SEQ ID NO:20), or
(b) the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0186] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1868 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1868
antibody.
[0187] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1868 polypeptide
by contacting the native PRO1868 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide.
[0188] In a still further embodiment, the invention concerns a
composition comprising a PRO1868 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
[0189] In another embodiment, the invention provides a composition
containing a PRO1868 polypeptide or an agonist or antagonist
antibody in admixture with a carrier or excipient. In one aspect,
the composition contains a therapeutically affective amount of the
peptide or antibody. In another aspect, when the composition
contains an inflammation stimulating molecule, the composition is
useful for: (a) increasing infiltration of inflammatory cells into
a tissue of a mammal in need thereof, (b) stimulating or enhancing
an immune response in a mammal in need thereof, or (c) increasing
the proliferation of T-lymphocytes in a mammal in need thereof in
response to an antigen. In a further aspect, when the composition
contains an inflammatory inhibiting molecule, the composition is
useful for: (a) decreasing infiltration of inflammatory cells into
a tissue of a mammal in need thereof, (b) inhibiting or reducing an
inflammatory response in a mammal in need thereof, or (c)
decreasing the proliferation of T-lymphocytes in a mammal in need
thereof in response to an antigen. In another aspect, the
composition contains a further active ingredient, which may, for
example, be a further antibody or a cytotoxic or chemotherapeutic
agent. Preferably, the composition is sterile.
[0190] In a further embodiment, the invention concerns nucleic acid
encoding an anti-PRO1868 antibody, and vectors and recombinant host
cells comprising such nucleic acid. In a still further embodiment,
the invention concerns a method for producing such an antibody by
culturing a host cell transformed with nucleic acid encoding the
antibody under conditions such that the antibody is expressed, and
recovering the antibody from the cell culture.
8. PRO3434
[0191] A cDNA clone (DNA77631-2537) has been identified that
encodes a novel polypeptide, designated in the present application
as "PRO3434."
[0192] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO3434
polypeptide.
[0193] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO3434 polypeptide
having the sequence of amino acid residues from 1 or about 17 to
about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or (b) the
complement of the DNA molecule of (a).
[0194] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO3434 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
residues 46 or about 94 and about 3132, inclusive, of FIG. 15 (SEQ
ID NO:21). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0195] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203651 (DNA77631-2537), or (b) the
complement of the DNA molecule of (a). In a preferred embodiment,
the nucleic acid comprises a DNA encoding the same mature
polypeptide encoded by the human protein cDNA in ATCC Deposit No.
203651 (DNA77631-2537).
[0196] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues from 1 or
about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or the
complement of the DNA of (a).
[0197] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least about 460 nucleotides and
produced by hybridizing a test DNA molecule under stringent
conditions with (a) a DNA molecule encoding a PRO3434 polypeptide
having the sequence of amino acid residues from 1 or about 17 to
about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or (b) the
complement of the DNA molecule of (a), and, if the DNA molecule has
at least about an 80% sequence identity, preferably at least about
an 85% sequence identity, more preferably at least about a 90%
sequence identity, most preferably at least about a 95% sequence
identity to (a) or (b), isolating the test DNA molecule.
[0198] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO3434
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, or is complementary to such encoding
nucleic acid molecule. The signal peptide has been tentatively
identified as extending from amino acid position 1 through about
amino acid position 16 in the sequence of FIG. 16 (SEQ ID
NO:22).
[0199] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 17 to about 1029,
inclusive of FIG. 16 (SEQ ID NO:22), or (b) the complement of the
DNA of (a).
[0200] Another embodiment is directed to fragments of a PRO3434
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length, and most preferably from about 20 to about
40 nucleotides in length.
[0201] In another embodiment, the invention provides isolated
PRO3434 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove defined.
[0202] In a specific aspect, the invention provides isolated native
sequence PRO3434 polypeptide, which in one embodiment, includes an
amino acid sequence comprising residues 1 or about 17 to 1029 of
FIG. 16 (SEQ ID NO:22).
[0203] In another aspect, the invention concerns an isolated
PRO3434 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 17 to about 1029,
inclusive of FIG. 16 (SEQ ID NO:22).
[0204] In a further aspect, the invention concerns an isolated
PRO3434 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 17 to 1029 of FIG. 16 (SEQ ID
NO:22).
[0205] In yet another aspect, the invention concerns an isolated
PRO3434 polypeptide, comprising the sequence of amino acid residues
1 or about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22),
or a fragment thereof sufficient to provide a binding site for an
anti-PRO3434tibody. Preferably, the PRO982 fragment retains a
qualitative biological activity of a native PRO3434
polypeptide.
[0206] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO3434
polypeptide having the sequence of amino acid residues from 1 or
about 17 to about 1029, inclusive of FIG. 16 (SEQ ID NO:22), or (b)
the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0207] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO3434 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO3434
antibody.
[0208] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO3434
polypeptide, by contacting the native PRO3434 polypeptide with a
candidate molecule and monitoring a biological activity mediated by
said polypeptide.
[0209] In a still further embodiment, the invention concerns a
composition comprising a PRO3434 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
9. PRO1927
[0210] A cDNA clone (DNA82307-2531) has been identified that
encodes a novel polypeptide having homology to
glycosyltransferases, and is designated in the present application
as "PRO1927".
[0211] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1927
polypeptide.
[0212] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1927 polypeptide
having the sequence of amino acid residues from 1 or about 24 to
about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b) the
complement of the DNA molecule of (a).
[0213] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1927 polypeptide comprising DNA
hybridizing to the complement of the nucleic acid between about
residues 120 and about 1694, inclusive, of FIG. 17 (SEQ ID NO:23).
Preferably, hybridization occurs under stringent hybridization and
wash conditions.
[0214] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 203537 (DNA82307-2531), or (b) the
complement of the DNA molecule of (a). In a preferred embodiment,
the nucleic acid comprises a DNA encoding the same mature
polypeptide encoded by the human protein cDNA in ATCC Deposit No.
203537 (DNA82307-2531).
[0215] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues from 1 or
about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or the
complement of the DNA of (a).
[0216] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least about 50 nucleotides, and
preferably at least about 100 nucleotides and produced by
hybridizing a test DNA molecule under stringent conditions with (a)
a DNA molecule encoding a PRO1927 polypeptide having the sequence
of amino acid residues from 1 or about 24 to about 548, inclusive
of FIG. 18 (SEQ ID NO:24), or (b) the complement of the DNA
molecule of (a), and, if the DNA molecule has at least about an 80%
sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), isolating the test DNA molecule.
[0217] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1927
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, and its soluble variants (i.e.
transmembrane domain deleted or inactivated), or is complementary
to such encoding nucleic acid molecule. The signal peptide has been
tentatively identified as extending from amino acid position 1
through about amino acid position 23 in the sequence of FIG. 18
(SEQ ID NO:24). A type II transmembrane domain has been tentatively
identified as extending from about amino acid position 6 to about
amino acid position 25 in the PRO1927 amino acid sequence (FIG. 18,
SEQ ID NO:24).
[0218] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 24 to about 548,
inclusive of FIG. 18 (SEQ ID NO:24), or (b) the complement of the
DNA of (a).
[0219] Another embodiment is directed to fragments of a PRO1927
polypeptide coding sequence that may find use as hybridization
probes. Such nucleic acid fragments are from about 20 to about 80
nucleotides in length, preferably from about 20 to about 60
nucleotides in length, more preferably from about 20 to about 50
nucleotides in length, and most preferably from about 20 to about
40 nucleotides in length.
[0220] In another embodiment, the invention provides isolated
PRO1927 polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove defined.
[0221] In a specific aspect, the invention provides isolated native
sequence PRO1927 polypeptide, which in one embodiment, includes an
amino acid sequence comprising residues 1 or about 24 to 548 of
FIG. 18 (SEQ ID NO:24).
[0222] In another aspect, the invention concerns an isolated
PRO1927 polypeptide, comprising an amino acid sequence having at
least about 80% sequence identity, preferably at least about 85%
sequence identity, more preferably at least about 90% sequence
identity, most preferably at least about 95% sequence identity to
the sequence of amino acid residues 1 or about 24 to about 548,
inclusive of FIG. 18 (SEQ ID NO:24).
[0223] In a further aspect, the invention concerns an isolated
PRO1927 polypeptide, comprising an amino acid sequence scoring at
least about 80% positives, preferably at least about 85% positives,
more preferably at least about 90% positives, most preferably at
least about 95% positives when compared with the amino acid
sequence of residues 1 or about 24 to 548 of FIG. 18 (SEQ ID
NO:24).
[0224] In yet another aspect, the invention concerns an isolated
PRO1927 polypeptide, comprising the sequence of amino acid residues
1 or about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or
a fragment thereof sufficient to provide a binding site for an
anti-PRO1927 antibody. Preferably, the PRO1927 fragment retains a
qualitative biological activity of a native PRO1927
polypeptide.
[0225] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1927
polypeptide having the sequence of amino acid residues from 1 or
about 24 to about 548, inclusive of FIG. 18 (SEQ ID NO:24), or (b)
the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, preferably at
least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), (ii) culturing a host cell
comprising the test DNA molecule under conditions suitable for
expression of the polypeptide, and (iii) recovering the polypeptide
from the cell culture.
[0226] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1927 polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-PRO1927
antibody.
[0227] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1927
polypeptide, by contacting the native PRO1927 polypeptide with a
candidate molecule and monitoring a biological activity mediated by
said polypeptide.
[0228] In a still further embodiment, the invention concerns a
composition comprising a PRO1927 polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier
10. Additional Embodiments
[0229] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described polypeptides. Host cell comprising any such vector are
also provided. By way of example, the host cells may be CHO cells,
E. coli, or yeast. A process for producing any of the herein
described polypeptides is further provided and comprises culturing
host cells under conditions suitable for expression of the desired
polypeptide and recovering the desired polypeptide from the cell
culture.
[0230] In other embodiments, the invention provides chimeric
molecules comprising any of the herein described polypeptides fused
to a heterologous polypeptide or amino acid sequence. Example of
such chimeric molecules comprise any of the herein described
polypeptides fused to an epitope tag sequence or a Fc region of an
immunoglobulin.
[0231] In another embodiment, the invention provides an antibody
which specifically binds to any of the above or below described
polypeptides. Optionally, the antibody is a monoclonal antibody,
humanized antibody, antibody fragment or single-chain antibody.
[0232] In yet other embodiments, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences or as antisense probes, wherein those probes
may be derived from any of the above or below described nucleotide
sequences.
[0233] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a PRO polypeptide.
[0234] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% sequence identity,
preferably at least about 81% sequence identity, more preferably at
least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence
identity, yet more preferably at least about 86% sequence identity,
yet more preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule encoding a PRO polypeptide having a full-length amino acid
sequence as disclosed herein, an amino acid sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a
transmembrane protein, with or without the signal peptide, as
disclosed herein or any other specifically defined fragment of the
full-length amino acid sequence as disclosed herein, or (b) the
complement of the DNA molecule of (a).
[0235] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% sequence
identity, preferably at least about 81% sequence identity, more
preferably at least about 82% sequence identity, yet more
preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule comprising the coding sequence of a full-length PRO
polypeptide cDNA as disclosed herein, the coding sequence of a PRO
polypeptide lacking the signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal peptide, as disclosed
herein or the coding sequence of any other specifically defined
fragment of the full-length amino acid sequence as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0236] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% sequence identity, preferably at least about 81%
sequence identity, more preferably at least about 82% sequence
identity, yet more preferably at least about 83% sequence identity,
yet more preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule that encodes the same mature polypeptide encoded by any of
the human protein cDNAs deposited with the ATCC as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0237] Another aspect the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated, or is complementary to such
encoding nucleotide sequence, wherein the transmembrane domain(s)
of such polypeptide are disclosed herein. Therefore, soluble
extracellular domains of the herein described PRO polypeptides are
contemplated.
[0238] Another embodiment is directed to fragments of a PRO
polypeptide coding sequence, or the complement thereof, that may
find use as, for example, hybridization probes, for encoding
fragments of a PRO polypeptide that may optionally encode a
polypeptide comprising a binding site for an anti-PRO antibody or
as antisense oligonucleotide probes. Such nucleic acid fragments
are usually at least about 20 nucleotides in length, preferably at
least about 30 nucleotides in length, more preferably at least
about 40 nucleotides in length, yet more preferably at least about
50 nucleotides in length, yet more preferably at least about 60
nucleotides in length, yet more preferably at least about 70
nucleotides in length, yet more preferably at least about 80
nucleotides in length, yet more preferably at least about 90
nucleotides in length, yet more preferably at least about 100
nucleotides in length, yet more preferably at least about 110
nucleotides in length, yet more preferably at least about 120
nucleotides in length, yet more preferably at least about 130
nucleotides in length, yet more preferably at least about 140
nucleotides in length, yet more preferably at least about 150
nucleotides in length, yet more preferably at least about 160
nucleotides in length, yet more preferably at least about 170
nucleotides in length, yet more preferably at least about 180
nucleotides in length, yet more preferably at least about 190
nucleotides in length, yet more preferably at least about 200
nucleotides in length, yet more preferably at least about 250
nucleotides in length, yet more preferably at least about 300
nucleotides in length, yet more preferably at least about 350
nucleotides in length, yet more preferably at least about 400
nucleotides in length, yet more preferably at least about 450
nucleotides in length, yet more preferably at least about 500
nucleotides in length, yet more preferably at least about 600
nucleotides in length, yet more preferably at least about 700
nucleotides in length, yet more preferably at least about 800
nucleotides in length, yet more preferably at least about 900
nucleotides in length and yet more preferably at least about 1000
nucleotides in length, wherein in this context the term "about"
means the referenced nucleotide sequence length plus or minus 10%
of that referenced length. It is noted that novel fragments of a
PRO polypeptide-encoding nucleotide sequence may be determined in a
routine manner by aligning the PRO polypeptide-encoding nucleotide
sequence with other known nucleotide sequences using any of a
number of well known sequence alignment programs and determining
which PRO polypeptide-encoding nucleotide sequence fragment(s) are
novel. All of such PRO polypeptide-encoding nucleotide sequences
are contemplated herein. Also contemplated are the PRO polypeptide
fragments encoded by these nucleotide molecule fragments,
preferably those PRO polypeptide fragments that comprise a binding
site for an anti-PRO antibody.
[0239] In another embodiment, the invention provides isolated PRO
polypeptide encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0240] In a certain aspect, the invention concerns an isolated PRO
polypeptide, comprising an amino acid sequence having at least
about 80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to a PRO
polypeptide having a full-length amino acid sequence as disclosed
herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular domain of a transmembrane
protein, with or without the signal peptide, as disclosed herein or
any other specifically defined fragment of the full-length amino
acid sequence as disclosed herein.
[0241] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence having at least about
80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to an amino acid
sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
[0242] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence scoring at least
about 80% positives, preferably at least about 81% positives, more
preferably at least about 82% positives, yet more preferably at
least about 83% positives, yet more preferably at least about 84%
positives, yet more preferably at least about 85% positives, yet
more preferably at least about 86% positives, yet more preferably
at least about 87% positives, yet more preferably at least about
88% positives, yet more preferably at least about 89% positives,
yet more preferably at least about 90% positives, yet more
preferably at least about 91% positives, yet more preferably at
least about 92% positives, yet more preferably at least about 93%
positives, yet more preferably at least about 94% positives, yet
more preferably at least about 95% positives, yet more preferably
at least about 96% positives, yet more preferably at least about
97% positives, yet more preferably at least about 98% positives and
yet more preferably at least about 99% positives when compared with
the amino acid sequence of a PRO polypeptide having a full-length
amino acid sequence as disclosed herein, an amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal
peptide, as disclosed herein or any other specifically defined
fragment of the full-length amino acid sequence as disclosed
herein.
[0243] In a specific aspect, the invention provides an isolated PRO
polypeptide without the N terminal signal sequence and/or the
initiating methionine and is encoded by a nucleotide sequence that
encodes such an amino acid sequence as hereinbefore described.
Processes for producing the same are also herein described, wherein
those processes comprise culturing a host cell comprising a vector
which comprises the appropriate encoding nucleic acid molecule
under conditions suitable for expression of the PRO polypeptide and
recovering the PRO polypeptide from the cell culture.
[0244] Another aspect the invention provides an isolated PRO
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated. Processes for producing the same
are also herein described, wherein those processes comprise
culturing a host cell comprising a vector which comprises the
appropriate encoding nucleic acid molecule under conditions
suitable for expression of the PRO polypeptide and recovering the
PRO polypeptide from the cell culture.
[0245] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO polypeptide as defined herein. In a
particular embodiment, the agonist or antagonist is an anti-PRO
antibody or a small molecule.
[0246] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists to a PRO polypeptide which
comprise contacting the PRO polypeptide with a candidate molecule
and monitoring a biological activity mediated by said PRO
polypeptide. Preferably, the PRO polypeptide is a native PRO
polypeptide.
[0247] In a still further embodiment, the invention concerns a
composition of matter comprising a PRO polypeptide, or an agonist
or antagonist of a PRO polypeptide as herein described, or an
anti-PRO antibody, in combination with a carrier. Optionally, the
carrier is a pharmaceutically acceptable carrier.
[0248] Another embodiment of the present invention is directed to
the use of a PRO polypeptide, or an agonist or antagonist thereof
as hereinbefore described, or an anti-PRO antibody, for the
preparation of a medicament useful in the treatment of a condition
which is responsive to the PRO polypeptide, an agonist or
antagonist thereof or an anti-PRO antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0249] FIG. 1 shows a nucleotide sequence (SEQ ID NO: 1) of a
native sequence PRO1800 cDNA, wherein SEQ ID NO: 1 is a clone
designated herein as "DNA35672-2508".
[0250] FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived
from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0251] FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a native
sequence PRO539 cDNA, wherein SEQ ID NO:6 is a clone designated
herein as "DNA47465-1561".
[0252] FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived
from the coding sequence of SEQ ID NO:6 shown in FIG. 3.
[0253] FIG. 5 shows a nucleotide sequence (SEQ ID NO:8) of a native
sequence PRO982 cDNA, wherein SEQ ID NO:8 is a clone designated
herein as "DNA57700-1408".
[0254] FIG. 6 shows the amino acid sequence (SEQ ID NO:9) derived
from the coding sequence of SEQ ID NO:8 shown in FIG. 5.
[0255] FIG. 7 shows a nucleotide sequence (SEQ ID NO: 12) of a
native sequence PRO1434 cDNA, wherein SEQ ID NO: 12 is a clone
designated herein as "DNA68818-2536".
[0256] FIG. 8 shows the amino acid sequence (SEQ ID NO: 13) derived
from the coding sequence of SEQ ID NO: 12 shown in FIG. 7.
[0257] FIG. 9 shows a nucleotide sequence (SEQ ID NO: 17) of a
native sequence PRO1863 cDNA, wherein SEQ ID NO:17 is a clone
designated herein as "DNA59847-2510".
[0258] FIG. 10 shows the amino acid sequence (SEQ ID NO: 18)
derived from the coding sequence of SEQ ID NO: 17 shown in FIG.
9.
[0259] FIG. 11 shows a nucleotide sequence (SEQ ID NO: 19) of a
native sequence PRO1917 cDNA, wherein SEQ ID NO: 19 is a clone
designated herein as "DNA76400-2528".
[0260] FIG. 12 shows the amino acid sequence (SEQ ID NO:20) derived
from the coding sequence of SEQ ID NO: 19 shown in FIG. 11.
[0261] FIG. 13 shows a nucleotide sequence (SEQ ID NO:21) of a
native sequence PRO1868 cDNA, wherein SEQ ID NO:21 is a clone
designated herein as "DNA77624-2515".
[0262] FIG. 14 shows the amino acid sequence (SEQ ID NO:22) derived
from the coding sequence of SEQ ID NO:21 shown in FIG. 13.
[0263] FIG. 15 shows a nucleotide sequence (SEQ ID NO:23) of a
native sequence PRO3434 cDNA, wherein SEQ ID NO:23 is a clone
designated herein as "DNA77631-2537".
[0264] FIG. 16 shows the amino acid sequence (SEQ ID NO:24) derived
from the coding sequence of SEQ ID NO:23 shown in FIG. 15.
[0265] FIG. 17 shows a nucleotide sequence (SEQ ID NO:25) of a
native sequence PRO1927 cDNA, wherein SEQ ID NO:25 is a clone
designated herein as "DNA82307-2531".
[0266] FIG. 18 shows the amino acid sequence (SEQ ID NO:26) derived
from the coding sequence of SEQ ID NO:25 shown in FIG. 17.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Definitions
[0267] The terms "PRO polypeptide" and "PRO" as used herein and
when immediately followed by a numerical designation refer to
various polypeptides, wherein the complete designation (i.e.,
PRO/number) refers to specific polypeptide sequences as described
herein. The terms "PRO/number polypeptide" and "PRO/number" wherein
the term "number" is provided as an actual numerical designation as
used herein encompass native sequence polypeptides and polypeptide
variants (which are further defined herein). The PRO polypeptides
described herein may be isolated from a variety of sources, such as
from human tissue types or from another source, or prepared by
recombinant or synthetic methods.
[0268] A "native sequence PRO polypeptide" comprises a polypeptide
having the same amino acid sequence as the corresponding PRO
polypeptide derived from nature. Such native sequence PRO
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated
or secreted forms of the specific PRO polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In various embodiments of the
invention, the native sequence PRO polypeptides disclosed herein
are mature or full-length native sequence polypeptides comprising
the full-length amino acids sequences shown in the accompanying
figures. Start and stop codons are shown in bold font and
underlined in the figures. However, while the PRO polypeptide
disclosed in the accompanying figures are shown to begin with
methionine residues designated herein as amino acid position 1 in
the figures, it is conceivable and possible that other methionine
residues located either upstream or downstream from the amino acid
position 1 in the figures may be employed as the starting amino
acid residue for the PRO polypeptides.
[0269] The PRO polypeptide "extracellular domain" or "ECD" refers
to a form of the PRO polypeptide which is essentially free of the
transmembrane and cytoplasmic domains. Ordinarily, a PRO
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of
such domains. It will be understood that any transmembrane domains
identified for the PRO polypeptides of the present invention are
identified pursuant to criteria routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain may vary but most likely by no more than
about 5 amino acids at either end of the domain as initially
identified herein. Optionally, therefore, an extracellular domain
of a PRO polypeptide may contain from about 5 or fewer amino acids
on either side of the transmembrane domain/extracellular domain
boundary as identified in the Examples or specification and such
polypeptides, with or without the associated signal peptide, and
nucleic acid encoding them, are comtemplated by the present
invention.
[0270] The approximate location of the "signal peptides" of the
various PRO polypeptides disclosed herein are shown in the present
specification and/or the accompanying figures. It is noted,
however, that the C-terminal boundary of a signal peptide may vary,
but most likely by no more than about 5 amino acids on either side
of the signal peptide C-terminal boundary as initially identified
herein, wherein the C-terminal boundary of the signal peptide may
be identified pursuant to criteria routinely employed in the art
for identifying that type of amino acid sequence element (e.g.,
Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al.,
Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from
a secreted polypeptide is not entirely uniform, resulting in more
than one secreted species. These polypeptides, where the signal
peptide is cleaved within no more than about 5 amino acids on
either side of the C-terminal boundary of the signal peptide as
identified herein, and the polynucleotides encoding them, are
contemplated by the present invention.
[0271] "PRO polypeptide variant" means an active PRO polypeptide as
defined above or below having at least about 80% amino acid
sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for instance, PRO polypeptides
wherein one or more amino acid residues are added, or deleted, at
the N- or C-terminus of the full-length native amino acid sequence.
Ordinarily, a PRO polypeptide variant will have at least about 80%
amino acid sequence identity, preferably at least about 81% amino
acid sequence identity, more preferably at least about 82% amino
acid sequence identity, more preferably at least about 83% amino
acid sequence identity, more preferably at least about 84% amino
acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more preferably at least about 86% amino
acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more preferably at least about 88% amino
acid sequence identity, more preferably at least about 89% amino
acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more preferably at least about 91% amino
acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more preferably at least about 93% amino
acid sequence identity, more preferably at least about 94% amino
acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more preferably at least about 96% amino
acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more preferably at least about 98% amino
acid sequence identity and most preferably at least about 99% amino
acid sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length PRO polypeptide sequence as
disclosed herein. Ordinarily, PRO variant polypeptides are at least
about 10 amino acids in length, often at least about 20 amino acids
in length, more often at least about 30 amino acids in length, more
often at least about 40 amino acids in length, more often at least
about 50 amino acids in length, more often at least about 60 amino
acids in length, more often at least about 70 amino acids in
length, more often at least about 80 amino acids in length, more
often at least about 90 amino acids in length, more often at least
about 100 amino acids in length, more often at least about 150
amino acids in length, more often at least about 200 amino acids in
length, more often at least about 300 amino acids in length, or
more.
[0272] "Percent (%) amino acid sequence identity" with respect to
the PRO polypeptide sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the specific PRO
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are generated using
the sequence comparison computer program ALIGN-2, wherein the
complete source code for the ALIGN-2 program is provided in Table 1
below. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Table 1
below has been filed with user documentation in the U.S. Copyright
Office, Wash. D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Table 1
below. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.OD. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0273] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. As examples of % amino acid
sequence identity calculations using this method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of
the amino acid sequence designated "Comparison Protein" to the
amino acid sequence designated "PRO", wherein "PRO" represents the
amino acid sequence of a hypothetical PRO polypeptide of interest,
"Comparison Protein" represents the amino acid sequence of a
polypeptide against which the "PRO" polypeptide of interest is
being compared, and "X, "Y" and "Z" each represent different
hypothetical amino acid residues.
[0274] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program. However, % amino acid sequence identity values may also be
obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid
sequence identity value is determined by dividing (a) the number of
matching identical amino acid residues between the amino acid
sequence of the PRO polypeptide of interest having a sequence
derived from the native PRO polypeptide and the comparison amino
acid sequence of interest (i.e., the sequence against which the PRO
polypeptide of interest is being compared which may be a PRO
variant polypeptide) as determined by WU-BLAST-2 by (b) the total
number of amino acid residues of the PRO polypeptide of interest.
For example, in the statement "a polypeptide comprising an the
amino acid sequence A which has or having at least 80% amino acid
sequence identity to the amino acid sequence B", the amino acid
sequence A is the comparison amino acid sequence of interest and
the amino acid sequence B is the amino acid sequence of the PRO
polypeptide of interest.
[0275] Percent amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program maybe downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0276] In situations where NCBI-BLAST2 is employed foramino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program NCBI-BLAST2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A.
[0277] "PRO variant polynucleotide" or "PRO variant nucleic acid
sequence" means a nucleic acid molecule which encodes an active PRO
polypeptide as defined below and which has at least about 80%
nucleic acid sequence identity with a nucleotide acid sequence
encoding a full-length native sequence PRO polypeptide sequence as
disclosed herein, a full-length native sequence PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about
80% nucleic acid sequence identity, more preferably at least about
81% nucleic acid sequence identity, more preferably at least about
82% nucleic acid sequence identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about
84% nucleic acid sequence identity, more preferably at least about
85% nucleic acid sequence identity, more preferably at least about
86% nucleic acid sequence identity, more preferably at least about
87% nucleic acid sequence identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about
90% nucleic acid sequence identity, more preferably at least about
91% nucleic acid sequence identity, more preferably at least about
92% nucleic acid sequence identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about
95% nucleic acid sequence identity, more preferably at least about
96% nucleic acid sequence identity, more preferably at least about
97% nucleic acid sequence identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least
about 99% nucleic acid sequence identity with a nucleic acid
sequence encoding a full-length native sequence PRO polypeptide
sequence as disclosed herein, a full-length native sequence PRO
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO polypeptide, with or
without the signal sequence, as disclosed herein or any other
fragment of a full-length PRO polypeptide sequence as disclosed
herein. Variants do not encompass the native nucleotide
sequence.
[0278] Ordinarily, PRO variant polynucleotides are at least about
30 nucleotides in length, often at least about 60 nucleotides in
length, more often at least about 90 nucleotides in length, more
often at least about 120 nucleotides in length, more often at least
about 150 nucleotides in length, more often at least about 180
nucleotides in length, more often at least about 210 nucleotides in
length, more often at least about 240 nucleotides in length, more
often at least about 270 nucleotides in length, more often at least
about 300 nucleotides in length, more often at least about 450
nucleotides in length, more often at least about 600 nucleotides in
length, more often at least about 900 nucleotides in length, or
more.
[0279] "Percent (%) nucleic acid sequence identity" with respect to
PRO-encoding nucleic acid sequences identified herein is defined as
the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the PRO nucleic acid sequence of
interest, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity.
Alignment for purposes of determining percent nucleic acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. For purposes herein, however, % nucleic acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc. and the
source code shown in Table 1 below has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif. or may be compiled
from the source code provided in Table 1 below. The ALIGN-2 program
should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.0D. All sequence comparison parameters are set by
the ALIGN-2 program and do not vary.
[0280] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "PRO-DNA" nucleic acid molecule of
interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides.
[0281] Unless specifically stated otherwise, all % nucleic acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program. However, % nucleic acid sequence identity values may also
be obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid
sequence identity value is determined by dividing (a) the number of
matching identical nucleotides between the nucleic acid sequence of
the PRO polypeptide-encoding nucleic acid molecule of interest
having a sequence derived from the native sequence PRO
polypeptide-encoding nucleic acid and the comparison nucleic acid
molecule of interest (i.e., the sequence against which the PRO
polypeptide-encoding nucleic acid molecule of interest is being
compared which may be a variant PRO polynucleotide) as determined
by WU-BLAST-2 by (b) the total number of nucleotides of the PRO
polypeptide-encoding nucleic acid molecule of interest. For
example, in the statement "an isolated nucleic acid molecule
comprising a nucleic acid sequence A which has or having at least
80% nucleic acid sequence identity to the nucleic acid sequence B",
the nucleic acid sequence A is the comparison nucleic acid molecule
of interest and the nucleic acid sequence B is the nucleic acid
sequence of the PRO polypeptide-encoding nucleic acid molecule of
interest.
[0282] Percent nucleic acid sequence identity may also be
determined using the sequence comparison program NCBI-BLAST2
(Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The
NCBI-BLAST2 sequence comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0283] In situations where NCBI-BLAST2 is employed for sequence
comparisons, the % nucleic acid sequence identity of a given
nucleic acid sequence C to, with, or against a given nucleic acid
sequence D (which can alternatively be phrased as a given nucleic
acid sequence C that has or comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program NCBI-BLAST2 in that program's
alignment of C and D, and where Z is the total number of
nucleotides in D. It will be appreciated that where the length of
nucleic acid sequence C is not equal to the length of nucleic acid
sequence D, the % nucleic acid sequence identity of C to D will not
equal the % nucleic acid sequence identity of D to C.
[0284] In other embodiments, PRO variant polynucleotides are
nucleic acid molecules that encode an active PRO polypeptide and
which are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to nucleotide sequences encoding
a full-length PRO polypeptide as disclosed herein. PRO variant
polypeptides may be those that are encoded by a PRO variant
polynucleotide.
[0285] The term "positives", in the context of sequence comparison
performed as described above, includes residues in the sequences
compared that are not identical but have similar properties (e.g.
as a result of conservative substitutions, see Table 6 below). For
purposes herein, the % value of positives is determined by dividing
(a) the number of amino acid residues scoring a positive value
between the PRO polypeptide amino acid sequence of interest having
a sequence derived from the native PRO polypeptide sequence and the
comparison amino acid sequence of interest (i.e., the amino acid
sequence against which the PRO polypeptide sequence is being
compared) as determined in the BLOSUM62 matrix of WU-BLAST-2 by (b)
the total number of amino acid residues of the PRO polypeptide of
interest.
[0286] Unless specifically stated otherwise, the % value of
positives is calculated as described in the immediately preceding
paragraph. However, in the context of the amino acid sequence
identity comparisons performed as described for ALIGN-2 and
NCBI-BLAST-2 above, includes amino acid residues in the sequences
compared that are not only identical, but also those that have
similar properties. Amino acid residues that score a positive value
to an amino acid residue of interest are those that are either
identical to the amino acid residue of interest or are a preferred
substitution (as defined in Table 6 below) of the amino acid
residue of interest.
[0287] For amino acid sequence comparisons using ALIGN-2 or
NCBI-BLAST2, the % value of positives of a given amino acid
sequence A to, with, or against a given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % positives to, with, or against
a given amino acid sequence B) is calculated as follows: 100 times
the fraction X/Y where X is the number of amino acid residues
scoring a positive value as defined above by the sequence alignment
program ALIGN-2 or NCBI-BLAST2 in that program's alignment of A and
B, and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence A
is not equal to the length of amino acid sequence B, the %
positives of A to B will not equal the % positives of B to A.
[0288] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the PRO
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0289] An "isolated" PRO polypeptide-encoding nucleic acid or other
polypeptide-encoding nucleic acid is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the polypeptide-encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the
form or setting in which it is found in nature. Isolated
polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-encoding nucleic acid
molecule as it exists in natural cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal location different from that of
natural cells.
[0290] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0291] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0292] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-PRO monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO antibody compositions with polyepitopic
specificity, single chain anti-PRO antibodies, and fragments of
anti-PRO antibodies (see below). The term "monoclonal antibody" as
used herein refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts.
[0293] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0294] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0295] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0296] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a PRO polypeptide fused to a "tag
polypeptide". The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide to
which it is fused. The tag polypeptide preferably also is fairly
unique so that the antibody does not substantially cross-react with
other epitopes. Suitable tag polypeptides generally have at least
six amino acid residues and usually between about 8 and 50 amino
acid residues (preferably, between about 10 and 20 amino acid
residues).
[0297] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0298] "Active" or "activity" for the purposes herein refers to
form(s) of a PRO polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring PRO,
wherein "biological" activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or
naturally-occurring PRO other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring PRO and an "immunological" activity
refers to the ability to induce the production of an antibody
against an antigenic epitope possessed by a native or
naturally-occurring PRO.
[0299] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native PRO polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native PRO polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native PRO polypeptides,
peptides, antisense oligonucleotides, small organic molecules, etc.
Methods for identifying agonists or antagonists of a PRO
polypeptide may comprise contacting a PRO polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable
change in one or more biological activities normally associated
with the PRO polypeptide.
[0300] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented.
[0301] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0302] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the
mammal is human.
[0303] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0304] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0305] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng. 8(10): 1057 1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0306] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0307] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0308] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0309] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa and lambda, based on the amino acid sequences
of their constant domains.
[0310] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0311] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0312] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0313] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0314] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0315] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0316] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a PRO polypeptide or antibody thereto)
to a mammal. The components of the liposome are commonly arranged
in a bilayer formation, similar to the lipid arrangement of
biological membranes.
[0317] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons. TABLE-US-00001 TABLE 2 PRO
XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison Protein
XXXXXYYYYYYY (Length = 12 amino acids) % amino acid sequence
identity = (the number of identically matching amino acid residues
between the two polypeptide sequences as determined by ALIGN-2)
divided by (the total number of amino acid residues of the PRO
polypeptide) = 5 divided by 15 = 33.3%
[0318] TABLE-US-00002 TABLE 3 PRO XXXXXXXXXX (Length = 10 amino
acids) Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity = (the number of identically
matching amino acid residues between the two polypeptide sequences
as determined by ALIGN-2) divided by (the total number of amino
acid residues of the PRO polypeptide) = 5 divided by 10 = 50%
[0319] TABLE-US-00003 TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16
nucleotides) % nucleic acid sequence identity = (the number of
identi- cally matching nucleotides between the two nucleic acid
sequences as determined by ALIGN-2) divided by (the total number of
nucleotides of the PRO-DNA nucleic acid sequence) = 6 divided by 14
= 42.9%
[0320] TABLE-US-00004 TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identi- cally
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
[0321] A. Full-Length PRO Polypeptides
[0322] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO polypeptides. In particular, cDNAs
encoding various PRO polypeptides have been identified and
isolated, as disclosed in further detail in the Examples below. It
is noted that proteins produced in separate expression rounds may
be given different PRO numbers but the UNQ number is unique for any
given DNA and the encoded protein, and will not be changed.
However, for sake of simplicity, in the present specification the
protein encoded by the full length native nucleic acid molecules
disclosed herein as well as all further native homologues and
variants included in the foregoing definition of PRO, will be
referred to as "PRO/number", regardless of their origin or mode of
preparation.
[0323] As disclosed in the Examples below, various cDNA clones have
been deposited with the ATCC. The actual nucleotide sequences of
those clones can readily be determined by the skilled artisan by
sequencing of the deposited clone using routine methods in the art.
The predicted amino acid sequence can be determined from the
nucleotide sequence using routine skill. For the PRO polypeptides
and encoding nucleic acids described herein, Applicants have
identified what is believed to be the reading frame best
identifiable with the sequence information available at the
time.
[0324] 1. Full-length PRO1800 Polypeptides
[0325] Using the WU-BLAST2 sequence alignment computer program, it
has been found that a portion of the full-length native sequence
PRO1800 (shown in FIG. 2 and SEQ ID NO:2) has certain amino acid
sequence identity with the human Hep27 protein (HE27_HUMAN).
Accordingly, it is presently believed that PRO1800 disclosed in the
present application is a newly identified Hep27 homolog and
possesses activity typical of that protein.
[0326] 2. Full-length PRO539 Polypeptides
[0327] Using the WU-BLAST2 sequence alignment computer program, it
has been found that a portion of the full-length native sequence
PRO539 (shown in FIG. 4 and SEQ ID NO:7) has certain amino acid
sequence identity with a portion of a kinesin-related protein from
Drosophila melanogaster (AF019250.sub.--1). Accordingly, it is
presently believed that PRO539 disclosed in the present application
is a newly identified member of the Hedgehog signaling pathway
protein family and possesses activity typical of the Drosophila
Costal-2 protein.
[0328] 3. Full-length PRO982 Polypeptides
[0329] As far as is known, the DNA57700-1408 sequence encodes a
novel secreted factor designated herein as PRO982. Although, using
WU-BLAST2 sequence alignment computer programs, some sequence
identities with known proteins were revealed.
[0330] 4. Full-length PRO1434 Polypeptides
[0331] Using the WU-BLAST2 sequence alignment computer program, it
has been found that a portion of the full-length native sequence
PRO1434 (shown in FIG. 10 and SEQ ID NO:13) has certain amino acid
sequence identity with the mouse nel protein precursor (NEL_MOUSE).
Accordingly, it is presently believed that PRO1434 disclosed in the
present application is a newly identified nel homolog and may
possess activity typical of the nel protein family.
[0332] 5. Full-length PRO1863 Polypeptides
[0333] The DNA59847-25 10 clone was isolated from a human prostate
tissue library. As far as is known, the DNA59847-2510 sequence
encodes a novel factor designated herein as PRO1863; using the
WU-BLAST2 sequence alignment computer program, no significant
sequence identities to any known proteins were revealed.
[0334] 6. Full-length PRO1917 Polypeptides
[0335] Using WU-BLAST2 sequence alignment computer programs, it has
been found that amino acids 41 to 487 of PRO1917 (shown in FIG. 14
and SEQ ID NO:20) has certain amino acid sequence identity with an
inositol phosphatase designated in the Dayhoff database as "AFO
12714.sub.--1". Accordingly, it is presently believed that PRO1917
disclosed in the present application is a newly identified member
of inositol phosphatase family and may possess enzymatic activity
typical of inositol phosphatases.
[0336] 7. Full-length PRO1868 Polypeptides
[0337] Using the WU-BLAST2 sequence alignment computer program, it
has been found that a portion of the full-length native sequence
PRO1868 (shown in FIG. 16 and SEQ ID NO:28) has certain amino acid
sequence identity with the human A33 antigen protein (P_W14146).
Accordingly, it is presently believed that PRO1868 disclosed in the
present application is a newly identified A33 antigen homolog which
may possess activity and/or expression patterns typical of the A33
antigen protein. The PRO1868 polypeptide may find use in the
therapeutic treatment of inflammatory diseases as described above
and colorectal cancer.
[0338] 8. Full-length PRO3434 Polypeptides
[0339] The DNA77631-2537 clone was isolated from a human aortic
tissue library using a trapping technique that selects for
nucleotide sequences encoding secreted proteins. As far as is
known, the DNA77631-2537 sequence encodes a novel factor designated
herein as PRO3434; using the WU-BLAST2 sequence alignment computer
program, no significant sequence identities to any known proteins
were revealed.
[0340] 9. Full-length PRO1927 Polypeptides
[0341] Using WU-BLAST2 sequence alignment computer programs, it has
been found that a full-length native sequence PRO1927 (FIG. 20; SEQ
ID NO:26) has certain amino acid sequence identity with the amino
acid sequence of the protein designated "AB000628.sub.--1" in the
Dayhoff database. Accordingly, it is presently believed that
PRO1927 disclosed in the present application is a newly identified
member of the glycosyltransferase family of proteins and may
possess glycosylation activity.
[0342] B. PRO Polypeptide Variants
[0343] In addition to the full-length native sequence PRO
polypeptides described herein, it is contemplated that PRO variants
can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide changes into the PRO DNA, and/or by
synthesis of the desired PRO polypeptide. Those skilled in the art
will appreciate that amino acid changes may alter
post-translational processes of the PRO, such as changing the
number or position of glycosylation sites or altering the membrane
anchoring characteristics.
[0344] Variations in the native full-length sequence PRO or in
various domains of the PRO described herein, can be made, for
example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the PRO that results in a change in the amino acid sequence of the
PRO as compared with the native sequence PRO. Optionally the
variation is by substitution of at least one amino acid with any
other amino acid in one or more of the domains of the PRO. Guidance
in determining which amino acid residue may be inserted,
substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the PRO with
that of homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high
homology. Amino acid substitutions can be the result of replacing
one amino acid with another amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine
with a serine, i.e., conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1
to 5 amino acids. The variation allowed may be determined by
systematically making insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for
activity exhibited by the full-length or mature native
sequence.
[0345] PRO polypeptide fragments are provided herein. Such
fragments may be truncated at the N terminus or C-terminus, or may
lack internal residues, for example, when compared with a full
length native protein. Certain fragments lack amino acid residues
that are not essential for a desired biological activity of the PRO
polypeptide.
[0346] PRO fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
PRO fragments by enzymatic digestion, e.g., by treating the protein
with an enzyme known to cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with
suitable restriction enzymes and isolating the desired fragment.
Yet another suitable technique involves isolating and amplifying a
DNA fragment encoding a desired polypeptide fragment, by polymerase
chain reaction (PCR). Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers
in the PCR. Preferably, PRO polypeptide fragments share at least
one biological and/or immunological activity with the native PRO
polypeptide disclosed herein.
[0347] In particular embodiments, conservative substitutions of
interest are shown in Table 1 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are introduced and the products
screened. TABLE-US-00005 TABLE 6 Original Exemplary Preferred
Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg
(R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu
glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro;
ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;
phe; leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe
Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V)
ile; leu; met; phe; leu ala; norleucine
[0348] Substantial modifications in function or immunological
identity of the PRO polypeptide are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
[0349] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0350] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the PRO variant DNA.
[0351] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W. H. Freeman
& Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)]. If
alanine substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0352] C. Modifications of PRO
[0353] Covalent modifications of PRO are included within the scope
of this invention. One type of covalent modification includes
reacting targeted amino acid residues of a PRO polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking PRO to a water-insoluble support matrix or surface
for use in the method for purifying anti-PRO antibodies, and
vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8 octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0354] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0355] Another type of covalent modification of the PRO polypeptide
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to
mean deleting one or more carbohydrate moieties found in native
sequence PRO (either by removing the underlying glycosylation site
or by deleting the glycosylation by chemical and/or enzymatic
means), and/or adding one or more glycosylation sites that are not
present in the native sequence PRO. In addition, the phrase
includes qualitative changes in the glycosylation of the native
proteins, involving a change in the nature and proportions of the
various carbohydrate moieties present.
[0356] Addition of glycosylation sites to the PRO polypeptide may
be accomplished by altering the amino acid sequence. The alteration
may be made, for example, by the addition -of, or substitution by,
one or more serine or threonine residues to the native sequence PRO
(for O-linked glycosylation sites). The PRO amino acid sequence may
optionally be altered through changes at the DNA level,
particularly by mutating the DNA encoding the PRO polypeptide at
preselected bases such that codons are generated that will
translate into the desired amino acids.
[0357] Another means of increasing the number of carbohydrate
moieties on the PRO polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0358] Removal of carbohydrate moieties present on the PRO
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52(1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0359] Another type of covalent modification of PRO comprises
linking the PRO polypeptide to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0360] The PRO of the present invention may also be modified in a
way to form a chimeric molecule comprising PRO fused to another,
heterologous polypeptide or amino acid sequence.
[0361] In one embodiment, such a chimeric molecule comprises a
fusion of the PRO with a tag polypeptide which provides an epitope
to which an anti-tag antibody can selectively bind. The epitope tag
is generally placed at the amino- or carboxyl-terminus of the PRO.
The presence of such epitope-tagged forms of the PRO can be
detected using an antibody against the tag polypeptide. Also,
provision of the epitope tag enables the PRO to be readily purified
by affinity purification using an anti-tag antibody or another type
of affinity matrix that binds to the epitope tag. Various tag
polypeptides and their respective antibodies are well known in the
art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; an .alpha.-tubulin epitope peptide [Skinner et al., J.
Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein
peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,
87:6393-6397 (1990)].
[0362] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the PRO with an imrnunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule (also referred to as an "immunoadhesin"), such a
fusion could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a PRO polypeptide in place
of at least one variable region within an Ig molecule. In a
particularly preferred embodiment, the immunoglobulin fusion
includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3
regions of an IgG1 molecule. For the production of immunoglobulin
fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0363] D. Preparation of PRO
[0364] The description below relates primarily to production of PRO
by culturing cells transformed or transfected with a vector
containing PRO nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed to prepare PRO. For instance, the PRO sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W. H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
PRO may be chemically synthesized separately and combined using
chemical or enzymatic methods to produce the full-length PRO.
[0365] 1. Isolation of DNA Encoding PRO
[0366] DNA encoding PRO may be obtained from a cDNA library
prepared from tissue believed to possess the PRO mRNA and to
express it at a detectable level. Accordingly, human PRO DNA can be
conveniently obtained from a cDNA library prepared from human
tissue, such as described in the Examples. The PRO-encoding gene
may also be obtained from a genomic library or by known synthetic
procedures (e.g., automated nucleic acid synthesis).
[0367] Libraries can be screened with probes (such as antibodies to
the PRO or oligonucleotides of at least about 20-80 bases) designed
to identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding PRO is to use PCR methodology [Sambrook
et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 1995)].
[0368] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0369] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0370] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0371] 2. Selection and Transformation of Host Cells
[0372] Host cells are transfected or transformed with expression or
cloning vectors described herein for PRO production and cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: a
Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook
et al., supra.
[0373] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.4,399,216.
Transformations into yeast are typically carried out according to
the method of Van Solingen et al., J. Bact., 130:946 (1977) and
Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979).
However, other methods for introducing DNA into cells, such as by
nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0374] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salinonella,
e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans,
and Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1 A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W31 10 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0375] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for PRO-encoding vectors. Saccharomyces cerevisiae is a commonly
used lower eukaryotic host microorganism. Others include
Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140
[19811; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S.
Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991))
such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et
al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC
12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178),
K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den
Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and
K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070;
Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]);
Candida; Trichoderna reesia (EP 244,234); Neurospora crassa (Case
et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);
Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538
published 31 Oct. 1990); and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10
Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et
al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et
al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci.
USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.,
4:475-479 [1985]). Methylotropic yeasts are suitable herein and
include, but are not limited to, yeast capable of growth on
methanol selected from the genera consisting of Hansenula, Candida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A
list of specific species that are exemplary of this class of yeasts
may be found in C. Anthony, The Biochemistry of Methylotrophs, 269
(1982).
[0376] Suitable host cells for the expression of glycosylated PRO
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod.,23:243 251(1980)); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT
060562, ATCC CCL5 1). The selection of the appropriate host cell is
deemed to be within the skill in the art.
[0377] 3. Selection and Use of a Replicable Vector
[0378] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. Various vectors are publicly
available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0379] The PRO may be produced recombinantly not only directly, but
also as a fusion polypeptide with a heterologous polypeptide, which
may be a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the PRO-encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published 4 Apr. 1990), or the signal described in WO 90/13646
published 15 Nov. 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0380] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu.plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0381] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0382] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the PRO-encoding nucleic acid, such as DHFR or thymidine
kinase. An appropriate host cell when wild-type DHFR is employed is
the CHO cell line deficient in DHFR activity, prepared and
propagated as described by Urlaub et al., Proc. Natl. Acad. Sci.
USA, 77:4216 (1980). A suitable selection gene for use in yeast is
the trp1 gene present in the yeast plasimid YRp7 [Stinchcomb et
al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);
Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a
selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1
[Jones, Genetics, 85:12 (1977)].
[0383] Expression and cloning vectors usually contain a promoter
operably linked to the PRO-encoding nucleic acid sequence to direct
mRNA synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the .beta.-lactamase and lactose promoter systems
[Changet al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)], alkaline phosphatase, atryptophan (trp) promoter
system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and
hybrid promoters such as the tac promoter [deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding PRO.
[0384] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0385] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, met allothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0386] PRO transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0387] Transcription of a DNA encoding the PRO by higher eukaryotes
may be increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp, that act on a promoter to increase its transcription. Many
enhancer sequences are now known from mammalian genes (globin,
elastase, albumin, .alpha.-fetoprotein, and insulin). Typically,
however, one will use an enhancer from a eukaryotic cell virus.
Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or 3' to the PRO coding sequence, but
is preferably located at a site 5' from the promoter.
[0388] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding PRO.
[0389] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of PRO in recombinant vertebrate cell
culture are described in Gething et al., Nature, 293:620-625
(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP
117,058.
[0390] 4. Detecting Gene Amplification/Expression
[0391] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0392] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence PRO polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to PRO DNA and encoding a specific antibody
epitope.
[0393] 5. Purification of Polypeptide
[0394] Forms of PRO may be recovered from culture medium or from
host cell lysates. If membrane-bound, it can be released from the
membrane using a suitable detergent solution (e.g. Triton-X 100) or
by enzymatic cleavage. Cells employed in expression of PRO can be
disrupted by various physical or chemical means, such as
freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents.
[0395] It may be desired to purify PRO from recombinant cell
proteins or polypeptides. The following procedures are exemplary of
suitable purification procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and met al
chelating columns to bind epitope-tagged forms of the PRO. Various
methods of protein purification may be employed and such methods
are known in the art and described for example in Deutscher,
Methods in Enzymology, 182 (1990); Scopes, Protein Purification:
Principles and Practice, Springer-Verlag, New York (1982). The
purification step(s) selected will depend, for example, on the
nature of the production process used and the particular PRO
produced.
[0396] E. Uses for PRO
[0397] Nucleotide sequences (or their complement) encoding PRO have
various applications in the art of molecular biology, including
uses as hybridization probes, in chromosome and gene mapping and in
the generation of anti-sense RNA and DNA. PRO nucleic acid will
also be useful for the preparation of PRO polypeptides by the
recombinant techniques described herein.
[0398] The full-length native sequence PRO gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length PRO cDNA or to isolate still other cDNAs
(for instance, those encoding naturally-occurring variants of PRO
or PRO from other species) which have a desired sequence identity
to the native PRO sequence disclosed herein. Optionally, the length
of the probes will be about 20 to about 50 bases. The hybridization
probes may be derived from at least partially novel regions of the
full length native nucleotide sequence wherein those regions may be
determined without undue experimentation or from genomic sequences
including promoters, enhancer elements and introns of native
sequence PRO. By way of example, a screening method will comprise
isolating the coding region of the PRO gene using the known DNA
sequence to synthesize a selected probe of about 40 bases.
Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the PRO gene of the present
invention can be used to screen libraries of human cDNA, genomic
DNA or mRNA to determine which members of such libraries the probe
hybridizes to. Hybridization techniques are described in further
detail in the Examples below.
[0399] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0400] Other useful fragments of the PRO nucleic acids include
antisense or sense oligonucleotides comprising a singe-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target PRO mRNA (sense) or PRO DNA (antisense) sequences. Antisense
or sense oligonucleotides, according to the present invention,
comprise a fragment of the coding region of PRO DNA. Such a
fragment generally comprises at least about 14 nucleotides,
preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense oligonucleotide, based upon a cDNA sequence
encoding a given protein is described in, for example, Stein and
Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.
(BioTechniques 6:958, 1988).
[0401] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. The antisense oligonucleotides thus may be used to block
expression of PRO proteins. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0402] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10048, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or met al complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0403] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0404] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0405] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0406] Antisense or sense RNA or DNA molecules are generally at
least about 5 bases in length, about 10 bases in length, about 15
bases in length, about 20 bases in length, about 25 bases in
length, about 30 bases in length, about 35 bases in length, about
40 bases in length, about 45 bases in length, about 50 bases in
length, about 55 bases in length, about 60 bases in length, about
65 bases in length, about 70 bases in length, about 75 bases in
length, about 80 bases in length, about 85 bases in length, about
90 bases in length, about 95 bases in length, about 100 bases in
length, or more.
[0407] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO coding sequences.
[0408] Nucleotide sequences encoding a PRO can also be used to
construct hybridization probes for mapping the gene which encodes
that PRO and for the genetic analysis of individuals with genetic
disorders. The nucleotide sequences provided herein may be mapped
to a chromosome and specific regions of a chromosome using known
techniques, such as in situ hybridization, linkage analysis against
known chromosomal markers, and hybridization screening with
libraries.
[0409] When the coding sequences for PRO encode a protein which
binds to another protein (example, where the PRO is a receptor),
the PRO can be used in assays to identify the other proteins or
molecules involved in the binding interaction. By such methods,
inhibitors of the receptor/ligand binding interaction can be
identified. Proteins involved in such binding interactions can also
be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction. Also, the receptor PRO can be
used to isolate correlative ligand(s). Screening assays can be
designed to find lead compounds that mimic the biological activity
of a native PRO or a receptor for PRO. Such screening assays will
include assays amenable to high-throughput screening of chemical
libraries, making them particularly suitable for identifying small
molecule drug candidates. Small molecules contemplated include
synthetic organic or inorganic compounds. The assays can be
performed in a variety of formats, including protein-protein
binding assays, biochemical screening assays, immunoassays and cell
based assays, which are well characterized in the art.
[0410] Nucleic acids which encode PRO or its modified forms can
also be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. A transgenic animal (e.g., a
mouse or rat) is an animal having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of
the animal at a prenatal, e.g., an embryonic stage. A transgene is
a DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, cDNA encoding PRO
can be used to clone genomic DNA encoding PRO in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
PRO. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for PRO
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding PRO introduced
into the germ line of the animal at an embryonic stage can be used
to examine the effect of increased expression of DNA encoding PRO.
Such animals can be used as tester animals for reagents thought to
confer protection from, for example, pathological conditions
associated with its overexpression. In accordance with this facet
of the invention, an animal is treated with the reagent and a
reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0411] Alternatively, non-human homologues of PRO can be used to
construct a PRO "knock out" animal which has a defective or altered
gene encoding PRO as a result of homologous recombination between
the endogenous gene encoding PRO and altered genomic DNA encoding
PRO introduced into an embryonic stem cell of the animal. For
example, cDNA encoding PRO can be used to clone genomic DNA
encoding PRO in accordance with established techniques. A portion
of the genomic DNA encoding PRO can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for
a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected [see
e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined-DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of the PRO polypeptide.
[0412] Nucleic acid encoding the PRO polypeptides may also be used
in gene therapy. In gene therapy applications, genes are introduced
into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0413] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262,4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87,3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0414] The PRO polypeptides described herein may also be employed
as molecular weight markers for protein electrophoresis purposes
and the isolated nucleic acid sequences may be used for
recombinantly expressing those markers.
[0415] The nucleic acid molecules encoding the PRO polypeptides or
fragments thereof described herein are useful for chromosome
identification. In this regard, there exists an ongoing need to
identify new chromosome markers, since relatively few chromosome
marking reagents, based upon actual sequence data are presently
available. Each PRO nucleic acid molecule of the present invention
can be used as a chromosome marker.
[0416] The PRO polypeptides and nucleic acid molecules of the
present invention may also be used for tissue typing, wherein the
PRO polypeptides of the present invention may be differentially
expressed in one tissue as compared to another. PRO nucleic acid
molecules will find use for generating probes for PCR, Northern
analysis, Southern analysis and Western analysis.
[0417] The PRO polypeptides described herein may also be employed
as therapeutic agents. The PRO polypeptides of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the PRO product
hereof is combined in admixture with a pharmaceutically acceptable
carrier vehicle. Therapeutic formulations are prepared for storage
by mixing the active ingredient having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate and other
organic acids; antioxidants including ascorbic acid; low molecular
weight (less than about 10 residues) polypeptides; proteins, such
as serum albumin, gelatin or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone, amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN.TM., PLURONICS.TM. or
PEG.
[0418] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to or following lyophilization
and reconstitution.
[0419] Therapeutic compositions herein 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.
[0420] The route of administration is in accord with known methods,
e.g. injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by sustained
release systems.
[0421] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0422] When in vivo administration of a PRO polypeptide or agonist
or antagonist thereof is employed, normal dosage amounts may vary
from about 10 ng/kg to up to 100 mg/kg of mammal body weight or
more per day, preferably about 1 .mu.g/kg/day to 10 mg/kg/day,
depending upon the route of administration. Guidance as to
particular dosages and methods of delivery is provided in the
literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344;
or 5,225,212. It is anticipated that different formulations will be
effective for different treatment compounds and different
disorders, that administration targeting one organ or tissue, for
example, may necessitate delivery in a manner different from that
to another organ or tissue.
[0423] Where sustained-release administration of a PRO polypeptide
is desired in a formulation with release characteristics suitable
for the treatment of any disease or disorder requiring
administration of the PRO polypeptide, microencapsulation of the
PRO polypeptide is contemplated. Microencapsulation of recombinant
proteins for sustained release has been successfully performed with
human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2,
and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda,
Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology,
8:755-758 (1990); Cleland, "Design and Production of Single
Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design: The Subunit and Adjuvant Approach,
Powell and Newman, eds, (Plenum Press: New York, 1995), pp.
439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No.
5,654,010.
[0424] The sustained-release formulations of these proteins were
developed using poly-lactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. Lewis, "Controlled release of
bioactive agents from lactide/glycolide polymer," in: M. Chasin and
R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems
(Marcel Dekker: New York, 1990), pp. 1-41.
[0425] This invention encompasses methods of screening compounds to
identify those that mimic the PRO polypeptide (agonists) or prevent
the effect of the PRO polypeptide (antagonists). Screening assays
for antagonist drug candidates are designed to identify compounds
that bind or complex with the PRO polypeptides encoded by the genes
identified herein, or otherwise interfere with the interaction of
the encoded polypeptides with other cellular proteins. Such
screening assays will include assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable
for identifying small molecule drug candidates.
[0426] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0427] All assays for antagonists are common in that they call for
contacting the drug candidate with a PRO polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0428] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the PRO polypeptide encoded by the gene
identified herein or the drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by
coating the solid surface with a solution of the PRO polypeptide
and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for the PRO polypeptide to be
immobilized can be used to anchor it to a solid surface. The assay
is performed by adding the non-immobilized component, which may be
labeled by a detectable label, to the immobilized component, e.g.,
the coated surface containing the anchored component. When the
reaction is complete, the non-reacted components are removed, e.g.,
by washing, and complexes anchored on the solid surface are
detected. When the originally non-immobilized component carries a
detectable label, the detection of label immobilized on the surface
indicates that complexing occurred. Where the originally
non-immobilized component does not carry a label, complexing can be
detected, for example, by using a labeled antibody specifically
binding the immobilized complex.
[0429] If the candidate compound interacts with but does not bind
to a particular PRO polypeptide encoded by a gene identified
herein, its interaction with that polypeptide can be assayed by
methods well known for detecting protein-protein interactions. Such
assays include traditional approaches, such as, e.g.,
cross-linking, co-immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers (Fields and Song, Nature
(London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans,
Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, consist of two
physically discrete modular domains, one acting as the DNA-binding
domain, the other one functioning as the transcription-activation
domain. The yeast expression system described in the foregoing
publications (generally referred to as the "two-hybrid system")
takes advantage of this property, and employs two hybrid proteins,
one in which the target protein is fused to the DNA-binding domain
of GAL4, and another, in which candidate activating proteins are
fused to the activation domain. The expression of a GAL1-lacZ
reporter gene under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0430] Compounds that interfere with the interaction of a gene
encoding a PRO polypeptide identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the product of the gene and
the intra- or extracellular component under conditions and for a
time allowing for the interaction and binding of the two products.
To test the ability of a candidate compound to inhibit binding, the
reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex
formation) between the test compound and the intra- or
extracellular component present in the mixture is monitored as
described hereinabove. The formation of a complex in the control
reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the
interaction of the test compound and its reaction partner.
[0431] To assay for antagonists, the PRO polypeptide may be added
to a cell along with the compound to be screened for a particular
activity and the ability of the compound to inhibit the activity of
interest in the presence of the PRO polypeptide indicates that the
compound is an antagonist to the PRO polypeptide. Alternatively,
antagonists may be detected by combining the PRO polypeptide and a
potential antagonist with membrane-bound PRO polypeptide receptors
or recombinant receptors under appropriate conditions for a
competitive inhibition assay. The PRO polypeptide can be labeled,
such as by radioactivity, such that the number of PRO polypeptide
molecules bound to the receptor can be used to determine the
effectiveness of the potential antagonist. The gene encoding the
receptor 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). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the PRO
polypeptide 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 PRO polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled PRO polypeptide. The
PRO polypeptide 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 autoradiographic analysis. Positive pools are
identified and sub-pools are prepared and re-transfected using an
interactive sub-pooling and re-screening process, eventually
yielding a single clone that encodes the putative receptor.
[0432] As an alternative approach for receptor identification,
labeled PRO polypeptide can be photoaffinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0433] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled PRO polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0434] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
PRO polypeptide, and, in particular, antibodies including, without
limitation, poly- and monoclonal antibodies and antibody fragments,
single-chain antibodies, anti-idiotypic antibodies, and chimeric or
humanized versions of such antibodies or fragments, as well as
human antibodies and antibody fragments. Alternatively, a potential
antagonist may be a closely related protein, for example, a mutated
form of the PRO polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of
the PRO polypeptide.
[0435] Another potential PRO polypeptide antagonist is an antisense
RNA or DNA construct prepared using antisense technology, where,
e.g., an antisense RNA or DNA molecule acts to block directly the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense technology can be used to control
gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are based on binding of a polynucleotide
to DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes the mature PRO polypeptides
herein, is used to design an antisense RNA oligonucleotide of from
about 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription (triple helix--see Lee et al., Nucl. Acids Res.,
6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et
al., Science, 251:1360 (1991)), thereby preventing transcription
and the production of the PRO polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the PRO polypeptide
(antisense--Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 1988). The oligonucleotides described above can also be
delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the PRO polypeptide.
When antisense DNA is used, oligodeoxyribonucleotides derived from
the translation-initiation site, e.g., between about -10 and +10
positions of the target gene nucleotide sequence, are
preferred.
[0436] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the PRO polypeptide, thereby
blocking the normal biological activity of the PRO polypeptide.
Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides,
and synthetic non-peptidyl organic or inorganic compounds.
[0437] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0438] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0439] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0440] F. Anti-PRO Antibodies
[0441] The present invention further provides anti-PRO antibodies.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies.
[0442] 1. Polyclonal Antibodies
[0443] The anti-PRO antibodies may comprise polyclonal antibodies.
Methods of preparing polyclonal antibodies are known to the skilled
artisan. Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the
PRO polypeptide or a fusion protein thereof. It may be useful to
conjugate the immunizing agent to a protein known to be immunogenic
in the mammal being immunized. Examples of such immunogenic
proteins include but are not limited to keyhole limpet hemocyanin,
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
Examples of adjuvants which may be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetic trehalose dicorynomycolate). The immunization protocol
may be selected by one skilled in the art without undue
experimentation.
[0444] 2. Monoclonal Antibodies
[0445] The anti-PRO antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunizedin vitro.
[0446] The immunizing agent will typically include the PRO
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0447] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0448] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against PRO. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0449] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0450] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0451] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra] or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0452] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0453] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0454] 3. Human and Humanized Antibodies
[0455] The anti-PRO antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab).sub.2 or other antigen-binding subsequences
of antibodies) which contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525(1986);
Riechmannet al, Nature,332:323-329(1988); and Presta, Curr. Op.
Struct. Biol., 2:593-596(1992)].
[0456] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0457] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0458] 4. Bispecific Antibodies
[0459] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the PRO, the other one is for any other
antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
[0460] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0461] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0462] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0463] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab').sub.2 fragments. These fragments are
reduced in the presence of the dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies produced can be used as agents
for the selective immobilization of enzymes.
[0464] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175:217-225 (1992) describe the production of a fully
humanized bispecific antibody F(ab').sub.2 molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0465] Various technique for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared. Tutt et al., J.
Immunol. 147:60 (1991).
[0466] Exemplary bispecific antibodies may bind to two different
epitopes on a given PRO polypeptide herein. Alternatively, an
anti-PRO polypeptide arm may be combined with an arm which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular PRO polypeptide. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a particular PRO polypeptide. These antibodies
possess a PRO-binding arm and an arm which binds a cytotoxic agent
or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the PRO polypeptide
and further binds tissue factor (TF).
[0467] 5. Heteroconjugate Antibodies
[0468] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No.4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0469] 6. Effector Function Engineering
[0470] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0471] 7. Immunoconjugates
[0472] The inventional also pertains to immunoconjugates comprising
an antibody conjugated to acytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0473] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudontonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0474] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0475] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
[0476] 8. Immunoliposomes
[0477] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwanget al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0478] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0479] 9. Pharmaceutical Compositions of Antibodies
[0480] Antibodies specifically binding a PRO polypeptide identified
herein, as well as other molecules identified by the screening
assays disclosed hereinbefore, can be administered for the
treatment of various disorders in the form of pharmaceutical
compositions.
[0481] If the PRO polypeptide is intracellular and whole antibodies
are used as inhibitors, internalizing antibodies are preferred.
However, lipofections or liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,
90: 7889-7893 (1993). The formulation herein may also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Alternatively, or in addition,
the composition may comprise an agent that enhances its function,
such as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0482] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
supra.
[0483] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0484] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S-S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0485] G. Uses for anti-PRO Antibodies
[0486] The anti-PRO antibodies of the invention have various
utilities. For example, anti-PRO antibodies may be used in
diagnostic assays for PRO, e.g., detecting its expression in
specific cells, tissues, or serum. Various diagnostic assay
techniques known in the art may be used, such as competitive
binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in either heterogeneous or
homogeneous phases [Zola, Monoclonal Antibodies: A Manual of
Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies
used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should be capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, .sup.35S, or .sup.125I, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for conjugating the antibody to the
detectable moiety may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982).
[0487] Anti-PRO antibodies also are useful for the affinity
purification of PRO from recombinant cell culture or natural
sources. In this process, the antibodies against PRO are
immobilized on a suitable support, such a Sephadex resin or filter
paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the PRO to be
purified, and thereafter the support is washed with a suitable
solvent that will remove substantially all the material in the
sample except the PRO, which is bound to the immobilized antibody.
Finally, the support is washed with another suitable solvent that
will release the PRO from the antibody.
[0488] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0489] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0490] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Extracellular Domain Homology Screening to Identify Novel
Polypeptides and cDNA Encoding Therefor
[0491] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included public databases (e.g.,
Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST-2 (Altschul et
al., Methods in Enzymology 266:460-480 (1996)) as a comparison of
the ECD protein sequences to a 6 frame translation of the EST
sequences. Those comparisons with a BLAST score of 70 (or in some
cases 90) or greater that did not encode known proteins were
clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.).
[0492] Using this extracellular domain homology screen, consensus
DNA sequences were assembled relative to the other identified EST
sequences using phrap. In addition, the consensus DNA sequences
obtained were often (but not always) extended using repeated cycles
of BLAST or BLAST-2 and phrap to extend the consensus sequence as
far as possible using the sources of EST sequences discussed
above.
[0493] Based upon the consensus sequences obtained as described
above, oligonucleotides were then synthesized and used to identify
by PCR a cDNA library that contained the sequence of interest and
for use as probes to isolate a clone of the full-length coding
sequence for a PRO polypeptide. Forward and reverse PCR primers
generally range from 20 to 30 nucleotides and are often designed to
give a PCR product of about 100-1000 bp in length. The probe
sequences are typically 40-55 bp in length. In some cases,
additional oligonucleotides are synthesized when the consensus
sequence is greater than about 1-1.5 kbp. In order to screen
several libraries for a full-length clone, DNA from the libraries
was screened by PCR amplification, as per Ausubel et al., Current
Protocols in Molecular Biology, with the PCR primer pair. A
positive library was then used to isolate clones encoding the gene
of interest using the probe oligonucleotide and one of the primer
pairs.
[0494] The cDNA libraries used to isolate the cDNA clones were
constructed by standard methods using commercially available
reagents such as those from Invitrogen, San Diego, Calif. The cDNA
was primed with oligo dT containing a NotI site, linked with blunt
to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
Example 2
Isolation of cDNA Clones by Amylase Screening
[0495] 1. Preparation of oligo dT primed cDNA library
[0496] mRNA was isolated from a human tissue of interest using
reagents and protocols from Invitrogen, San Diego, Calif. (Fast
Track 2). This RNA was used to generate an oligo dT primed cDNA
library in the vector pRK5D using reagents and protocols from Life
Technologies, Gaithersburg, Md. (Super Script Plasmid System). In
this procedure, the double stranded cDNA was sized to greater than
1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotI
cleaved vector. pRK5D is a cloning vector that has an sp6
transcription initiation site followed by an SfiI restriction
enzyme site preceding the XhoI/NotI cDNA cloning sites.
[0497] 2. Preparation of random primed cDNA library
[0498] A secondary cDNA library was generated in order to
preferentially represent the 5' ends of the primary cDNA clones.
Sp6 RNA was generated from the primary library (described above),
and this RNA was used to generate a random primed cDNA library in
the vector pSST-AMY.0 using reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In
this procedure the double stranded cDNA was sized to 500-1000 bp,
linkered with blunt to NotI adaptors, cleaved with SfiI, and cloned
into SfiI/NotI cleaved vector. pSST-AMY.0 is a cloning vector that
has a yeast alcohol dehydrogenase promoter preceding the cDNA
cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal) followed by the yeast alcohol
dehydrogenase terminator, after the cloning sites. Thus, cDNAs
cloned into -this vector that are fused in frame with amylase
sequence will lead to the secretion of amylase from appropriately
transfected yeast colonies.
[0499] 3. Transformation and Detection
[0500] DNA from the library described in paragraph 2 above was
chilled on ice to which was added electrocompetent DHIOB bacteria
(Life Technologies, 20 ml). The bacteria and vector mixture was
then electroporated as recommended by the manufacturer.
Subsequently, SOC media (Life Technologies, 1 ml) was added and the
mixture was incubated at 37.degree. C. for 30 minutes. The
transformants were then plated onto 20 standard 150 mm LB plates
containing ampicillin and incubated for 16 hours (37.degree. C.).
Positive colonies were scraped off the plates and the DNA was
isolated from the bacterial pellet using standard protocols, e.g.
CsCl-gradient. The purified DNA was then carried on to the yeast
protocols below.
[0501] The yeast methods were divided into three categories: (1)
Transformation of yeast with the plasmid/cDNA combined vector; (2)
Detection and isolation of yeast clones secreting amylase; and (3)
PCR amplification of the insert directly from the yeast colony and
purification of the DNA for sequencing and further analysis.
[0502] The yeast strain used was HD56-5A (ATCC-90785). This strain
has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112,
his3-11, his3-15, MAL.sup.+, SUC.sup.+, GAL.sup.+. Preferably,
yeast mutants can be employed that have deficient
post-translational pathways. Such mutants may have translocation
deficient alleles in sec71, sec72, sec62, with truncated sec71
being most preferred. Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the
normal operation of these genes, other proteins implicated in this
post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p,
TDJlp or SSA1p-4p) or the complex formation of these proteins may
also be preferably employed in combination with the
amylase-expressing yeast.
[0503] Transformation was performed based on the protocol outlined
by Gietz et al., Nucl. Acid. Res., 20: 1425 (1992). Transformed
cells were then inoculated from agar into YEPD complex media broth
(100 ml) and grown overnight at 30.degree. C. The YEPD broth was
prepared as described in Kaiser et al., Methods in Yeast Genetics,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 207 (1994).
The overnight culture was then diluted to about 2.times.10.sup.6
cells/ml (approx. OD.sub.600=0.1) into fresh YEPD broth (500 ml)
and regrown to 1.times.10.sup.7 cells/ml (approx.
OD.sub.600=0.4-0.5).
[0504] The cells were then harvested and prepared for
transformation by transfer into GS3 rotor bottles in a Sorval GS3
rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water, and centrifuged again in 50 ml
falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The
supernatant was discarded and the cells were subsequently washed
with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM
Li.sub.2OOCCH.sub.3), and resuspended into LiAc/TE (2.5 ml).
[0505] Transformation took place by mixing the prepared cells (100
.mu.l) with freshly denatured single stranded salmon testes DNA
(Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 .mu.g,
vol.<10 .mu.l) in microfuge tubes. The mixture was mixed briefly
by vortexing, then 40% PEG/TE (600 .mu.l, 40% polyethylene
glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li.sub.2OOCCH.sub.3,
pH 7.5) was added. This mixture was gently mixed and incubated at
30.degree. C. while agitating for 30 minutes. The cells were then
heat shocked at 42.degree. C. for 15 minutes, and the reaction
vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds,
decanted and resuspended into TE (500 .mu.l, 10 mM Tris-HCl, 1 mM
EDTA pH 7.5) followed by recentrifugation. The cells were then
diluted into TE (1 ml) and aliquots (200 .mu.l) were spread onto
the selective media previously prepared in 150 mm growth plates
(VWR).
[0506] Alternatively, instead of multiple small reactions, the
transformation was performed using a single, large scale reaction,
wherein reagent amounts were scaled up accordingly.
[0507] The selective media used was a synthetic complete dextrose
agar lacking uracil (SCD-Ura) prepared as described in Kaiser et
al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at
30.degree. C. for 2-3 days.
[0508] The detection of colonies secreting amylase was performed by
including red starch in the selective growth media. Starch was
coupled to the red dye (Reactive Red-120, Sigma) as per the
procedure described by Biely et al., Anal. Biochem., 172:176-179
(1988). The coupled starch was incorporated into the SCD-Uraagar
plates at a final concentration of 0.15% (w/v), and was buffered
with potassium phosphate to a pH of 7.0 (50-100 mM final
concentration).
[0509] The positive colonies were picked and streaked across fresh
selective media (onto 150 mm plates) in order to obtain well
isolated and identifiable single colonies. Well isolated single
colonies positive for amylase secretion were detected by direct
incorporation of red starch into buffered SCD-Ura agar. Positive
colonies were determined by their ability to break down starch
resulting in a clear halo around the positive colony visualized
directly.
[0510] 4. Isolation of DNA by PCR Amplification
[0511] When a positive colony was isolated, a portion of it was
picked by a toothpick and diluted into sterile water (30 .mu.l) in
a 96 well plate. At this time, the positive colonies were either
frozen and stored for subsequent analysis or immediately amplified.
An aliquot of cells (5 .mu.l) was used as a template for the PCR
reaction in a 25 .mu.l volume containing: 0.5 .mu.l Klentaq
(Clontech, Palo Alto, Calif.); 4.0 .mu.l 10 mM dNTP's (Perkin
Elmer-Cetus); 2.5 .mu.l Kentaq buffer (Clontech); 0.25 .mu.l
forward oligo 1; 0.25 .mu.l reverse oligo 2; 12.5 .mu.l distilled
water. The sequence of the forward oligonucleotide 1 was:
[0512] 5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID
NO:25) The sequence of reverse oligonucleotide 2 was:
[0513] 5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO:
26)
[0514] PCR was then performed as follows: TABLE-US-00006 a.
Denature 92.degree. C., 5 minutes b. 3 cycles of: Denature
92.degree. C., 30 seconds Anneal 59.degree. C., 30 seconds Extend
72.degree. C., 60 seconds c. 3 cycles of: Denature 92.degree. C.,
30 seconds Anneal 57.degree. C., 30 seconds Extend 72.degree. C.,
60 seconds d. 25 cycles of: Denature 92.degree. C., 30 seconds
Anneal 55.degree. C., 30 seconds Extend 72.degree. C., 60 seconds
e. Hold 4.degree. C.
[0515] The underlined regions of the oligonucleotides annealed to
the ADH promoter region and the amylase region, respectively, and
amplified a 307 bp region from vector pSST-AMY.0 when no insert was
present. Typically, the first 18 nucleotides of the 5' end of these
oligonucleotides contained annealing sites for the sequencing
primers. Thus, the total product of the PCR reaction from an empty
vector was 343 bp. However, signal sequence-fused cDNA resulted in
considerably longer nucleotide sequences.
[0516] Following the PCR, an aliquot of the reaction (5 .mu.l) was
examined by agarose gel electrophoresis in a 1% agarose gel using a
Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et
al., supra. Clones resulting in a single strong PCR product larger
than 400 bp were further analyzed by DNA sequencing after
purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, Calif.).
Example 3
Isolation of cDNA Clones Using Signal Algorithm Analysis
[0517] Various polypeptide-encoding nucleic acid sequences were
identified by applying a proprietary signal sequence finding
algorithm developed by Genentech, Inc. (South San Francisco,
Calif.) upon ESTs as well as clustered and assembled EST fragments
from public (e.g., GenBank) and/or private (LIFESEQ.RTM., Incyte
Pharmaceuticals, Inc., Palo Alto, Calif.) databases. The signal
sequence algorithm computes a secretion signal score based on the
character of the DNA nucleotides surrounding the first and
optionally the second methionine codon(s) (ATG) at the 5'-end of
the sequence or sequence fragment under consideration. The
nucleotides following the first ATG must code for at least 35
unambiguous amino acids without any stop codons. If the first ATG
has the required amino acids, the second is not examined. If
neither meets the requirement, the candidate sequence is not
scored. In order to determine whether the EST sequence contains an
authentic signal sequence, the DNA and corresponding amino acid
sequences surrounding the ATG codon are scored using a set of seven
sensors (evaluation parameters) known to be associated with
secretion signals. Use of this algorithm resulted in the
identification of numerous polypeptide-encoding nucleic acid
sequences.
Example 4
Isolation of cDNA clones Encoding Human PRO1800
[0518] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30934. Based on the
DNA30934 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO1800.
[0519] PCR primers (forward and reverse) were synthesized:
TABLE-US-00007 (SEQ ID NO:3) forward PCR primer (30934.f1)
5'-GCATAATGGATGTCACTGAGG-3' (SEQ ID NO:4) reverse PCR primer
(30934.r1) 5'-AGAACAATCCTGCTGAAAGCTAG-3'
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30934 sequence which had the
following nucleotide sequence hybridization probe (30934.p1)
5'-GAAACGAGGAGGCGGCTCAGTGGTGATCGTGTCTTCCATAGCAGCC-3' (SEQ ID
NO:5)
[0520] RNA for construction of the cDNA libraries was isolated from
human fet al liver tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO1800
(designated herein as DNA35672-2508 [FIG. 1, SEQ ID NO: 1]; and the
derived protein sequence for PRO1800.
[0521] The entire nucleotide sequence of DNA35672-2508 is shown in
FIG. 1 (SEQ ID NO: 1). Clone DNA35672-2508 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 36-38 and ending at the stop codon at
nucleotide positions 870-872 (FIG. 1). The predicted polypeptide
precursor is 278 amino acids long (FIG. 2). The full-length PRO1800
protein shown in FIG. 2 has an estimated molecular weight of about
29,537 daltons and a pI of about 8.97. Analysis of the full-length
PRO1800 sequence shown in FIG. 2 (SEQ ID NO:2) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 15, a potential N-glycosylation site from about
amino acid 183 to about amino acid 186, potential N-myristolation
sites from about amino acid 43 to about amino acid 48, from about
amino acid 80 to about amino acid 85, from about amino acid 191 to
about amino acid 196, from about amino acid 213 to about amino acid
218 and from about amino acid 272 to about amino acid 277 and a
microbodies C-terminal targeting signal from about amino acid 276
to about amino acid 278. Clone DNA35672-2508 has been deposited
with ATCC on Dec. 15, 1998 and is assigned ATCC deposit no.
203538.
[0522] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 2 (SEQ ID NO:2), evidenced
significant homology between the PRO1800 amino acid sequence and
the following Dayhoff sequences: HE27_HUMAN, CELF36H9.sub.--1,
CEF54F3.sub.--3, A69621, AP000007.sub.--227, UCPA_ECOLI, F69868,
Y4LA_RHISN, DHK2_STRVN and DHG1_BACME.
Example 5
Isolation of cDNA Clones Encoding Human PRO539
[0523] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1. This consensus
sequence is herein designated DNA41882. Based on the DNA41882
consensus sequence shown, oligonucleotides were synthesized: 1) to
identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO539.
[0524] RNA for construction of the cDNA libraries was isolated from
human fet al kidney tissue. DNA sequencing of the clones isolated
as described above gave the full-length DNA sequence for PRO539
(designated herein as DNA47465-1561 [FIG. 3, SEQ ID NO:6]; and the
derived protein sequence for PRO539.
[0525] The entire nucleotide sequence of DNA47465-1561 is shown in
FIG. 3 (SEQ ID NO:6). Clone DNA47465-1561 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 186-188 and ending at the stop codon at
nucleotide positions 2676-2678 (FIG. 3). The predicted polypeptide
precursor is 830 amino acids long (FIG. 4). The full-length PRO539
protein shown in FIG. 4 has an estimated molecular weight of about
95,029 daltons and a pI of about 8.26. Analysis of the full-length
PRO539 sequence shown in FIG. 4 (SEQ ID NO:7) evidences the
presence of the following: leucine zipper pattern sequences from
about amino acid 557 to about amino acid 578 and from about amino
acid 794 to about amino acid 815, potential N-glycosylation sites
from about amino acid 133 to about amino acid 136 and from about
amino acid 383 to about amino acid 386 and a kinesin-related
protein Kif-4 coiled coil domain from about amino acid 231 to about
amino acid 672. Clone DNA47465-1561 has been deposited with ATCC on
Feb. 9, 1999 and is assigned ATCC deposit no. 203661.
[0526] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 4 (SEQ ID NO:7), evidenced
homology between the PRO539 amino acid sequence and the following
Dayhoff sequences: AF019250.sub.--1, KIF4_MOUSE, TRHY_HUMAN,
A56514, G02520, MYSP_HUMAN, AF041382.sub.--1, A45592,
HS125H2.sub.--1 and HS68O2.sub.--2.
Example 6
Isolation of cDNA Clones Encoding Human PRO982
[0527] Use of the signal sequence algorithm described in Example 3
above allowed identification of a single Incyte EST sequence
designated herein as Incyte EST cluster sequence no. 43715. This
EST sequence was compared to a variety of EST databases which
included public EST databases (e.g., GenBank) and a proprietary EST
DNA database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto,
Calif.) to identify existing homologies. The homology search was
performed using the computer program BLAST or BLAST2 (Altshul et
al., Methods in Enzymology 266:460-480 (1996)). Those comparisons
resulting in a BLAST score of 70 (or in some cases 90) or greater
that did not encode known proteins were clustered and assembled
into a consensus DNA sequence with the program "phrap" (Phil Green,
University of Washington, Seattle, Wash.). The consensus sequence
obtained therefrom is designated DNA56095.
[0528] In light of an observed sequence homology between DNA56095
and Merck EST no. AA024389, Merck EST clone AA024389 was obtained
and sequenced. The sequence, designated DNA57700-1408 (SEQ ID
NO:8), is shown in FIG. 5. It is the full-length DNA sequence for
PRO982.
[0529] The full length clone shown in FIG. 5 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 26-28 and ending at the stop codon found at
nucleotide positions 401-403 (SEQ ID NO:8). The predicted
polypeptide precursor is 125 amino acids long, has a calculated
molecular weight of approximately 14,198 daltons and an estimated
pI of approximately 9.01. Analysis of the full-length PRO982
sequence shown in FIG. 6 (SEQ ID NO:9) evidences the presence of a
signal peptide from about amino acid 1 to about amino acid 21 and
potential anaphylatoxin domain from about amino acid 50 to about
amino acid 59. An analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced homology between the PRO982 amino acid
sequence and the following Dayhoff sequences: RNTMDCV.sub.--1;
A48151; WAP_RAT; S24596; A53640; MT4_HUMAN; U93486.sub.--1;
SYNBILGFG.sub.--1; P_R49917; and P_R41880. Clone DNA57700-1408 was
deposited with the ATCC on Jan. 12, 1999 and is assigned ATCC
deposit no. 203583.
Example 7
Isolation of cDNA Clones Encoding Human PRO1434
[0530] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA54187. Based on the
DNA54187 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO1434.
[0531] PCR primers (forward and reverse) were synthesized:
TABLE-US-00008 (SEQ ID NO:12) forward PCR primer
5'-GAGGTGTCGCTGTGAAGCCAACGG-3' (SEQ ID NO:13) reverse PCR primer
5'-CGCTCGATTCTCCATGTGCCTTCC-3'
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA54187 sequence which had the
following nucleotide sequence hybridization probe
5'-GACGGAGTGTGTGGACCCTGTGTACGAGCCTGATCAGTGCTGTCC-3' (SEQ ID NO:
14)
[0532] RNA for construction of the cDNA libraries was isolated from
human retina tissue (LIB94). DNA sequencing of the clones isolated
as described above gave the full-length DNA sequence for PRO1434
(designated herein as DNA68818-2536 [FIG. 7, SEQ ID NO:10]; and the
derived protein sequence for PRO1434.
[0533] The entire nucleotide sequence of DNA68818-2536 is shown in
FIG. 7 (SEQ ID NO: 10). Clone DNA68818-2536 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 581-583 and ending at the stop codon at
nucleotide positions 1556-1558 (FIG. 7). The predicted polypeptide
precursor is 325 amino acids long (FIG. 8). The full-length PRO1434
protein shown in FIG. 8 has an estimated molecular weight of about
35,296 daltons and a pI of about 5.37. Analysis of the full-length
PRO1434 sequence shown in FIG. 8 (SEQ ID NO: 11) evidences the
presence of a variety of important protein domains as shown in FIG.
8. Clone DNA68818-2536 has been deposited with ATCC on Feb. 9, 1999
and is assigned ATCC deposit no. 203657.
[0534] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 8 (SEQ ID NO: 11), evidenced
significant homology between the PRO1434 amino acid sequence and
the following Dayhoff sequences: NEL_MOUSE, APMU_PIG,
P_W37501,NEL_RAT, TSP1_CHICK, P_W37500, NEL2_HUMAN,
MMU010792.sub.--1, D86983.sub.--1 and 10 MUCS_BOVIN.
Example 8
Isolation of cDNA Clones Encoding Human PRO1863
[0535] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
Incyte database, designated Incyte EST cluster sequence no. 82468.
This EST cluster sequence was then compared to a variety of
expressed sequence tag (EST) databases which included public EST
databases (e.g., GenBank) and a proprietary EST DNA database
(Lifeseq.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.) to
identify existing homologies. The homology search was performed
using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.). The consensus sequence obtained
therefrom is herein designated DNA56029.
[0536] In light of the sequence homology between the DNA56029
sequence and an EST sequence contained within the Incyte EST clone
no. 2186536, the Incyte ESTclone no. 2186536 was purchased and the
cDNA insert was obtained and sequenced. The sequence of this cDNA
insert is shown in FIG. 9 and is herein designated as
DNA59847-2510.
[0537] Clone DNA59847-2510 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 17-19 and ending at the stop codon at nucleotide
positions 1328-1330 (FIG. 9). The predicted polypeptide precursor
is 437 amino acids long (FIG. 10). The full-length PRO1863 protein
shown in FIG. 10 has an estimated molecular weight of about 46,363
daltons and a pI of about 6.22. Analysis of the full-length PRO1863
sequence shown in FIG. 10 (SEQ ID NO: 16) evidences the presence of
the following: a signal peptide from about amino acid 1 to about
amino acid 15, a transmembrane domain from about amino acid 243 to
about amino acid 260, potential N-glycosylation sites from about
amino acid 46 to about amino acid 49, from about amino acid 189 to
about amino acid 192 and from about amino acid 382 to about amino
acid 385, glycosaminoglycan attachment sites from about amino acid
51 to about amino acid 54 and from about amino acid 359 to about
amino acid 362 and potential N-myristolation sites from about amino
acid 54 to about amino acid 59, from about amino acid 75 to about
amino acid 80, from about amino acid 141 to about amino acid 146,
from about amino acid 154 to about amino acid 159, from about amino
acid 168 to about amino acid 173, from about amino acid 169 to
about amino acid 174, from about amino acid 198 to about amino acid
203, from about amino acid 254 to about amino acid 259, from about
amino acid 261 to about amino acid 266, from about amino acid 269
to about amino acid 274, from about amino acid 284 to about amino
acid 289, from about amino acid 333 to about amino acid 338, from
about amino acid 347 to about amino acid 352, from about amino acid
360 to about amino acid 365, from about amino acid 361 to about
amino acid 366, from about amino acid 388 to about amino acid 393,
from about amino acid 408 to about amino acid 413 and from about
amino acid 419 to about amino acid 424. Clone DNA59847-25 10 has
been deposited with ATCC on Jan. 12, 1999 and is assigned ATCC
deposit no.203576.
[0538] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 10 (SEQ ID NO:16), evidenced
homology between the PRO1863 amino acid sequence and the following
Dayhoff sequences: AF041083.sub.--1, P_W26579, HSA223603.sub.--1,
MMU97068, RNMAGPIAN.sub.--1, CAHX_FLABR, S61882, AB007899.sub.--1,
CAH1_FLALI and P_W13386.
Example 9
Isolation of cDNA Clones Encoding Human PRO1917
[0539] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
LIFESEQ.RTM. database, designated EST cluster no. 85496. This EST
cluster sequence was then compared to a the EST databases listed
above to identify existing homologies. The homology search was
performed using the computer program BLAST or BLAST2 (Altshul et
al., Methods in Enzymology 266:460-480 (1996)). Those comparisons
resulting in a BLAST score of 70 (or in some cases 90) or greater
that did not encode known proteins were clustered and assembled
into a consensus DNA sequence with the program "phrap" (Phil Green,
University of Washington, Seattle, Wash.). The consensus sequence
obtained therefrom is herein designated DNA56415.
[0540] In light of the sequence homology between the DNA56415
sequence and an EST sequence contained within EST no.3255033, the
EST clone, which derived from an ovarian tumor library, was
purchased and the cDNA insert was obtained and sequenced. The
sequence of this cDNA insert is shown in FIG. 11 and is herein
designated as DNA76400-2528.
[0541] The full length clone shown in FIG. 11 contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 6-9 and ending at the stop codon found at
nucleotide positions 1467-1469 (FIG. 11; SEQ ID NO: 17). The
predicted polypeptide precursor (FIG. 12, SEQ ID NO: 18) is 487
amino acids long. PRO1917 has a calculated molecular weight of
approximately 55,051 daltons and an estimated pI of approximately
8.14. Additional features include: a signal peptide at about amino
acids 1-30; potential N-glycosylation sites at about amino acids
242-245 and 481-484, protein kinase C phosphorylation sites at
about amino acids 95-97, 182-184, and 427-429; N-myristoylation
sites at about amino acids 107-112, 113-118, 117-122, 118-123, and
128-133; and an endoplasmic reticulum targeting sequence at about
amino acids 484-487.
[0542] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 12 (SEQ ID NO:18), revealed
significant homology between the PRO1917 amino acid sequence and
Dayhoff sequence AF012714.sub.--1. Significant homology was also
revealed between the PRO1917 amino acid sequence and the sequence
of a chondrocyte protein, designated "P_W52286" on the Dayhoff
database, which has been reported to be involved in the transition
of chondrocytes from proliferate to hypertrophic states
(International Patent Application Publication No. W09801468-A1).
Homology was also revealed between the PRO1917 amino acid sequence
and the following additional Dayhoff sequences: P_W52286,
GGU59420.sub.--1, P_R25597, PPA3_YEAST, PPA1_SCHPO, PPA2_SCHPO,
A46783.sub.--1, DMC165H7.sub.--1, and AST8_DROME.
[0543] Clone DNA76400-2528 was deposited with the ATCC on Jan. 12,
1999, and is assigned ATCC deposit no. 203573.
Example 10
Isolation of cDNA Clones Encoding Human PRO1868
[0544] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA49803. Based up an
observed homology between the DNA49803 consensus sequence and an
EST sequence contained within the Incyte EST clone no. 2994689,
Incyte EST clone no. 2994689 was purchased and its insert obtained
and sequenced. The sequence of that insert is shown in FIG. 13 and
is herein designated DNA77624-2515.
[0545] The entire nucleotide sequence of DNA77624-2515 is shown in
FIG. 13 (SEQ ID NO: 19). Clone DNA77624-2515 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 51-53 and ending at the stop codon at
nucleotide positions 981-983 (FIG. 13). The predicted polypeptide
precursor is 310 amino acids long (FIG. 14). The full-length
PRO1868 protein shown in FIG. 14 has an estimated molecular weight
of about 35,020 daltons and a pI of about 7.90. Analysis of the
full-length PRO1868 sequence shown in FIG. 14 (SEQ ID NO:20)
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 30, a transmembrane domain
from about amino acid 243 to about amino acid 263, potential
N-glycosylation sites from about amino acid 104 to about amino acid
107 and from about amino acid 192 to about amino acid 195, a cAMP-
and cGMP-dependent protein kinase phosphorylation site from about
amino acid 107 to about amino acid 110, casein kinase II
phosphorylation sites from about amino acid 106 to about amino acid
109 and from about amino acid 296 to about amino acid 299, a
tyrosine kinase phosphorylation site from about amino acid 69 to
about amino acid 77 and potential N-myristolation sites from about
amino acid 26 to about amino acid 31, from about amino acid 215 to
about amino acid 220, from about amino acid 226 to about amino acid
231, from about amino acid 243 to about amino acid 248, from about
amino acid 244 to about amino acid 249 and from about amino acid
262 to about amino acid 267. Clone DNA77624-2515 has been deposited
with ATCC on Dec. 22, 1998 and is assigned ATCC deposit no.
203553.
[0546] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 14 (SEQ ID NO:20), evidenced
significant homology between the PRO1868 amino acid sequence and
the following Dayhoff sequences: HGS_RC75, P_W61379, A33_HUMAN,
P_W14146, P_W14158, AMAL_DROME, P_R77437, I38346, NCM2_HUMAN and
PTPD_HUMAN.
Example 11
Isolation of cDNA Clones Encoding Human PRO3434
[0547] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
Incyte database. This EST cluster sequence was then compared to a
variety of expressed sequence tag (EST) databases which included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (Lifeseq.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.)
to identify existing homologies. The homology search was performed
using the computer program BLAST or BLAST (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.). The consensus sequence obtained
therefrom is herein designated DNA56009.
[0548] In light of the sequence homology between the DNA56009
sequence and an EST sequence contained within the Incyte EST clone
no.3327089, the Incyte EST clone no.3327089 was purchased and the
cDNA insert was obtained and sequenced. The sequence of this cDNA
insert is shown in FIG. 15 and is herein designated as
DNA77631-2537.
[0549] Clone DNA77631-2537 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 46-48 and ending at the stop codon at nucleotide
positions 3133-3135 (FIG. 15). The predicted polypeptide precursor
is 1029 amino acids long (FIG. 16). The full-length PRO3434 protein
shown in FIG. 16 has an estimated molecular weight of about 114,213
daltons and a pI of about 6.42. Analysis of the full-length PRO3434
sequence shown in FIG. 16 (SEQ ID NO:22) evidences the presence of
very important polypeptide domains as shown in FIG. 16. Clone
DNA77631-2537 has been deposited with ATCC on Feb. 9, 1999 and is
assigned ATCC deposit no. 203651.
[0550] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 16 (SEQ ID NO:22), evidenced
homology between the PRO3434 amino acid sequence and the following
Dayhoff sequences: VATX_YEAST, P_R51171, POLS_IBDVP,
IBDVORF.sub.--2. JC5043, IBDVPIV.sub.--1, VE7_HPV11, GEN14220,
MUTS_THETH and COAC_CHICK.
Example 12
Isolation of cDNA Clones Encoding Human PRO1927
[0551] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
LIFESEQ.RTM. database, designated EST Cluster No. 1913. This EST
cluster sequence was then compared to a variety of expressed
sequence tag (EST) databases which included the databases listed
above, including an additional proprietary EST DNA database
(Genentech, South San Francisco, Calif.) to identify existing
homologies. The homology search was performed using the computer
program BLAST or BLAST2 (Altshul et al., Methods in Enzymology
266:460-480 (1996)). Those comparisons resulting in a BLAST score
of 70 (or in some cases 90) or greater that did not encode known
proteins were clustered and assembled into a consensus DNA sequence
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.). The consensus sequence obtained therefrom is
herein designated DNA73896.
[0552] In light of the sequence homology between the DNA73896
sequence and an EST sequence contained within EST no. 3326981H1,
EST clone no. 3326981H1, which was obtained from a library
constructed from RNA isolated from aortic tissue, was purchased and
the cDNA insert was obtained and sequenced. The sequence of this
cDNA insert is shown in FIG. 17 and is herein designated as
"DNA82307-2531".
[0553] The full length clone shown in FIG. 17 contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 51-53 and ending at the stop codon found at
nucleotide positions 1695-1697 (FIG. 17; SEQ ID NO:23). The
predicted polypeptide precursor (FIG. 18, SEQ ID NO:24) is 548
amino acids long. PRO1927 has a calculated molecular weight of
approximately 63,198 daltons and an estimated pI of approximately
8.10. Additional features include: a signal peptide at about amino
acids 1-23; a putative transmembrane domain at about amino acids
6-25; potential N-glycosylation sites at about amino acids 5-8,
87-90, 103-106, and 465-469; potential N-myristoylation sites at
about amino acids 6-11, 136-141, 370-375, and 509-514.
[0554] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 18 (SEQ ID NO:24), revealed
significant homology between the PRO1927 amino acid sequence and
Dayhoff sequence AB000628.sub.--1. Homology was also revealed
between the PRO1927 amino acid sequence and the following
additional Dayhoff sequences: HGS_A251, HGS_A197,
CELC50H11.sub.--2, CPXM_BACSU, VF03_VACCC, VF03_VACCV, DYHA_CHLRE,
C69084, and A64315.
[0555] Clone DNA82307-2531 was deposited with the ATCC on Dec. 15,
1998, and is assigned ATCC deposit no. 203537.
Example 13
Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay
67)
[0556] This example shows that one or more of the polypeptides of
the invention are active as inhibitors of the proliferation of
stimulated T-lymphocytes. Compounds which inhibit proliferation of
lymphocytes are useful therapeutically where suppression of an
immune response is beneficial.
[0557] The basic protocol for this assay is described in Current
Protocols in Immunology, unit 3.12; edited by J E Coligan, A M
Kruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes
of Health, Published by John Wiley & Sons, Inc.
[0558] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fet al bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fet al
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate). The stimulator PBMCs
are prepared by irradiating the cells (about 3000 Rads).
[0559] The assay is prepared by plating in triplicate wells a
mixture of:
[0560] 100:1 of test sample diluted to 1% or to 0.1%,
[0561] 50:1 of irradiated stimulator cells, and
[0562] 50:1 of responder PBMC cells.
[0563] 100 microliters of cell culture media or 100 microliter of
CD4-IgG is used as the control. The wells are then incubated at
37.degree. C., 5% CO.sub.2 for 4 days. On day 5, each well is
pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6
hours the cells are washed 3 times and then the uptake of the label
is evaluated.
[0564] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fet al
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PB MCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[0565] Any decreases below control is considered to be a positive
result for an inhibitory compound, with decreases of less than or
equal to 80% being preferred. However, any value less than control
indicates an inhibitory effect for the test protein.
[0566] The following polypeptides tested positive in this assay:
PRO1917 and PRO1868.
Example 14
Skin Vascular Permeability Assay (Assay 64)
[0567] This assay shows that certain polypeptides of the invention
stimulate an immune response and induce inflammation by inducing
mononuclear cell, eosinophil and PMN infiltration at the site of
injection of the animal. Compounds which stimulate an immune
response are useful therapeutically where stimulation of an immune
response is beneficial. This skin vascular permeability assay is
conducted as follows. Hairless guinea pigs weighing 350 grams or
more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg
xylazine intramuscularly (IM). A sample of purified polypeptide of
the invention or a conditioned media test sample is injected
intradermally onto the backs of the test animals with 100 .mu.l per
injection site. It is possible to have about 10-30, preferably
about 16-24, injection sites per animal. One .mu.l of Evans blue
dye (I % in physiologic buffered saline) is injected
intracardially. Blemishes at the injection sites are then measured
(mm diameter) at 1 hr and 6 hr post injection. Animals were
sacrificed at 6 hrs after injection. Each skin injection site is
biopsied and fixed in formalin. The skins are then prepared for
histopathologic evaluation. Each site is evaluated for inflammatory
cell infiltration into the skin. Sites with visible inflammatory
cell inflammation are scored as positive. Inflammatory cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic. At least a
minimal perivascular infiltrate at the injection site is scored as
positive, no infiltrate at the site of injection is scored as
negative.
[0568] The following polypeptides tested positive in this assay:
PRO1434.
Example 15
Proliferation of Rat Utricular Supporting Cells (Assay 54)
[0569] This assay shows that certain polypeptides of the invention
act as potent mitogens for inner ear supporting cells which are
auditory hair cell progenitors and, therefore, are useful for
inducing the regeneration of auditory hair cells and treating
hearing loss in mammals. The assay is performed as follows. Rat
UEC-4 utricular epithelial cells are aliquoted into 96 well plates
with a density of 3000 cells/well in 200 .mu.l of serum-containing
medium at 33.degree. C. The cells are cultured overnight and are
then switched to serum-free medium at 37.degree. C. Various
dilutions of PRO polypeptides (or nothing for a control) are then
added to the cultures and the cells are incubated for 24 hours.
After the 24 hour incubation, .sup.3H-thymidine (1 .mu.Ci/well) is
added and the cells are then cultured for an additional 24 hours.
The cultures are then washed to remove unincorporated radiolabel,
the cells harvested and Cpm per well determined. Cpm of at least
30% or greater in the PRO polypeptide treated cultures as compared
to the control cultures is considered a positive in the assay.
[0570] The following polypeptides tested positive in this assay:
PRO982.
Example 16
Gene Amplification
[0571] This example shows that the PRO1800-, PRO539-, PRO3434- and
PRO1927-encoding genes are amplified in the genome of certain human
lung, colon and/or breast cancers and/or cell lines. Amplification
is associated with overexpression of the gene product, indicating
that the polypeptides are useful targets for therapeutic
intervention in certain cancers such as colon, lung, breast and
other cancers and diagnostic determination of the presence of those
cancers. Therapeutic agents may take the form of antagonists of
PRO1800, PRO539, PRO3434 or PRO1927 polypeptide, for example,
murine-human chimeric, humanized or human antibodies against a
PRO1800, PRO539, PRO3434 or PRO1927 polypeptide.
[0572] The starting material for the screen was genomic DNA
isolated from a variety cancers. The DNA is quantitated precisely,
e.g., fluorometrically. As a negative control, DNA was isolated
from the cells of ten normal healthy individuals which was pooled
and used as assay controls for the gene copy in healthy individuals
(not shown). The 5' nuclease assay (for example, TaqMan.TM.) and
real-time quantitative PCR (for example, ABI Prizm 7700 Sequence
Detection System.TM. (Perkin Elmer, Applied Biosystems Division,
Foster City, Calif.)), were used to find genes potentially
amplified in certain cancers. The results were used to determine
whether the DNA encoding PRO1800, PRO539, PRO3434 or PRO1927 is
over-represented in any of the primary lung or colon cancers or
cancer cell lines or breast cancer cell lines that were screened.
The primary lung cancers were obtained from individuals with tumors
of the type and stage as indicated in Table 6. An explanation of
the abbreviations used for the designation of the primary tumors
listed in Table 6 and the primary tumors and cell lines referred to
throughout this example are given below.
[0573] The results of the TaqMan.TM. are reported in delta
(.DELTA.) Ct units. One unit corresponds to 1 PCR cycle or
approximately a 2-fold amplification relative to normal, two units
corresponds to 4-fold, 3 units to 8-fold amplification and so on.
Quantitation was obtained using primers and a TaqMan.TM.
fluorescent probe derived from the PRO1800-, PRO539-, PRO3434- or
PRO1927-encoding gene. Regions of PRO1800, PRO539, PRO3434 or
PRO1927 which are most likely to contain unique nucleic acid
sequences and which are least likely to have spliced out introns
are preferred for the primer and probe derivation, e.g.,
3'-untranslated regions. The sequences for the primers and probes
(forward, reverse and probe) used for the PRO1800, PRO539, PRO3434
or PRO1927 gene amplification analysis were as follows:
[0574] PRO1800 (DNA35672-2508) TABLE-US-00009 (SEQ ID NO:27)
forward 5'-ACTCGGGATTCCTGCTGTT-3' (SEQ ID NO:28) probe
5'-AGGCCTTTACCCAAGGCCACAAC-3' (SEQ ID NO:29) reverse
5'-GGCCTGTCCTGTGTTCTCA-3'
[0575] PRO539 (DNA47465-1561) TABLE-US-00010 (SEQ ID NO:30) forward
5'-TCCCACCACTTACTTCCATGAA-3' (SEQ ID NO:31) probe
5'-CTGTGGTACCCAATTGCCGCCTTGT-3' (SEQ ID NO:32) reverse
5'-ATTGTCCTGAGATTCGAGCAAGA-3'
[0576] PRO3434 (DNA77631-2537) TABLE-US-00011 (SEQ ID NO:33)
forward 5'-GTCCAGCAAGCCCTCATT-3' (SEQ ID NO:34) probe
5'-CTTCTGGGCCACAGCCCTGC-3' (SEQ ID NO:35) reverse
5'-CAGTTCAGGTCGTTTCATTCA-3'
[0577] PRO1927 (DNA82307-2531) TABLE-US-00012 (SEQ ID NO:36)
forward 5'-CCAGTCAGGCCGTTTTAGA-3' (SEQ ID NO:37) probe
5'-CGGGCGCCCAAGTAAAAGCTC-3' (SEQ ID NO:38) reverse
5'-CATAAAGTAGTATATGCATTCCAGTGTT-3'
[0578] The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor amplification in real time. Two
oligonucleotide primers are used to generate an amplicon typical of
a PCR reaction. A third oligonucleotide, or probe, is designed to
detect nucleotide sequence located between the two PCR primers. The
probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the amplification reaction, the
Taq DNA polymerase enzyme cleaves the probe in a template-dependent
manner. The resultant probe fragments disassociate in solution, and
signal from the released reporter dye is free from the quenching
effect of the second fluorophore. One molecule of reporter dye is
liberated for each new molecule synthesized, and detection of the
unquenched reporter dye provides the basis for quantitative
interpretation of the data.
[0579] The 5' nuclease procedure is run on a real time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0580] 5' Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular target sequence
in a nucleic acid sample when comparing cancer DNA results to
normal human DNA results.
[0581] Table 6 describes the stage, T stage and N stage of various
primary tumors which were used to screen the PRO1800, PRO539,
PRO3434 and PRO1927 compounds of the invention. TABLE-US-00013
TABLE 6 Primary Lung and Colon Tumor Profiles Primary Tumor Stage
Stage Other Stage Dukes Stage T Stage N Stage Human lung tumor
AdenoCa (SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725)
[LT1a] IIB T3 N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0
Human lung tumor AdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung
tumor AdenoCa (SRCC728) [LT4] IB T2 N0 Human lung tumor SqCCa
(SRCC729) [LT6] IB T2 N0 Human lung tumor Aden/SqCCa (SRCC730)
[LT7] IA T1 N0 Human lung tumor AdenoCa (SRCC731) [LT9] IB T2 N0
Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 N1 Human lung tumor
SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumor AdenoCa (SRCC734)
[LT12] IV T2 N0 Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2
N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2 N0 Human lung
tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa
(SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18]
IB T2 N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human
lung tumor LCCa (SRCC741) [LT21] IIB T3 N1 Human lung AdenoCa
(SRCC811) [LT22] 1A T1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D
pT4 N0 Human colon AdenoCa (SRCC743) [CT3] B pT3 N0 Human colon
AdenoCa (SRCC744) [CT8] B T3 N0 Human colon AdenoCa (SRCC745)
[CT10] A pT2 N0 Human colon AdenoCa (SRCC746) [CT12] MO, R1 B T3 N0
Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pN0 Human colon
AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2 Human colon AdenoCa
(SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa (SRCC750) [CT17]
C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 B pT3 N0
Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa
(SRCC753) [CT5] G2 C1 pT3 pN0 Human colon AdenoCa (SRCC754) [CT6]
pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0
Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon
AdenoCa (SRCC757) [CT11] B T3 N0 Human colon AdenoCa (SRCC758)
[CT18] MO, RO B pT3 pN0
DNA Preparation:
[0582] DNA was prepared from cultured cell lines, primary tumors,
normal human blood. The isolation was performed using purification
kit, buffer set and protease and all from Quiagen, according to the
manufacturer's instructions and the description below.
[0583] Cell culture lysis:
[0584] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifuging at 1000 rpm
for 5 minutes at 4.degree. C., followed by washing again with 1/2
volume of PBS recentrifugation. The pellets were washed a third
time, the suspended cells collected and washed 2.times. with PBS.
The cells were then suspended into 10 ml PBS. Buffer C1 was
equilibrated at 4.degree. C. Qiagen protease #19155 was diluted
into 6.25 ml cold ddH.sub.2O to a final concentration of 20 mg/ml
and equilibrated at 4.degree. C. 10 ml of G2 Buffer was prepared by
diluting Qiagen RNAse A stock (100 mg/ml) to a final concentration
of 200 .mu.g/ml.
[0585] Buffer C1 (10 ml, 4.degree. C.) and ddH20 (40 ml, 4.degree.
C.) were then added to the 10 ml of cell suspension, mixed by
inverting and incubated on ice for 10 minutes. The cell nuclei were
pelleted by centrifuging in a Beckman swinging bucket rotor at 2500
rpm at 4.degree. C. for 15 minutes. The supernatant was discarded
and the nuclei were suspended with a vortex into 2 ml Buffer C1 (at
4.degree. C.) and 6 ml ddH.sub.2O, followed by a second 4.degree.
C. centrifugation at 2500 rpm for 15 minutes. The nuclei were then
resuspended into the residual buffer using 200 .mu.l per tip. G2
buffer (10 ml) was added to the suspended nuclei while gentle
vortexing was applied. Upon completion of buffer addition, vigorous
vortexing was applied for 30 seconds. Quiagen protease (200 .mu.l,
prepared as indicated above) was added and incubated at 50.degree.
C. for 60 minutes. The incubation and centrifugation was repeated
until the lysates were clear (e.g., incubating additional 30-60
minutes, pelleting at 3000.times.g for 10 min., 4.degree. C.).
[0586] Solid human tumor sample preparation and lysis:
[0587] Tumor samples were weighed and placed into 50 ml conical
tubes and held on ice. Processing was limited to no more than 250
mg tissue per preparation (1 tip/preparation). The protease
solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNAse A to
a final concentration of 200 mg/ml (from 100 mg/ml stock). The
tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood in
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2L ddH.sub.2O, followed by G2 buffer
(50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[0588] Quiagen protease (prepared as indicated above, 1.0 ml) was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation was repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times.g for 10 min., 4.degree. C.).
[0589] Human blood preparation and lysis:
[0590] Blood was drawn from healthy volunteers using standard
infectious agent protocols and citrated into 10 ml samples per tip.
Quiagen protease was freshly prepared by dilution into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer was prepared by diluting RNAse A to a final
concentration of 200 .mu.g/ml from 100 mg/ml stock. The blood (10
ml) was placed into a 50 ml conical tube and 10 ml C1 buffer and 30
ml ddH.sub.2O (both previously equilibrated to 4.degree. C.) were
added, and the components mixed by inverting and held on ice for 10
minutes. The nuclei were pelleted with a Beckman swinging bucket
rotor at 2500 rpm, 4.degree. C. for 15 minutes and the supernatant
discarded. With a vortex, the nuclei were suspended into 2 ml C1
buffer (4.degree. C.) and 6 ml ddH.sub.2O (4.degree. C.). Vortexing
was repeated until the pellet was white. The nuclei were then
suspended into the residual buffer using a 200 .mu.l tip. G2 buffer
(10 ml) were added to the suspended nuclei while gently vortexing,
followed by vigorous vortexing for 30 seconds. Quiagen protease was
added (200 .mu.l) and incubated at 50.degree. C. for 60 minutes.
The incubation and centrifugation was repeated until the lysates
were clear (e.g., incubating additional 30-60 minutes, pelleting at
3000.times.g for 10 min., 4.degree. C.).
[0591] Purification of cleared lysates:
[0592] (1) Isolation of genomic DNA:
[0593] Genomic DNA was equilibrated (1 sample per maxi tip
preparation) with 10 ml QBT buffer. QF elution buffer was
equilibrated at 50.degree. C. The samples were vortexed for 30
seconds, then loaded onto equilibrated tips and drained by gravity.
The tips were washed with 2.times.15 ml QC buffer. The DNA was
eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15
ml QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, the tubes covered with parafin and mixed by repeated
inversion until the DNA precipitated. Samples were pelleted by
centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at
4.degree. C. The pellet location was marked, the supernatant
discarded, and 10 ml 70% ethanol (4.degree. C.) was added. Samples
were pelleted again by centrifugation on the SS-34 rotor at 10,000
rpm for 10 minutes at 4.degree. C. The pellet location was marked
and the supernatant discarded. The tubes were then placed on their
side in a drying rack and dried 10 minutes at 37.degree. C., taking
care not to overdry the samples.
[0594] After drying, the pellets were dissolved into 1.0 ml TE (pH
8.5) and placed at 50.degree. C. for 1-2 hours. Samples were held
overnight at 4.degree. C. as dissolution continued. The DNA
solution was then transferred to 1.5 ml tubes with a 26 gauge
needle on a tuberculin syringe. The transfer was repeated 5.times.
in order to shear the DNA. Samples were then placed at 50.degree.
C. for 1-2 hours.
[0595] (2) Quantitation of genomic DNA and preparation for gene
amplification assay:
[0596] The DNA levels in each tube were quantified by standard
A.sub.260, A.sub.280 spectrophotometry on a 1:20 dilution (5 .mu.l
DNA+95 .mu.l ddH.sub.2O) using the 0.1 ml quartz cuvetts in the
Beckman DU640 spectrophotometer. A.sub.260/A.sub.280 ratios were in
the range of 1.8-1.9. Each DNA samples was then diluted further to
approximately 200 ng/ml in TE (pH 8.5). If the original material
was highly concentrated (about 700 ng/.mu.l), the material was
placed at 50.degree. C. for several hours until resuspended.
[0597] Fluorometric DNA quantitation was then performed on the
diluted material (20-600 ng/ml) using the manufacturer'sguidelines
as modified below. This was accomplished by allowing a Hoeffer DyNA
Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst
dye working solution (#H33258, 10 .mu.l, prepared within 12 hours
of use) was diluted into 100 ml 1.times.TNE buffer. A 2 ml cuvette
was filled with the fluorometer solution, placed into the machine,
and the machine was zeroed. pGEM 3Zf(+) (2 .mu.l, lot #360851026)
was added to 2 ml of fluorometer solution and calibrated at 200
units. An additional 2 .mu.l of pGEM 3Zf(+) DNA was then tested and
the reading confirmed at 400 +/-10 units. Each sample was then read
at least in triplicate. When 3 samples were found to be within 10%
of each other, their average was taken and this value was used as
the quantification value.
[0598] The fluorometrically determined concentration was then used
to dilute each sample to 10 ng/.mu.l in ddH.sub.2O. This was done
simultaneously on all template samples for a single TaqMan plate
assay, and with enough material to run 500-1000 assays. The samples
were tested in triplicate with Taqman.TM. primers and probe both
B-actin and GAPDH on a single plate with normal human DNA and
no-template controls. The diluted samples were used provided that
the CT value of normal human DNA subtracted from test DNA was +/-1
Ct. The diluted, lot-qualified genomic DNA was stored in 1.0 ml
aliquots at -80.degree. C. Aliquots which were subsequently to be
used in the gene amplification assay were stored at 4.degree. C.
Each 1 ml aliquot is enough for 8-9 plates or 64 tests.
[0599] Gene amplification assay:
[0600] The PRO1800, PRO539, PRO3434 and PRO1927 compounds of the
invention were screened in the following primary tumors and the
resulting .DELTA.Ct values greater than or equal to 1.0 are
reported in Table 7 below. TABLE-US-00014 TABLE 7 (.DELTA.Ct values
in lung and colon primary tumor models) Primary Tumor PRO1800
PRO539 PRO3434 PRO1927 LT11 1.65, 1.59, 1.03 LT12 1.34, 2.28, 2.03
1.25 LT13 1.27, 2.18 1.64, 1.08 5.24, 4.47 4.38, 4.80 LT15 1.70,
2.23, 1.93 1.78, 1.10 1.24 1.00 LT16 1.00, 1.05, 1.09 3.65, 3.19
2.73, 2.74 LT17 1.94, 1.63 1.94, 1.01 LT18 1.12 LT19 2.51, 2.18
1.16 LT21 1.30 1.32 CT2 1.50 CT3 1.17 CT10 1.16 CT12 1.19 CT14 1.62
CT15 1.48, 1.08 1.03 1.19, 1.40 1.10, 1.30 CT5 1.10 CT11 1.20 1.12
Colo-320 1.16 1.78, 1.76, 1.74 1.51 (colon tumor cell line)
HF-00084(lung tumor cell line) 2.20 2.41 HCT-116(colon tumor cell
line) 2.15, 2.22 1.41, 1.47 HF-00129 1.00, 1.17, 4.64 2.31, 5.14
(lung tumor cell line) 1.11 2.40 SW-620(colon tumor cell line) 1.30
HT-29(colon tumor cell line) 1.64 SW-403(colon tumor cell line)
1.75 LS174T(colon tumor cell line) 1.42 HCC-2998(colon tumor cell
line) 1.15 A549(lung tumor cell line) 1.51, 1.09 Calu-6(lung tumor
cell line) 1.60, 1.22 H157(lung tumor cell line) 1.61 H441(lung
tumor cell line) 1.07, 1.15 H460(lung tumor cell line) 1.01
SKMES1(lung tumor cell line) 1.02 H810(lung tumor cell line) 1.20,
1.54
Example 17
Induction of Pancreatic .beta.-Cell Precursor Proliferation (Assay
117)
[0601] This assay shows that certain polypeptides of the invention
act to induce an increase in the number of pancreatic .beta.-cell
precursor cells and, therefore, are useful for treating various
insulin deficient states in mammals, including diabetes mellitus.
The assay is performed as follows. The assay uses a primary culture
of mouse fet al pancreatic cells and the primary readout is an
alteration in the expression of markers that represent either
.beta.-cell precursors or mature .beta.-cells. Marker expression is
measured by real time quantitative PCR (RTQ-PCR); wherein the
marker being evaluated is a transcription factor called Pdx1.
[0602] The pancreata are dissected from E14 embryos (CD1 mice). The
pancreata are then digested with collagenase/dispase in F12/DMEM at
37.degree. C. for 40 to 60 minutes (collagenase/dispase, 1.37
mg/ml, Boehringer Mannheim, #1097113). The digestion is then
neutralized with an equal volume of 5% BSA and the cells are washed
once with RPMI1640. At day 1, the cells are seeded into 12-well
tissue culture plates (pre-coated with laminin, 20 .mu.g/ml in PBS,
Boehringer Mannheim, #124317). Cells from pancreata from 1-2
embryos are distributed per well. The culture medium for this
primary cuture is 14F/1640. At day 2, the media is removed and the
attached cells washed with RPMI11640. Two mls of minimal media are
added in addition to the protein to be tested. At day 4, the media
is removed and RNA prepared from the cells and marker expression
analyzed by real time quantitative RT-PCR. A protein is considered
to be active in the assay if it increases the expression of the
relevant .beta.-cell marker as compared to untreated controls.
14F/1640 is RPMI1640 (Gibco) plus the following:
[0603] group A 1:1000
[0604] group B 1:1000
[0605] recombinant human insulin 10 .mu.g/ml
[0606] Aprotinin (50 .mu.g/ml) 1:2000 (Boehringer manheim
#981532)
[0607] Bovine pituitary extract (BPE) 60 .mu.g/ml
[0608] Gentamycin 100 ng/ml
Group A: (in 10 ml PBS)
[0609] Transferrin, 100 mg (Sigma T2252)
[0610] Epidermal Growth Factor, 100 .mu.g (BRL 100004)
[0611] Triiodothyronine, 10 .mu.l of 5.times.10.sup.-6 M (Sigma
T5516)
[0612] Ethanolamine, 100 .mu.l of 10.sup.-1 M (Sigma E0135)
[0613] Phosphoethalamine, 100 .mu.l of 10.sup.-1 M (Sigma
P0503)
[0614] Selenium, 4 .mu.l of 10.sup.-1 M (Aesar #12574)
Group C: (in 10 ml 100% ethanol)
[0615] Hydrocortisone, 2 .mu.l of 5.times.10.sup.-3 M (Sigma
#H0135)
[0616] Progesterone, 100 .mu.t of 1.times.10.sup.-3 M (Sigma
#P6149)
[0617] Forskolin, 500 .mu.l of 20 mM (Calbiochem #344270)
Minimal media:
[0618] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml), aprotinin (50 .mu.g/ml) and BPE
(15 .mu.g/ml).
Defined media:
[0619] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml) and aprotinin (50 .mu.g/ml).
[0620] The following polypeptide was positive in this assay:
PRO1868.
Example 18
Induction of Pancreatic .beta.-Cell Precursor Differentiation
(Assay 89)
[0621] This assay shows that certain polypeptides of the invention
act to induce differentiation of pancreatic .beta.-cell precursor
cells into mature pancreatic .beta.-cells and, therefore, are
useful for treating various insulin deficient states in mammals,
including diabetes mellitus. The assay is performed as follows. The
assay uses a primary culture of mouse fet al pancreatic cells and
the primary readout is an alteration in the expression of markers
that represent either .beta.-cell precursors or mature
.beta.-cells. Marker expression is measured by real time
quantitative PCR (RTQ-PCR); wherein the marker being evaluated is
insulin.
[0622] The pancreata are dissected from E14 embryos (CD1 mice). The
pancreata are then digested with collagenase/dispase in F12/DMEM at
37.degree. C. for 40 to 60 minutes (collagenase/dispase, 1.37
mg/ml, Boehringer Mannheim, #1097113). The digestion is then
neutralized with an equal volume of 5% BSA and the cells are washed
once with RPM11640. At day 1, the cells are seeded into 12-well
tissue culture plates (pre-coated with laminin, 20 .mu.g/ml in PBS,
Boehringer Mannheim, #124317). Cells from pancreata from 1-2
embryos are distributed per well. The culture medium for this
primary cuture is 14F/1640. At day 2, the media is removed and the
attached cells washed with RPMI/1640. Two mls of minimal media are
added in addition to the protein to be tested. At day 4, the media
is removed and RNA prepared from the cells and marker expression
analyzed by real time quantitative RT-PCR. A protein is considered
to be active in the assay if it increases the expression of the
relevant P-cell marker as compared to untreated controls.
14F/1640 is RPMI1640 (Gibco) plus the following:
[0623] group A 1:1000
[0624] group B 1:1000
[0625] recombinant human insulin 10 .mu.g/ml
[0626] Aprotinin (50 .mu.g/ml) 1:2000 (Boehringer manheim
#981532)
[0627] Bovine pituitary extract (BPE) 60 .mu.g/ml
[0628] Gentamycin 100 ng/ml
Group A: (in 10 ml PBS)
[0629] Transferrin, 100 mg (Sigma T2252)
[0630] Epidermal Growth Factor, 100 .mu.g (BRL 100004)
[0631] Triiodothyronine, 10 .mu.l of 5.times.10.sup.-6 M (Sigma
T5516)
[0632] Ethanolamine, 100 .mu.l of 10.sup.-1 M (Sigma E0135)
[0633] Phosphoethalamine, 100 .mu.l of 10.sup.-1 M (Sigma
P0503)
[0634] Selenium, 4 .mu.l of 10.sup.-1 M (Aesar #12574)
Group C: (in 10 ml 100% ethanol)
[0635] Hydrocortisone, 2 .mu.l of 5.times.10.sup.-3 M (Sigma
#H0135)
[0636] Progesterone, 100 .mu.l of 1.times.10.sup.-3 M (Sigma
#P6149)
[0637] Forskolin, 500 .mu.l of 20 mM (Calbiochem #344270)
Minimal media:
[0638] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml), aprotinin (50 .mu.g/ml) and BPE
(15 .mu.g/ml).
Defined media:
[0639] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml) and aprotinin (50 .mu.g/ml).
[0640] The following polypeptide was positive in this assay:
PRO1863.
Example 19
Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)
[0641] This assay shows that certain polypeptides of the invention
act to induce proliferation of mammalian kidney mesangial cells
and, therefore, are useful for treating kidney disorders associated
with decreased mesangial cell function such as Berger disease or
other nephropathies associated with Schonlein-Henoch purpura,
celiac disease, dermatitis herpetiformis or Crohn disease. The
assay is performed as follows. On day one, mouse kidney mesangial
cells are plated on a 96 well plate in growth media (3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95% fet al
bovine serum, 5% supplemented with 14 mM HEPES) and grown
overnight. On day 2, PRO polypeptides are diluted at 2
concentrations(1% and 0.1%) in serum-free medium and added to the
cells. Control samples are serum-free medium alone. On day 4, 20
.mu.l of the Cell Titer 96 Aqueous one solution reagent (Progema)
was added to each well and the colormetric reaction was allowed to
proceed for 2 hours. The absorbance (OD) is then measured at 490
nm. A positive in the assay is anything that gives an absorbance
reading which is at least 15% above the control reading.
[0642] The following polypeptide tested positive in this assay:
PRO1917.
Example 20
Fibroblast (BHK-21) Proliferation (Assay 98)
[0643] This assay shows that certain polypeptides of the invention
act to induce proliferation of mammalian fibroblast cells in
culture and, therefore, function as useful growth factors in
mammalian systems. The assay is performed as follows. BHK-21
fibroblast cells plated in standard growth medium at 2500
cells/well in a total volume of 100 .mu.l. The PRO polypeptide,
.beta.-FGF (positive control) or nothing (negative control) are
then added to the wells in the presence of 1 .mu.g/ml of heparin
for a total final volume of 200 .mu.l. The cells are then incubated
at 37.degree. C. for 6 to 7 days. After incubation, the media is
removed, the cells are washed with PBS and then an acid phosphatase
substrate reaction mixture (100 .mu.l/well) is added. The cells are
then incubated at 37.degree. C. for 2 hours. 10 .mu.l per well of
1N NaOH is then added to stop the acid phosphatase reaction. The
plates are then read at OD 405 nm. A positive in the assay is acid
phosphatase activity which is at least 50% above the negative
control.
[0644] The following polypeptide tested positive in this assay:
PRO982.
Example 21
Chondrocyte Re-differentiation Assay (Assay 110)
[0645] This assay shows that certain polypeptides of the invention
act to induce redifferentiation of chondrocytes, therefore, are
expected to be useful for the treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and
arthritis. The assay is performed as follows. Porcine chondrocytes
are isolated by overnight collagenase digestion of articulary
cartilage of metacarpophalangeal joints of 4-6 month old female
pigs. The isolated cells are then seeded at 25,000 cells/cm.sup.2
in Ham F-12 containing 10% FBS and 4 .mu.g/ml gentamycin. The
culture media is changed every third day and the cells are then
seeded in 96 well plates at 5,000 cells/well in 100 .mu.l of the
same media without serum and 100 .mu.l of the test PRO polypeptide,
5 nM staurosporin (positive control) or medium alone (negative
control) is added to give a final volume of 200 .mu.l/well. After 5
days of incubation at 37.degree. C., a picture of each well is
taken and the differentiation state of the chondrocytes is
determined. A positive result in the assay occurs when the
redifferentiation of the chondrocytes is determined to be more
similar to the positive control than the negative control.
[0646] The following polypeptide tested positive in this assay:
PRO1863.
Example 22
Use of PRO as a Hybridization Probe
[0647] The following method describes use of a nucleotide sequence
encoding PRO as a hybridization probe.
[0648] DNA comprising the coding sequence of full-length or mature
PRO as disclosed herein is employed as a probe to screen for
homologous DNAs (such as those encoding naturally-occurring
variants of PRO) in human tissue cDNA libraries or human tissue
genomic libraries.
[0649] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO-derived probe to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times.Denhardt's solution, and 10% dextran sulfate at
42.degree. C. for 20 hours. Washing of the filters is performed in
an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree.
C.
[0650] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO can then be identified
using standard techniques known in the art.
Example 23
Expression of PRO in E. coli
[0651] This example illustrates preparation of an unglycosylated
form of PRO by recombinant expression in E. coli.
[0652] The DNA sequence encoding PRO is initially amplified using
selected PCR primers. The primers should contain restriction enzyme
sites which correspond to the restriction enzyme sites on the
selected expression vector. A variety of expression vectors may be
employed. An example of a suitable vector is pBR322 (derived from
E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains
genes for ampicillin and tetracycline resistance. The vector is
digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector
will preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the PRO coding region, lambda transcriptional terminator,
and an argU gene.
[0653] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0654] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0655] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized PRO protein can then be purified using a
met al chelating column under conditions that allow tight binding
of the protein.
[0656] PRO may be expressed in E. coli in a poly-His tagged form,
using the following procedure. The DNA encoding PRO is initially
amplified using selected PCR primers. The primers will contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector, and other useful sequences
providing for efficient and reliable translation initiation, rapid
purification on a met al chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(laclq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g 15 Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking. Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[0657] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02M, respectively, and the
solution is stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of met al chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA met al
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0658] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0659] Fractions containing the desired folded PRO polypeptide are
pooled and the acetonitrile removed using a gentle stream of
nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile
filtered.
[0660] Many of the PRO polypeptides disclosed herein were
successfully expressed as described above.
Example 24
Expression of PRO in Mammalian Cells
[0661] This example illustrates preparation of a potentially
glycosylated form of PRO by recombinant expression in mammalian
cells.
[0662] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the PRO DNA using ligation methods such as described
in Sambrook et al., supra. The resulting vector is called
pRK5-PRO.
[0663] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fet al calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-PRO DNA is mixed with about 1 .mu.g DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved
in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To
this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed
to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0664] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.C.i/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of PRO polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0665] In an alternative technique, PRO may be introduced into 293
cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-PRO DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed PRO can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0666] In another embodiment, PRO can be expressed in CHO cells.
The pRK5-PRO can be transfected into CHO cells using known reagents
such as CaPO.sub.4 or DEAE-dextran. As described above, the cell
cultures can be incubated, and the medium replaced with culture
medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of PRO
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed PRO can then be concentrated and purified by any
selected method.
[0667] Epitope-tagged PRO may also be expressed in host CHO cells.
The PRO may be subcloned out of the pRK5 vector. The subclone
insert can undergo PCR to fuse in frame with a selected epitope tag
such as a poly-his tag into a Baculovirus expression vector. The
poly-his tagged PRO insert can then be subcloned into a SV40 driven
vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells can be transfected (as
described above) with the SV40 driven vector. Labeling may be
performed, as described above, to verify expression. The culture
medium containing the expressed poly-His tagged PRO can then be
concentrated and purified by any selected method, such as by
Ni.sup.2+-chelate affinity chromatography.
[0668] PRO may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[0669] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0670] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0671] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.-7 cells are frozen in an ampule for further growth
and production as described below.
[0672] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fet al bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH i.e.
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0673] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0674] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH,6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
[0675] Many of the PRO polypeptides disclosed herein were
successfully expressed as described above.
Example 25
Expression of PRO in Yeast
[0676] The following method describes recombinant expression of PRO
in yeast.
[0677] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO from the ADH2/GAPDH
promoter. DNA encoding PRO and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of PRO. For secretion, DNA encoding PRO
can be cloned into the selected plasmid, together with DNA encoding
the ADH2/GAPDH1 promoter, a native PRO signal peptide or other
mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signal/leader sequence, and linker sequences
(if needed) for expression of PRO.
[0678] Yeast cells, such as yeast strain AB 110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0679] Recombinant PRO can subsequently be isolated and purified by
removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing PRO may further be
purified using selected column chromatography resins.
[0680] Many of the PRO polypeptides disclosed herein were
successfully expressed as described above.
Example 26
Expression of PRO in Baculovirus-Infected Insect Cells
[0681] The following method describes recombinant expression of PRO
in Baculovirus-infected insect cells.
[0682] The sequence coding for PRO is fused upstream of an epitope
tag contained within a baculovirus expression vector. Such epitope
tags include poly-his tags and immunoglobulin tags (like Fc regions
of IgG). A variety of plasmids may be employed, including plasmids
derived from commercially available plasmids such as pVL1393
(Novagen). Briefly, the sequence encoding PRO or the desired
portion of the coding sequence of PRO such as the sequence encoding
the extracellular domain of a transmembrane protein or the sequence
encoding the mature protein if the protein is extracellular is
amplified by PCR with primers complementary to the 5' and 3'
regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product is then digested with those
selected restriction enzymes and subcloned into the expression
vector.
[0683] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0684] Expressed poly-his tagged PRO can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-179 (1993). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL
Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1%
NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45, m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280baseline again, the column is developed with a 0 to 500 mM
Imidazole gradient in the secondary wash buffer. One mL fractions
are collected and analyzed by SDS-PAGE and silver staining or
Western blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His.sub.10-tagged PRO are
pooled and dialyzed against loading buffer.
[0685] Alternatively, purification of the IgG tagged (or Fc tagged)
PRO can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
[0686] Many of the PRO polypeptides disclosed herein were
successfully expressed as described above.
Example 27
Preparation of Antibodies that Bind PRO
[0687] This example illustrates preparation of monoclonal
antibodies which can specifically bind PRO.
[0688] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified PRO, fusion
proteins containing PRO, and cells expressing recombinant PRO on
the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0689] Mice, such as Balb/c, are immunized with the PRO immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-PRO antibodies.
[0690] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0691] The hybridoma cells will be screened in an ELISA for
reactivity against PRO. Determination of "positive" hybridoma cells
secreting the desired monoclonal antibodies against PRO is within
the skill in the art.
[0692] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or
protein G can be employed.
Example 28
Purification of PRO Polypeptides Using Specific Antibodies
[0693] Native or recombinant PRO polypeptides may be purified by a
variety of standard techniques in the art of protein purification.
For example, pro-PRO polypeptide, mature PRO polypeptide, or
pre-PRO polypeptide is purified by immunoaffinity chromatography
using antibodies specific for the PRO polypeptide of interest. In
general, an immunoaffinity column is constructed by covalently
coupling the anti-PRO polypeptide antibody to an activated
chromatographic resin.
[0694] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0695] Such an immunoaffinity column is utilized in the
purification of PRO polypeptide by preparing a fraction from cells
containing PRO polypeptide in a soluble form. This preparation is
derived by solubilization of the whole cell or of a subcellular
fraction obtained via differential centrifugation by the addition
of detergent or by other methods well known in the art.
Alternatively, soluble PRO polypeptide containing a signal sequence
may be secreted in useful quantity into the medium in which the
cells are grown.
[0696] A soluble PRO polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of PRO
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/PRO polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and PRO polypeptide is
collected.
Example 29
Drug Screening
[0697] This invention is particularly useful for screening
compounds by using PRO polypeptides or binding fragment thereof in
any of a variety of drug screening techniques. The PRO polypeptide
or fragment employed in such a test may either be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened against such transformed cells in
competitive binding assays. Such cells, either in viable or fixed
form, can be used for standard binding assays. One may measure, for
example, the formation of complexes between PRO polypeptide or a
fragment and the agent being tested. Alternatively, one can examine
the diminution in complex formation between the PRO polypeptide and
its target cell or target receptors caused by the agent being
tested.
[0698] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an PRO polypeptide or fragment
thereof and assaying (I) for the presence of a complex between the
agent and the PRO polypeptide or fragment, or (ii) for the presence
of a complex between the PRO polypeptide or fragment and the cell,
by methods well known in the art. In such competitive binding
assays, the PRO polypeptide or fragment is typically labeled. After
suitable incubation, free PRO polypeptide or fragment is separated
from that present in bound form, and the amount of free or
uncomplexed label is a measure of the ability of the particular
agent to bind to PRO polypeptide or to interfere with the PRO
polypeptide/cell complex.
[0699] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on Sep. 13, 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to a PRO polypeptide, the peptide test compounds are reacted with
PRO polypeptide and washed. Bound PRO polypeptide is detected by
methods well known in the art. Purified PRO polypeptide can also be
coated directly onto plates for use in the aforementioned drug
screening techniques. In addition, non-neutralizing antibodies can
be used to capture the peptide and immobilize it on the solid
support.
[0700] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO polypeptide specifically compete with a test compound
for binding to PRO polypeptide or fragments thereof. In this
manner, the antibodies can be used to detect the presence of any
peptide which shares one or more antigenic determinants with PRO
polypeptide.
Example 30
Rational Drug Design
[0701] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a PRO
polypeptide) or of small molecules with which they interact, e.g.,
agonists, antagonists, or inhibitors. Any of these examples can be
used to fashion drugs which are more active or stable forms of the
PRO polypeptide or which enhance or interfere with the function of
the PRO polypeptide in vivo (cf, Hodgson, Bio/Technoloy, 9: 19-21
(1991)).
[0702] In one approach, the three-dimensional structure of the PRO
polypeptide, or of an PRO polypeptide-inhibitor complex, is
determined by x-ray crystallography, by computer modeling or, most
typically, by a combination of the two approaches. Both the shape
and charges of the PRO polypeptide must be ascertained to elucidate
the structure and to determine active site(s) of the molecule. Less
often, useful information regarding the structure of the PRO
polypeptide may be gained by modeling based on the structure of
homologous proteins. In both cases, relevant structural information
is used to design analogous PRO polypeptide-like molecules or to
identify efficient inhibitors. Useful examples of rational drug
design may include molecules which have improved activity or
stability as shown by Braxton and Wells, Biochemistry. 31:7796 7801
(1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by Athauda et al., J. Biochem.,
113:742-746 (1993).
[0703] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The anti id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides would then
act as the pharmacore.
[0704] By virtue of the present invention, sufficient amounts of
the PRO polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the PRO polypeptide amino acid sequence provided herein will
provide guidance to those employing computer modeling techniques in
place of or in addition to x-ray crystallography.
Deposit of Material
[0705] The following materials have been deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., USA (ATCC): TABLE-US-00015 Material ATCC Dep. No. Deposit Date
DNA35672-2508 203538 Dec. 15, 1998 DNA47465-1561 203661 Feb. 9,
1999 DNA57700-1408 203583 Jan. 12, 1999 DNA68818-2536 203657 Feb.
9, 1999 DNA59847-2510 203576 Jan. 12, 1999 DNA76400-2528 203573
Jan. 12, 1999 DNA77624-2515 203553 Dec. 22, 1998 DNA77631-2537
203651 Feb. 9, 1999 DNA82307-2531 203537 Dec. 15, 1998
[0706] These deposit were made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 886 OG 638).
[0707] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0708] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
38 1 1283 DNA Homo sapiens 1 cggacgcgtg ggacccatac ttgctggtct
gatccatgca caaggcgggg 50 ctgctaggcc tctgtgcccg ggcttggaat
tcggtgcgga tggccagctc 100 cgggatgacc cgccgggacc cgctcgcaaa
taaggtggcc ctggtaacgg 150 cctccaccga cgggatcggc ttcgccatcg
cccggcgttt ggcccaggac 200 ggggcccatg tggtcgtcag cagccggaag
cagcagaatg tggaccaggc 250 ggtggccacg ctgcaggggg aggggctgag
cgtgacgggc accgtgtgcc 300 atgtggggaa ggcggaggac cgggagcggc
tggtggccac ggctgtgaag 350 cttcatggag gtatcgatat cctagtctcc
aatgctgctg tcaacccttt 400 ctttggaagc ataatggatg tcactgagga
ggtgtgggac aagactctgg 450 acattaatgt gaaggcccca gccctgatga
caaaggcagt ggtgccagaa 500 atggagaaac gaggaggcgg ctcagtggtg
atcgtgtctt ccatagcagc 550 cttcagtcca tctcctggct tcagtcctta
caatgtcagt aaaacagcct 600 tgctgggcct gaccaagacc ctggccatag
agctggcccc aaggaacatt 650 agggtgaact gcctagcacc tggacttatc
aagactagct tcagcaggat 700 gctctggatg gacaaggaaa aagaggaaag
catgaaagaa accctgcgga 750 taagaaggtt aggcgagcca gaggattgtg
ctggcatcgt gtctttcctg 800 tgctctgaag atgccagcta catcactggg
gaaacagtgg tggtgggtgg 850 aggaaccccg tcccgcctct gaggaccggg
agacagccca caggccagag 900 ttgggctcta gctcctggtg ctgttcctgc
attcacccac tggcctttcc 950 cacctctgct caccttactg ttcacctcat
caaatcagtt ctgccctgtg 1000 aaaagatcca gccttccctg ccgtcaaggt
ggcgtcttac tcgggattcc 1050 tgctgttgtt gtggccttgg gtaaaggcct
cccctgagaa cacaggacag 1100 gcctgctgac aaggctgagt ctaccttggc
aaagaccaag atattttttc 1150 ctgggccact ggtgaatctg aggggtgatg
ggagagaagg aacctggagt 1200 ggaaggagca gagttgcaaa ttaacagctt
gcaaatgagg tgcaaataaa 1250 atgcagatga ttgcgcggct ttgaaaaaaa aaa
1283 2 278 PRT Homo sapiens 2 Met His Lys Ala Gly Leu Leu Gly Leu
Cys Ala Arg Ala Trp Asn 1 5 10 15 Ser Val Arg Met Ala Ser Ser Gly
Met Thr Arg Arg Asp Pro Leu 20 25 30 Ala Asn Lys Val Ala Leu Val
Thr Ala Ser Thr Asp Gly Ile Gly 35 40 45 Phe Ala Ile Ala Arg Arg
Leu Ala Gln Asp Gly Ala His Val Val 50 55 60 Val Ser Ser Arg Lys
Gln Gln Asn Val Asp Gln Ala Val Ala Thr 65 70 75 Leu Gln Gly Glu
Gly Leu Ser Val Thr Gly Thr Val Cys His Val 80 85 90 Gly Lys Ala
Glu Asp Arg Glu Arg Leu Val Ala Thr Ala Val Lys 95 100 105 Leu His
Gly Gly Ile Asp Ile Leu Val Ser Asn Ala Ala Val Asn 110 115 120 Pro
Phe Phe Gly Ser Ile Met Asp Val Thr Glu Glu Val Trp Asp 125 130 135
Lys Thr Leu Asp Ile Asn Val Lys Ala Pro Ala Leu Met Thr Lys 140 145
150 Ala Val Val Pro Glu Met Glu Lys Arg Gly Gly Gly Ser Val Val 155
160 165 Ile Val Ser Ser Ile Ala Ala Phe Ser Pro Ser Pro Gly Phe Ser
170 175 180 Pro Tyr Asn Val Ser Lys Thr Ala Leu Leu Gly Leu Thr Lys
Thr 185 190 195 Leu Ala Ile Glu Leu Ala Pro Arg Asn Ile Arg Val Asn
Cys Leu 200 205 210 Ala Pro Gly Leu Ile Lys Thr Ser Phe Ser Arg Met
Leu Trp Met 215 220 225 Asp Lys Glu Lys Glu Glu Ser Met Lys Glu Thr
Leu Arg Ile Arg 230 235 240 Arg Leu Gly Glu Pro Glu Asp Cys Ala Gly
Ile Val Ser Phe Leu 245 250 255 Cys Ser Glu Asp Ala Ser Tyr Ile Thr
Gly Glu Thr Val Val Val 260 265 270 Gly Gly Gly Thr Pro Ser Arg Leu
275 3 21 DNA Artificial Sequence Synthetic Oligonucleotide Probe 3
gcataatgga tgtcactgag g 21 4 23 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 4 agaacaatcc tgctgaaagc tag 23 5 46 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 5 gaaacgagga
ggcggctcag tggtgatcgt gtcttccata gcagcc 46 6 3121 DNA Homo sapiens
6 gcgccctgag ctccgcctcc gggcccgata gcggcatcga gagcgcctcc 50
gtcgaggacc aggcggcgca gggggccggc gggcgaaagg aggatgaggg 100
ggcgcagcag ctgctgaccc tgcagaacca ggtggcgcgg ctggaggagg 150
agaaccgaga ctttctggct gcgctggagg acgccatgga gcagtacaaa 200
ctgcagagcg accggctgcg tgagcagcag gaggagatgg tggaactgcg 250
gctgcggtta gagctggtgc ggccaggctg ggggggcctg cggctcctga 300
atggcctgcc tcccgggtcc tttgtgcctc gacctcatac agcccccctg 350
gggggtgccc acgcccatgt gctgggcatg gtgccgcctg cctgcctccc 400
tggagatgaa gttggctctg agcagagggg agagcaggtg acaaatggca 450
gggaggctgg agctgagttg ctgactgagg tgaacaggct gggaagtggc 500
tcttcagctg cttcagagga ggaagaggag gaggaggagc cgcccaggcg 550
gaccttacac ctgcgcagaa ataggatcag caactgcagt cagagggcgg 600
gggcacgccc agggagtctg ccagagagga agggcccaga gctttgcctt 650
gaggagttgg atgcagccat tccagggtcc agagcagttg gtgggagcaa 700
ggcccgagtt caggcccgcc aggtcccccc tgccacagcc tcagagtggc 750
ggctggccca ggcccagcag aagatccggg agctggctat caacatccgc 800
atgaaggagg agcttattgg cgagctggtc cgcacaggaa aggcagctca 850
ggccctgaac cgccagcaca gccagcgtat ccgggagctg gagcaggagg 900
cagagcaggt gcgggccgag ctgagtgaag gccagaggca gctgcgggag 950
ctcgagggca aggagctcca ggatgctggc gagcggtctc ggctccagga 1000
gttccgcagg agggtcgctg cggcccagag ccaggtgcag gtgctgaagg 1050
agaagaagca ggctacggag cggctggtgt cactgtcggc ccagagtgag 1100
aagcgactgc aggagctcga gcggaacgtg cagctcatgc ggcagcagca 1150
gggacagctg cagaggcggc ttcgcgagga gacggagcag aagcggcgcc 1200
tggaggcaga aatgagcaag cggcagcacc gcgtcaagga gctggagctg 1250
aagcatgagc aacagcagaa gatcctgaag attaagacgg aagagatcgc 1300
ggccttccag aggaagaggc gcagtggcag caacggctct gtggtcagcc 1350
tggaacagca gcagaagatt gaggagcaga agaagtggct ggaccaggag 1400
atggagaagg tgctacagca gcggcgggcg ctggaggagc tgggggagga 1450
gctccacaag cgggaggcca tcctggccaa gaaggaggcc ctgatgcagg 1500
agaagacggg gctggagagc aagcgcctga gatccagcca ggccctcaac 1550
gaggacatcg tgcgagtgtc cagccggctg gagcacctgg agaaggagct 1600
gtccgagaag agcgggcagc tgcggcaggg cagcgcccag agccagcagc 1650
agatccgcgg ggagatcgac agcctgcgcc aggagaagga ctcgctgctc 1700
aagcagcgcc tggagatcga cggcaagctg aggcagggga gtctgctgtc 1750
ccccgaggag gagcggacgc tgttccagtt ggatgaggcc atcgaggccc 1800
tggatgctgc cattgagtat aagaatgagg ccatcacatg ccgccagcgg 1850
gtgcttcggg cctcagcctc gttgctgtcc cagtgcgaga tgaacctcat 1900
ggccaagctc agctacctct catcctcaga gaccagagcc ctcctctgca 1950
agtattttga caaggtggtg acgctccgag aggagcagca ccagcagcag 2000
attgccttct cggaactgga gatgcagctg gaggagcagc agaggctggt 2050
gtactggctg gaggtggccc tggagcggca gcgcctggag atggaccgcc 2100
agctgaccct gcagcagaag gagcacgagc agaacatgca gctgctcctg 2150
cagcagagtc gagaccacct cggtgaaggg ttagcagaca gcaggaggca 2200
gtatgaggcc cggattcaag ctctggagaa ggaactgggc cgttacatgt 2250
ggataaacca ggaactgaaa cagaagctcg gcggtgtgaa cgctgtaggc 2300
cacagcaggg gtggggagaa gaggagcctg tgctcggagg gcagacaggc 2350
tcctggaaat gaagatgagc tccacctggc acccgagctt ctctggctgt 2400
cccccctcac tgagggggcc ccccgcaccc gggaggagac gcgggacttg 2450
gtccacgctc cgttaccctt gacctggaaa cgctcgagcc tgtgtggtga 2500
ggagcagggg tcccccgagg aactgaggca gcgggaggcg gctgagcccc 2550
tggtggggcg ggtgcttcct gtgggtgagg caggcctgcc ctggaacttt 2600
gggcctttgt ccaagccccg gcgggaactg cgacgagcca gcccggggat 2650
gattgatgtc cggaaaaacc ccctgtaagc cctcggggca gaccctgcct 2700
tggagggaga ctccgagcct gctgaaaggg gcagctgcct gttttgcttc 2750
tgtgaagggc agtccttacc gcacacccta aatccaggcc ctcatctgta 2800
ccctcactgg gatcaacaaa tttgggccat ggcccaaaag aactggaccc 2850
tcatttaaca aaataatatg caaattccca ccacttactt ccatgaagct 2900
gtggtaccca attgccgcct tgtgtcttgc tcgaatctca ggacaattct 2950
ggtttcaggc gtaaatggat gtgcttgtag ttcaggggtt tggccaagaa 3000
tcatcacgaa agggtcggtg gcaaccaggt tgtggtttaa atggtcttat 3050
gtatataggg gaaactggga gactttagga tcttaaaaaa ccatttaata 3100
aaaaaaaatc tttgaaggga c 3121 7 830 PRT Homo sapiens 7 Met Glu Gln
Tyr Lys Leu Gln Ser Asp Arg Leu Arg Glu Gln Gln 1 5 10 15 Glu Glu
Met Val Glu Leu Arg Leu Arg Leu Glu Leu Val Arg Pro 20 25 30 Gly
Trp Gly Gly Leu Arg Leu Leu Asn Gly Leu Pro Pro Gly Ser 35 40 45
Phe Val Pro Arg Pro His Thr Ala Pro Leu Gly Gly Ala His Ala 50 55
60 His Val Leu Gly Met Val Pro Pro Ala Cys Leu Pro Gly Asp Glu 65
70 75 Val Gly Ser Glu Gln Arg Gly Glu Gln Val Thr Asn Gly Arg Glu
80 85 90 Ala Gly Ala Glu Leu Leu Thr Glu Val Asn Arg Leu Gly Ser
Gly 95 100 105 Ser Ser Ala Ala Ser Glu Glu Glu Glu Glu Glu Glu Glu
Pro Pro 110 115 120 Arg Arg Thr Leu His Leu Arg Arg Asn Arg Ile Ser
Asn Cys Ser 125 130 135 Gln Arg Ala Gly Ala Arg Pro Gly Ser Leu Pro
Glu Arg Lys Gly 140 145 150 Pro Glu Leu Cys Leu Glu Glu Leu Asp Ala
Ala Ile Pro Gly Ser 155 160 165 Arg Ala Val Gly Gly Ser Lys Ala Arg
Val Gln Ala Arg Gln Val 170 175 180 Pro Pro Ala Thr Ala Ser Glu Trp
Arg Leu Ala Gln Ala Gln Gln 185 190 195 Lys Ile Arg Glu Leu Ala Ile
Asn Ile Arg Met Lys Glu Glu Leu 200 205 210 Ile Gly Glu Leu Val Arg
Thr Gly Lys Ala Ala Gln Ala Leu Asn 215 220 225 Arg Gln His Ser Gln
Arg Ile Arg Glu Leu Glu Gln Glu Ala Glu 230 235 240 Gln Val Arg Ala
Glu Leu Ser Glu Gly Gln Arg Gln Leu Arg Glu 245 250 255 Leu Glu Gly
Lys Glu Leu Gln Asp Ala Gly Glu Arg Ser Arg Leu 260 265 270 Gln Glu
Phe Arg Arg Arg Val Ala Ala Ala Gln Ser Gln Val Gln 275 280 285 Val
Leu Lys Glu Lys Lys Gln Ala Thr Glu Arg Leu Val Ser Leu 290 295 300
Ser Ala Gln Ser Glu Lys Arg Leu Gln Glu Leu Glu Arg Asn Val 305 310
315 Gln Leu Met Arg Gln Gln Gln Gly Gln Leu Gln Arg Arg Leu Arg 320
325 330 Glu Glu Thr Glu Gln Lys Arg Arg Leu Glu Ala Glu Met Ser Lys
335 340 345 Arg Gln His Arg Val Lys Glu Leu Glu Leu Lys His Glu Gln
Gln 350 355 360 Gln Lys Ile Leu Lys Ile Lys Thr Glu Glu Ile Ala Ala
Phe Gln 365 370 375 Arg Lys Arg Arg Ser Gly Ser Asn Gly Ser Val Val
Ser Leu Glu 380 385 390 Gln Gln Gln Lys Ile Glu Glu Gln Lys Lys Trp
Leu Asp Gln Glu 395 400 405 Met Glu Lys Val Leu Gln Gln Arg Arg Ala
Leu Glu Glu Leu Gly 410 415 420 Glu Glu Leu His Lys Arg Glu Ala Ile
Leu Ala Lys Lys Glu Ala 425 430 435 Leu Met Gln Glu Lys Thr Gly Leu
Glu Ser Lys Arg Leu Arg Ser 440 445 450 Ser Gln Ala Leu Asn Glu Asp
Ile Val Arg Val Ser Ser Arg Leu 455 460 465 Glu His Leu Glu Lys Glu
Leu Ser Glu Lys Ser Gly Gln Leu Arg 470 475 480 Gln Gly Ser Ala Gln
Ser Gln Gln Gln Ile Arg Gly Glu Ile Asp 485 490 495 Ser Leu Arg Gln
Glu Lys Asp Ser Leu Leu Lys Gln Arg Leu Glu 500 505 510 Ile Asp Gly
Lys Leu Arg Gln Gly Ser Leu Leu Ser Pro Glu Glu 515 520 525 Glu Arg
Thr Leu Phe Gln Leu Asp Glu Ala Ile Glu Ala Leu Asp 530 535 540 Ala
Ala Ile Glu Tyr Lys Asn Glu Ala Ile Thr Cys Arg Gln Arg 545 550 555
Val Leu Arg Ala Ser Ala Ser Leu Leu Ser Gln Cys Glu Met Asn 560 565
570 Leu Met Ala Lys Leu Ser Tyr Leu Ser Ser Ser Glu Thr Arg Ala 575
580 585 Leu Leu Cys Lys Tyr Phe Asp Lys Val Val Thr Leu Arg Glu Glu
590 595 600 Gln His Gln Gln Gln Ile Ala Phe Ser Glu Leu Glu Met Gln
Leu 605 610 615 Glu Glu Gln Gln Arg Leu Val Tyr Trp Leu Glu Val Ala
Leu Glu 620 625 630 Arg Gln Arg Leu Glu Met Asp Arg Gln Leu Thr Leu
Gln Gln Lys 635 640 645 Glu His Glu Gln Asn Met Gln Leu Leu Leu Gln
Gln Ser Arg Asp 650 655 660 His Leu Gly Glu Gly Leu Ala Asp Ser Arg
Arg Gln Tyr Glu Ala 665 670 675 Arg Ile Gln Ala Leu Glu Lys Glu Leu
Gly Arg Tyr Met Trp Ile 680 685 690 Asn Gln Glu Leu Lys Gln Lys Leu
Gly Gly Val Asn Ala Val Gly 695 700 705 His Ser Arg Gly Gly Glu Lys
Arg Ser Leu Cys Ser Glu Gly Arg 710 715 720 Gln Ala Pro Gly Asn Glu
Asp Glu Leu His Leu Ala Pro Glu Leu 725 730 735 Leu Trp Leu Ser Pro
Leu Thr Glu Gly Ala Pro Arg Thr Arg Glu 740 745 750 Glu Thr Arg Asp
Leu Val His Ala Pro Leu Pro Leu Thr Trp Lys 755 760 765 Arg Ser Ser
Leu Cys Gly Glu Glu Gln Gly Ser Pro Glu Glu Leu 770 775 780 Arg Gln
Arg Glu Ala Ala Glu Pro Leu Val Gly Arg Val Leu Pro 785 790 795 Val
Gly Glu Ala Gly Leu Pro Trp Asn Phe Gly Pro Leu Ser Lys 800 805 810
Pro Arg Arg Glu Leu Arg Arg Ala Ser Pro Gly Met Ile Asp Val 815 820
825 Arg Lys Asn Pro Leu 830 8 662 DNA Homo sapiens 8 attctcctag
agcatctttg gaagcatgag gccacgatgc tgcatcttgg 50 ctcttgtctg
ctggataaca gtcttcctcc tccagtgttc aaaaggaact 100 acagacgctc
ctgttggctc aggactgtgg ctgtgccagc cgacacccag 150 gtgtgggaac
aagatctaca acccttcaga gcagtgctgt tatgatgatg 200 ccatcttatc
cttaaaggag acccgccgct gtggctccac ctgcaccttc 250 tggccctgct
ttgagctctg ctgtcccgag tcttttggcc cccagcagaa 300 gtttcttgtg
aagttgaggg ttctgggtat gaagtctcag tgtcacttat 350 ctcccatctc
ccggagctgt accaggaaca ggaggcacgt cctgtaccca 400 taaaaacccc
aggctccact ggcagacggc agacaagggg agaagagacg 450 aagcagctgg
acatcggaga ctacagttga acttcggaga gaagcaactt 500 gacttcagag
ggatggctca atgacatagc tttggagagg agcccagctg 550 gggatggcca
gacttcaggg gaagaatgcc ttcctgcttc atcccctttc 600 cagctcccct
tcccgctgag agccactttc atcggcaata aaatccccca 650 catttaccat ct 662 9
125 PRT Homo sapiens 9 Met Arg Pro Arg Cys Cys Ile Leu Ala Leu Val
Cys Trp Ile Thr 1 5 10 15 Val Phe Leu Leu Gln Cys Ser Lys Gly Thr
Thr Asp Ala Pro Val 20 25 30 Gly Ser Gly Leu Trp Leu Cys Gln Pro
Thr Pro Arg Cys Gly Asn 35 40 45 Lys Ile Tyr Asn Pro Ser Glu Gln
Cys Cys Tyr Asp Asp Ala Ile 50 55 60 Leu Ser Leu Lys Glu Thr Arg
Arg Cys Gly Ser Thr Cys Thr Phe 65 70 75 Trp Pro Cys Phe Glu Leu
Cys Cys Pro Glu Ser Phe Gly Pro Gln 80 85 90 Gln Lys Phe Leu Val
Lys Leu Arg Val Leu Gly Met Lys Ser Gln 95 100 105 Cys His Leu Ser
Pro Ile Ser Arg Ser Cys Thr Arg Asn Arg Arg 110 115 120 His Val Leu
Tyr Pro 125 10 1942 DNA Homo sapiens 10 cccacgcgtc cgcccacgcg
tccgggtgcc actcgcgcgc cggccgcgct 50 ccgggcttct cttttccctc
cgacgcgcca cggctgccca gacattccgg 100 ctgccgggtc tggagagctc
cccgaacccc tccgcggaga ggagcgaggc 150 ggcgccaggg tggcccccgg
ggcgcgcttg gtctcggaga agcggggacg 200 aggccggagg atgagcgact
gagggcgacg cgggcactga cgcgagttgg 250 ggccgcgact
accggcagct gacagcgcga tgagcgactc cccagagacg 300 ccctagcccg
gtgtgcgcgc caggcggagc gcgcaggtgg ggctgggctg 350 ttagtggtcc
gccccacgcg ggtcgccggc cggcccagga tgggcgctgg 400 caacccgggc
ccgcgcccgc cgctgctacc cctgcgcccg ctgcgagccc 450 ggcgtccggc
ccgcgccctg cgctcatgga cggcggctcc cggctggcgg 500 cggcgcgccc
ccgggctgtg aatgcgactc gcccctcggc cgcgctcccc 550 gcccgcccgc
ccgccgggac gtggtagggg atgcccagct ccactgcgat 600 ggcagttggc
gcgctctcca gttccctcct ggtcacctgc tgcctgatgg 650 tggctctgtg
cagtccgagc atcccgctgg agaagctggc ccaggcacca 700 gagcagccgg
gccaggagaa gcgtgagcac gccactcggg acggcccggg 750 gcgggtgaac
gagctcgggc gcccggcgag ggacgagggc ggcagcggcc 800 gggactggaa
gagcaagagc ggccgtgggc tcgccggccg tgagccgtgg 850 agcaagctga
agcaggcctg ggtctcccag ggcgggggcg ccaaggccgg 900 ggatctgcag
gtccggcccc gcggggacac cccgcaggcg gaagccctgg 950 ccgcagccgc
ccaggacgcg attggcccgg aactcgcgcc cacgcccgag 1000 ccacccgagg
agtacgtgta cccggactac cgtggcaagg gctgcgtgga 1050 cgagagcggc
ttcgtgtacg cgatcgggga gaagttcgcg ccgggcccct 1100 cggcctgccc
gtgcctgtgc accgaggagg ggccgctgtg cgcgcagccc 1150 gagtgcccga
ggctgcaccc gcgctgcatc cacgtcgaca cgagccagtg 1200 ctgcccgcag
tgcaaggaga ggaagaacta ctgcgagttc cggggcaaga 1250 cctatcagac
tttggaggag ttcgtggtgt ctccatgcga gaggtgtcgc 1300 tgtgaagcca
acggtgaggt gctatgcaca gtgtcagcgt gtccccagac 1350 ggagtgtgtg
gaccctgtgt acgagcctga tcagtgctgt cccatctgca 1400 aaaatggtcc
aaactgcttt gcagaaaccg cggtgatccc tgctggcaga 1450 gaagtgaaga
ctgacgagtg caccatatgc cactgtactt atgaggaagg 1500 cacatggaga
atcgagcggc aggccatgtg cacgagacat gaatgcaggc 1550 aaatgtagac
gcttcccaga acacaaactc tgactttttc tagaacattt 1600 tactgatgtg
aacattctag atgactctgg gaactatcag tcaaagaaga 1650 cttttgatga
ggaataatgg aaaattgttg gtacttttcc ttttcttgat 1700 aacagttact
acaacagaag gaaatggata tatttcaaaa catcaacaag 1750 aactttgggc
ataaaatcct tctctaaata aatgtgctat tttcacagta 1800 agtacacaaa
agtacactat tatatatcaa atgtatttct ataatccctc 1850 cattagagag
cttatataag tgttttctat agatgcagat taaaaatgct 1900 gtgttgtcaa
ccgtcaaaaa aaaaaaaaaa aaaaaaaaaa aa 1942 11 325 PRT Homo sapiens 11
Met Pro Ser Ser Thr Ala Met Ala Val Gly Ala Leu Ser Ser Ser 1 5 10
15 Leu Leu Val Thr Cys Cys Leu Met Val Ala Leu Cys Ser Pro Ser 20
25 30 Ile Pro Leu Glu Lys Leu Ala Gln Ala Pro Glu Gln Pro Gly Gln
35 40 45 Glu Lys Arg Glu His Ala Thr Arg Asp Gly Pro Gly Arg Val
Asn 50 55 60 Glu Leu Gly Arg Pro Ala Arg Asp Glu Gly Gly Ser Gly
Arg Asp 65 70 75 Trp Lys Ser Lys Ser Gly Arg Gly Leu Ala Gly Arg
Glu Pro Trp 80 85 90 Ser Lys Leu Lys Gln Ala Trp Val Ser Gln Gly
Gly Gly Ala Lys 95 100 105 Ala Gly Asp Leu Gln Val Arg Pro Arg Gly
Asp Thr Pro Gln Ala 110 115 120 Glu Ala Leu Ala Ala Ala Ala Gln Asp
Ala Ile Gly Pro Glu Leu 125 130 135 Ala Pro Thr Pro Glu Pro Pro Glu
Glu Tyr Val Tyr Pro Asp Tyr 140 145 150 Arg Gly Lys Gly Cys Val Asp
Glu Ser Gly Phe Val Tyr Ala Ile 155 160 165 Gly Glu Lys Phe Ala Pro
Gly Pro Ser Ala Cys Pro Cys Leu Cys 170 175 180 Thr Glu Glu Gly Pro
Leu Cys Ala Gln Pro Glu Cys Pro Arg Leu 185 190 195 His Pro Arg Cys
Ile His Val Asp Thr Ser Gln Cys Cys Pro Gln 200 205 210 Cys Lys Glu
Arg Lys Asn Tyr Cys Glu Phe Arg Gly Lys Thr Tyr 215 220 225 Gln Thr
Leu Glu Glu Phe Val Val Ser Pro Cys Glu Arg Cys Arg 230 235 240 Cys
Glu Ala Asn Gly Glu Val Leu Cys Thr Val Ser Ala Cys Pro 245 250 255
Gln Thr Glu Cys Val Asp Pro Val Tyr Glu Pro Asp Gln Cys Cys 260 265
270 Pro Ile Cys Lys Asn Gly Pro Asn Cys Phe Ala Glu Thr Ala Val 275
280 285 Ile Pro Ala Gly Arg Glu Val Lys Thr Asp Glu Cys Thr Ile Cys
290 295 300 His Cys Thr Tyr Glu Glu Gly Thr Trp Arg Ile Glu Arg Gln
Ala 305 310 315 Met Cys Thr Arg His Glu Cys Arg Gln Met 320 325 12
24 DNA Artificial Sequence Synthetic Oligonucleotide Probe 12
gaggtgtcgc tgtgaagcca acgg 24 13 24 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 13 cgctcgattc tccatgtgcc ttcc 24 14
45 DNA Artificial Sequence Synthetic Oligonucleotide Probe 14
gacggagtgt gtggaccctg tgtacgagcc tgatcagtgc tgtcc 45 15 1587 DNA
Homo sapiens 15 cagccacaga cgggtcatga gcgcggtatt actgctggcc
ctcctggggt 50 tcatcctccc actgccagga gtgcaggcgc tgctctgcca
gtttgggaca 100 gttcagcatg tgtggaaggt gtccgaccta ccccggcaat
ggacccctaa 150 gaacaccagc tgcgacagcg gcttggggtg ccaggacacg
ttgatgctca 200 ttgagagcgg accccaagtg agcctggtgc tctccaaggg
ctgcacggag 250 gccaaggacc aggagccccg cgtcactgag caccggatgg
gccccggcct 300 ctccctgatc tcctacacct tcgtgtgccg ccaggaggac
ttctgcaaca 350 acctcgttaa ctccctcccg ctttgggccc cacagccccc
agcagaccca 400 ggatccttga ggtgcccagt ctgcttgtct atggaaggct
gtctggaggg 450 gacaacagaa gagatctgcc ccaaggggac cacacactgt
tatgatggcc 500 tcctcaggct caggggagga ggcatcttct ccaatctgag
agtccaggga 550 tgcatgcccc agccaggttg caacctgctc aatgggacac
aggaaattgg 600 gcccgtgggt atgactgaga actgcaatag gaaagatttt
ctgacctgtc 650 atcgggggac caccattatg acacacggaa acttggctca
agaacccact 700 gattggacca catcgaatac cgagatgtgc gaggtggggc
aggtgtgtca 750 ggagacgctg ctgctcatag atgtaggact cacatcaacc
ctggtgggga 800 caaaaggctg cagcactgtt ggggctcaaa attcccagaa
gaccaccatc 850 cactcagccc ctcctggggt gcttgtggcc tcctataccc
acttctgctc 900 ctcggacctg tgcaatagtg ccagcagcag cagcgttctg
ctgaactccc 950 tccctcctca agctgcccct gtcccaggag accggcagtg
tcctacctgt 1000 gtgcagcccc ttggaacctg ttcaagtggc tccccccgaa
tgacctgccc 1050 caggggcgcc actcattgtt atgatgggta cattcatctc
tcaggaggtg 1100 ggctgtccac caaaatgagc attcagggct gcgtggccca
accttccagc 1150 ttcttgttga accacaccag acaaatcggg atcttctctg
cgcgtgagaa 1200 gcgtgatgtg cagcctcctg cctctcagca tgagggaggt
ggggctgagg 1250 gcctggagtc tctcacttgg ggggtggggc tggcactggc
cccagcgctg 1300 tggtggggag tggtttgccc ttcctgctaa ctctattacc
cccacgattc 1350 ttcaccgctg ctgaccaccc acactcaacc tccctctgac
ctcataacct 1400 aatggccttg gacaccagat tctttcccat tctgtccatg
aatcatcttc 1450 cccacacaca atcattcata tctactcacc taacagcaac
actggggaga 1500 gcctggagca tccggacttg ccctatggga gaggggacgc
tggaggagtg 1550 gctgcatgta tctgataata cagaccctgt cctttca 1587 16
437 PRT Homo sapiens 16 Met Ser Ala Val Leu Leu Leu Ala Leu Leu Gly
Phe Ile Leu Pro 1 5 10 15 Leu Pro Gly Val Gln Ala Leu Leu Cys Gln
Phe Gly Thr Val Gln 20 25 30 His Val Trp Lys Val Ser Asp Leu Pro
Arg Gln Trp Thr Pro Lys 35 40 45 Asn Thr Ser Cys Asp Ser Gly Leu
Gly Cys Gln Asp Thr Leu Met 50 55 60 Leu Ile Glu Ser Gly Pro Gln
Val Ser Leu Val Leu Ser Lys Gly 65 70 75 Cys Thr Glu Ala Lys Asp
Gln Glu Pro Arg Val Thr Glu His Arg 80 85 90 Met Gly Pro Gly Leu
Ser Leu Ile Ser Tyr Thr Phe Val Cys Arg 95 100 105 Gln Glu Asp Phe
Cys Asn Asn Leu Val Asn Ser Leu Pro Leu Trp 110 115 120 Ala Pro Gln
Pro Pro Ala Asp Pro Gly Ser Leu Arg Cys Pro Val 125 130 135 Cys Leu
Ser Met Glu Gly Cys Leu Glu Gly Thr Thr Glu Glu Ile 140 145 150 Cys
Pro Lys Gly Thr Thr His Cys Tyr Asp Gly Leu Leu Arg Leu 155 160 165
Arg Gly Gly Gly Ile Phe Ser Asn Leu Arg Val Gln Gly Cys Met 170 175
180 Pro Gln Pro Gly Cys Asn Leu Leu Asn Gly Thr Gln Glu Ile Gly 185
190 195 Pro Val Gly Met Thr Glu Asn Cys Asn Arg Lys Asp Phe Leu Thr
200 205 210 Cys His Arg Gly Thr Thr Ile Met Thr His Gly Asn Leu Ala
Gln 215 220 225 Glu Pro Thr Asp Trp Thr Thr Ser Asn Thr Glu Met Cys
Glu Val 230 235 240 Gly Gln Val Cys Gln Glu Thr Leu Leu Leu Ile Asp
Val Gly Leu 245 250 255 Thr Ser Thr Leu Val Gly Thr Lys Gly Cys Ser
Thr Val Gly Ala 260 265 270 Gln Asn Ser Gln Lys Thr Thr Ile His Ser
Ala Pro Pro Gly Val 275 280 285 Leu Val Ala Ser Tyr Thr His Phe Cys
Ser Ser Asp Leu Cys Asn 290 295 300 Ser Ala Ser Ser Ser Ser Val Leu
Leu Asn Ser Leu Pro Pro Gln 305 310 315 Ala Ala Pro Val Pro Gly Asp
Arg Gln Cys Pro Thr Cys Val Gln 320 325 330 Pro Leu Gly Thr Cys Ser
Ser Gly Ser Pro Arg Met Thr Cys Pro 335 340 345 Arg Gly Ala Thr His
Cys Tyr Asp Gly Tyr Ile His Leu Ser Gly 350 355 360 Gly Gly Leu Ser
Thr Lys Met Ser Ile Gln Gly Cys Val Ala Gln 365 370 375 Pro Ser Ser
Phe Leu Leu Asn His Thr Arg Gln Ile Gly Ile Phe 380 385 390 Ser Ala
Arg Glu Lys Arg Asp Val Gln Pro Pro Ala Ser Gln His 395 400 405 Glu
Gly Gly Gly Ala Glu Gly Leu Glu Ser Leu Thr Trp Gly Val 410 415 420
Gly Leu Ala Leu Ala Pro Ala Leu Trp Trp Gly Val Val Cys Pro 425 430
435 Ser Cys 17 2387 DNA Homo sapiens 17 cgacgatgct acgcgcgccc
ggctgcctcc tccggacctc cgtagcgcct 50 gccgcggccc tggctgcggc
gctgctctcg tcgcttgcgc gctgctctct 100 tctagagccg agggacccgg
tggcctcgtc gctcagcccc tatttcggca 150 ccaagactcg ctacgaggat
gtcaaccccg tgctattgtc gggccccgag 200 gctccgtggc gggaccctga
gctgctggag gggacctgca ccccggtgca 250 gctggtcgcc ctcattcgcc
acggcacccg ctaccccacg gtcaaacaga 300 tccgcaagct gaggcagctg
cacgggttgc tgcaggcccg cgggtccagg 350 gatggcgggg ctagtagtac
cggcagccgc gacctgggtg cagcgctggc 400 cgactggcct ttgtggtacg
cggactggat ggacgggcag ctagtagaga 450 agggacggca ggatatgcga
cagctggcgc tgcgtctggc ctcgctcttc 500 ccggcccttt tcagccgtga
gaactacggc cgcctgcggc tcatcaccag 550 ttccaagcac cgctgcatgg
atagcagcgc cgccttcctg caggggctgt 600 ggcagcacta ccaccctggc
ttgccgccgc cggacgtcgc agatatggag 650 tttggacctc caacagttaa
tgataaacta atgagatttt ttgatcactg 700 tgagaagttt ttaactgaag
tagaaaaaaa tgctacagct ctttatcacg 750 tggaagcctt caaaactgga
ccagaaatgc agaacatttt aaaaaaagtt 800 gcagctactt tgcaagtgcc
agtaaatgat ttaaatgcag atttaattca 850 agtagccttt ttcacctgtt
catttgacct ggcaattaaa ggtgttaaat 900 ctccttggtg tgatgttttt
gacatagatg atgcaaaggt attagaatat 950 ttaaatgatc tgaaacaata
ttggaaaaga ggatatgggt atactattaa 1000 cagtcgatcc agctgcacct
tgtttcagga tatctttcag cacttggaca 1050 aagcagttga acagaaacaa
aggtctcagc caatttcttc tccagtcatc 1100 ctccagtttg gtcatgcaga
gactcttctt ccactgcttt ctctcatggg 1150 ctacttcaaa gacaaggaac
ccctaacagc gtacaattac aaaaaacaaa 1200 tgcatcggaa gttccgaagt
ggtctcattg taccttatgc ctcgaacctg 1250 atatttgtgc tttaccactg
tgaaaatgct aagactccta aagaacaatt 1300 ccgagtgcag atgttattaa
atgaaaaggt gttacctttg gcttactcac 1350 aagaaactgt ttcattttat
gaagatctga agaaccacta caaggacatc 1400 cttcagagtt gtcaaaccag
tgaagaatgt gaattagcaa gggctaacag 1450 tacatctgat gaactatgag
taactgaaga acatttttaa ttctttagga 1500 atctgcaatg agtgattaca
tgcttgtaat aggtaggcaa ttccttgatt 1550 acaggaagct tttatattac
ttgagtattt ctgtcttttc acagaaaaac 1600 attgggtttc tctctgggtt
tggacatgaa atgtaagaaa agatttttca 1650 ctggagcagc tctcttaagg
agaaacaaat ctatttagag aaacagctgg 1700 ccctgcaaat gtttacagaa
atgaaattct tcctacttat ataagaaatc 1750 tcacactgag atagaattgt
gatttcataa taacacttga aaagtgctgg 1800 agtaacaaaa tatctcagtt
ggaccatcct taacttgatt gaactgtcta 1850 ggaactttac agattgttct
gcagttctct cttcttttcc tcaggtagga 1900 cagctctagc attttcttaa
tcaggaatat tgtggtaagc tgggagtatc 1950 actctggaag aaagtaacat
ctccagatga gaatttgaaa caagaaacag 2000 agtgttgtaa aaggacacct
tcactgaagc aagtcggaaa gtacaatgaa 2050 aataaatatt tttggtattt
atttatgaaa tatttgaaca ttttttcaat 2100 aattcctttt tacttctagg
aagtctcaaa agaccatctt aaattattat 2150 atgtttggac aattagcaac
aagtcagata gttagaatcg aagtttttca 2200 aatccattgc ttagctaact
ttttcattct gtcacttggc ttcgattttt 2250 atattttcct attatatgaa
atgtatcttt tggttgtttg atttttcttt 2300 ctttctttgt aaatagttct
gagttctgtc aaatgccgtg aaagtatttg 2350 ctataataaa gaaaattctt
gtgactttaa aaaaaaa 2387 18 487 PRT Homo sapiens 18 Met Leu Arg Ala
Pro Gly Cys Leu Leu Arg Thr Ser Val Ala Pro 1 5 10 15 Ala Ala Ala
Leu Ala Ala Ala Leu Leu Ser Ser Leu Ala Arg Cys 20 25 30 Ser Leu
Leu Glu Pro Arg Asp Pro Val Ala Ser Ser Leu Ser Pro 35 40 45 Tyr
Phe Gly Thr Lys Thr Arg Tyr Glu Asp Val Asn Pro Val Leu 50 55 60
Leu Ser Gly Pro Glu Ala Pro Trp Arg Asp Pro Glu Leu Leu Glu 65 70
75 Gly Thr Cys Thr Pro Val Gln Leu Val Ala Leu Ile Arg His Gly 80
85 90 Thr Arg Tyr Pro Thr Val Lys Gln Ile Arg Lys Leu Arg Gln Leu
95 100 105 His Gly Leu Leu Gln Ala Arg Gly Ser Arg Asp Gly Gly Ala
Ser 110 115 120 Ser Thr Gly Ser Arg Asp Leu Gly Ala Ala Leu Ala Asp
Trp Pro 125 130 135 Leu Trp Tyr Ala Asp Trp Met Asp Gly Gln Leu Val
Glu Lys Gly 140 145 150 Arg Gln Asp Met Arg Gln Leu Ala Leu Arg Leu
Ala Ser Leu Phe 155 160 165 Pro Ala Leu Phe Ser Arg Glu Asn Tyr Gly
Arg Leu Arg Leu Ile 170 175 180 Thr Ser Ser Lys His Arg Cys Met Asp
Ser Ser Ala Ala Phe Leu 185 190 195 Gln Gly Leu Trp Gln His Tyr His
Pro Gly Leu Pro Pro Pro Asp 200 205 210 Val Ala Asp Met Glu Phe Gly
Pro Pro Thr Val Asn Asp Lys Leu 215 220 225 Met Arg Phe Phe Asp His
Cys Glu Lys Phe Leu Thr Glu Val Glu 230 235 240 Lys Asn Ala Thr Ala
Leu Tyr His Val Glu Ala Phe Lys Thr Gly 245 250 255 Pro Glu Met Gln
Asn Ile Leu Lys Lys Val Ala Ala Thr Leu Gln 260 265 270 Val Pro Val
Asn Asp Leu Asn Ala Asp Leu Ile Gln Val Ala Phe 275 280 285 Phe Thr
Cys Ser Phe Asp Leu Ala Ile Lys Gly Val Lys Ser Pro 290 295 300 Trp
Cys Asp Val Phe Asp Ile Asp Asp Ala Lys Val Leu Glu Tyr 305 310 315
Leu Asn Asp Leu Lys Gln Tyr Trp Lys Arg Gly Tyr Gly Tyr Thr 320 325
330 Ile Asn Ser Arg Ser Ser Cys Thr Leu Phe Gln Asp Ile Phe Gln 335
340 345 His Leu Asp Lys Ala Val Glu Gln Lys Gln Arg Ser Gln Pro Ile
350 355 360 Ser Ser Pro Val Ile Leu Gln Phe Gly His Ala Glu Thr Leu
Leu 365 370 375 Pro Leu Leu Ser Leu Met Gly Tyr Phe Lys Asp Lys Glu
Pro Leu 380 385 390 Thr Ala Tyr Asn Tyr Lys Lys Gln Met His Arg Lys
Phe Arg Ser 395 400 405 Gly Leu Ile Val Pro Tyr Ala Ser Asn Leu Ile
Phe Val Leu Tyr 410 415 420 His Cys Glu Asn Ala Lys Thr
Pro Lys Glu Gln Phe Arg Val Gln 425 430 435 Met Leu Leu Asn Glu Lys
Val Leu Pro Leu Ala Tyr Ser Gln Glu 440 445 450 Thr Val Ser Phe Tyr
Glu Asp Leu Lys Asn His Tyr Lys Asp Ile 455 460 465 Leu Gln Ser Cys
Gln Thr Ser Glu Glu Cys Glu Leu Ala Arg Ala 470 475 480 Asn Ser Thr
Ser Asp Glu Leu 485 19 3554 DNA Homo sapiens 19 gggactacaa
gccgcgccgc gctgccgctg gcccctcagc aaccctcgac 50 atggcgctga
ggcggccacc gcgactccgg ctctgcgctc ggctgcctga 100 cttcttcctg
ctgctgcttt tcaggggctg cctgataggg gctgtaaatc 150 tcaaatccag
caatcgaacc ccagtggtac aggaatttga aagtgtggaa 200 ctgtcttgca
tcattacgga ttcgcagaca agtgacccca ggatcgagtg 250 gaagaaaatt
caagatgaac aaaccacata tgtgtttttt gacaacaaaa 300 ttcagggaga
cttggcgggt cgtgcagaaa tactggggaa gacatccctg 350 aagatctgga
atgtgacacg gagagactca gccctttatc gctgtgaggt 400 cgttgctcga
aatgaccgca aggaaattga tgagattgtg atcgagttaa 450 ctgtgcaagt
gaagccagtg acccctgtct gtagagtgcc gaaggctgta 500 ccagtaggca
agatggcaac actgcactgc caggagagtg agggccaccc 550 ccggcctcac
tacagctggt atcgcaatga tgtaccactg cccacggatt 600 ccagagccaa
tcccagattt cgcaattctt ctttccactt aaactctgaa 650 acaggcactt
tggtgttcac tgctgttcac aaggacgact ctgggcagta 700 ctactgcatt
gcttccaatg acgcaggctc agccaggtgt gaggagcagg 750 agatggaagt
ctatgacctg aacattggcg gaattattgg gggggttctg 800 gttgtccttg
ctgtactggc cctgatcacg ttgggcatct gctgtgcata 850 cagacgtggc
tacttcatca acaataaaca ggatggagaa agttacaaga 900 acccagggaa
accagatgga gttaactaca tccgcactga cgaggagggc 950 gacttcagac
acaagtcatc gtttgtgatc tgagacccgc ggtgtggctg 1000 agagcgcaca
gagcgcacgt gcacatacct ctgctagaaa ctcctgtcaa 1050 ggcagcgaga
gctgatgcac tcggacagag ctagacactc attcagaagc 1100 ttttcgtttt
ggccaaagtt gaccactact cttcttactc taacaagcca 1150 catgaataga
agaattttcc tcaagatgga cccggtaaat ataaccacaa 1200 ggaagcgaaa
ctgggtgcgt tcactgagtt gggttcctaa tctgtttctg 1250 gcctgattcc
cgcatgagta ttagggtgat cttaaagagt ttgctcacgt 1300 aaacgcccgt
gctgggccct gtgaagccag catgttcacc actggtcgtt 1350 cagcagccac
gacagcacca tgtgagatgg cgaggtggct ggacagcacc 1400 agcagcgcat
cccggcggga acccagaaaa ggcttcttac acagcagcct 1450 tacttcatcg
gcccacagac accaccgcag tttcttctta aaggctctgc 1500 tgatcggtgt
tgcagtgtcc attgtggaga agctttttgg atcagcattt 1550 tgtaaaaaca
accaaaatca ggaaggtaaa ttggttgctg gaagagggat 1600 cttgcctgag
gaaccctgct tgtccaacag ggtgtcagga tttaaggaaa 1650 accttcgtct
taggctaagt ctgaaatggt actgaaatat gcttttctat 1700 gggtcttgtt
tattttataa aattttacat ctaaattttt gctaaggatg 1750 tattttgatt
attgaaaaga aaatttctat ttaaactgta aatatattgt 1800 catacaatgt
taaataacct atttttttaa aaaagttcaa cttaaggtag 1850 aagttccaag
ctactagtgt taaattggaa aatatcaata attaagagta 1900 ttttacccaa
ggaatcctct catggaagtt tactgtgatg ttccttttct 1950 cacacaagtt
ttagcctttt tcacaaggga actcatactg tctacacatc 2000 agaccatagt
tgcttaggaa acctttaaaa attccagtta agcaatgttg 2050 aaatcagttt
gcatctcttc aaaagaaacc tctcaggtta gctttgaact 2100 gcctcttcct
gagatgacta ggacagtctg tacccagagg ccacccagaa 2150 gccctcagat
gtacatacac agatgccagt cagctcctgg ggttgcgcca 2200 ggcgcccccg
ctctagctca ctgttgcctc gctgtctgcc aggaggccct 2250 gccatccttg
ggccctggca gtggctgtgt cccagtgagc tttactcacg 2300 tggcccttgc
ttcatccagc acagctctca ggtgggcact gcagggacac 2350 tggtgtcttc
catgtagcgt cccagctttg ggctcctgta acagacctct 2400 ttttggttat
ggatggctca caaaataggg cccccaatgc tatttttttt 2450 ttttaagttt
gtttaattat ttgttaagat tgtctaaggc caaaggcaat 2500 tgcgaaatca
agtctgtcaa gtacaataac atttttaaaa gaaaatggat 2550 cccactgttc
ctctttgcca cagagaaagc acccagacgc cacaggctct 2600 gtcgcatttc
aaaacaaacc atgatggagt ggcggccagt ccagcctttt 2650 aaagaacgtc
aggtggagca gccaggtgaa aggcctggcg gggaggaaag 2700 tgaaacgcct
gaatcaaaag cagttttcta attttgactt taaatttttc 2750 atccgccgga
gacactgctc ccatttgtgg ggggacatta gcaacatcac 2800 tcagaagcct
gtgttcttca agagcaggtg ttctcagcct cacatgccct 2850 gccgtgctgg
actcaggact gaagtgctgt aaagcaagga gctgctgaga 2900 aggagcactc
cactgtgtgc ctggagaatg gctctcacta ctcaccttgt 2950 ctttcagctt
ccagtgtctt gggtttttta tactttgaca gctttttttt 3000 aattgcatac
atgagactgt gttgactttt tttagttatg tgaaacactt 3050 tgccgcaggc
cgcctggcag aggcaggaaa tgctccagca gtggctcagt 3100 gctccctggt
gtctgctgca tggcatcctg gatgcttagc atgcaagttc 3150 cctccatcat
tgccaccttg gtagagaggg atggctcccc accctcagcg 3200 ttggggattc
acgctccagc ctccttcttg gttgtcatag tgatagggta 3250 gccttattgc
cccctcttct tataccctaa aaccttctac actagtgcca 3300 tgggaaccag
gtctgaaaaa gtagagagaa gtgaaagtag agtctgggaa 3350 gtagctgcct
ataactgaga ctagacggaa aaggaatact cgtgtatttt 3400 aagatatgaa
tgtgactcaa gactcgaggc cgatacgagg ctgtgattct 3450 gcctttggat
ggatgttgct gtacacagat gctacagact tgtactaaca 3500 caccgtaatt
tggcatttgt ttaacctcat ttataaaagc ttcaaaaaaa 3550 ccca 3554 20 310
PRT Homo sapiens 20 Met Ala Leu Arg Arg Pro Pro Arg Leu Arg Leu Cys
Ala Arg Leu 1 5 10 15 Pro Asp Phe Phe Leu Leu Leu Leu Phe Arg Gly
Cys Leu Ile Gly 20 25 30 Ala Val Asn Leu Lys Ser Ser Asn Arg Thr
Pro Val Val Gln Glu 35 40 45 Phe Glu Ser Val Glu Leu Ser Cys Ile
Ile Thr Asp Ser Gln Thr 50 55 60 Ser Asp Pro Arg Ile Glu Trp Lys
Lys Ile Gln Asp Glu Gln Thr 65 70 75 Thr Tyr Val Phe Phe Asp Asn
Lys Ile Gln Gly Asp Leu Ala Gly 80 85 90 Arg Ala Glu Ile Leu Gly
Lys Thr Ser Leu Lys Ile Trp Asn Val 95 100 105 Thr Arg Arg Asp Ser
Ala Leu Tyr Arg Cys Glu Val Val Ala Arg 110 115 120 Asn Asp Arg Lys
Glu Ile Asp Glu Ile Val Ile Glu Leu Thr Val 125 130 135 Gln Val Lys
Pro Val Thr Pro Val Cys Arg Val Pro Lys Ala Val 140 145 150 Pro Val
Gly Lys Met Ala Thr Leu His Cys Gln Glu Ser Glu Gly 155 160 165 His
Pro Arg Pro His Tyr Ser Trp Tyr Arg Asn Asp Val Pro Leu 170 175 180
Pro Thr Asp Ser Arg Ala Asn Pro Arg Phe Arg Asn Ser Ser Phe 185 190
195 His Leu Asn Ser Glu Thr Gly Thr Leu Val Phe Thr Ala Val His 200
205 210 Lys Asp Asp Ser Gly Gln Tyr Tyr Cys Ile Ala Ser Asn Asp Ala
215 220 225 Gly Ser Ala Arg Cys Glu Glu Gln Glu Met Glu Val Tyr Asp
Leu 230 235 240 Asn Ile Gly Gly Ile Ile Gly Gly Val Leu Val Val Leu
Ala Val 245 250 255 Leu Ala Leu Ile Thr Leu Gly Ile Cys Cys Ala Tyr
Arg Arg Gly 260 265 270 Tyr Phe Ile Asn Asn Lys Gln Asp Gly Glu Ser
Tyr Lys Asn Pro 275 280 285 Gly Lys Pro Asp Gly Val Asn Tyr Ile Arg
Thr Asp Glu Glu Gly 290 295 300 Asp Phe Arg His Lys Ser Ser Phe Val
Ile 305 310 21 3437 DNA Homo sapiens 21 caggaccagg tcttcctacg
ctggagcagc ggggagacag ccaccatgca 50 catcctcgtg gtccatgcca
tggtgatcct gctgacgctg ggcccgcctc 100 gagccgacga cagcgagttc
caggcgctgc tggacatctg gtttccggag 150 gagaagccac tgcccaccgc
cttcctggtg gacacatcgg aggaggcgct 200 gctgcttcct gactggctga
agctgcgcat gatccgttct gaggtgctcc 250 gcctggtgga cgccgccctg
caggacctgg agccgcagca gctgctgctg 300 ttcgtgcagt cgtttggcat
ccccgtgtcc agcatgagca aactcctcca 350 gttcctggac caggcagtgg
cccacgaccc ccagactctg gagcagaaca 400 tcatggacaa gaattacatg
gcccacctgg tggaggtcca gcatgagcgc 450 ggcgcctccg gaggccagac
tttccactcc ttgctcacag cctccctgcc 500 gccccgccga gacagcacag
aggcacccaa accaaagagc agcccagagc 550 agcccatagg ccagggccgg
attcgggtgg ggacccagct ccgggtgctg 600 ggccctgagg acgacctggc
tggcatgttc ctccagattt tcccgctcag 650 cccggaccct cggtggcaga
gctccagtcc ccgccccgtg gccctcgccc 700 tgcagcaggc cctgggccag
gagctggccc gcgtcgtcca gggcagcccc 750 gaggtgccgg gcatcacggt
gcgtgtcctg caggccctcg ccaccctgct 800 cagctcccca cacggcggtg
ccctggtgat gtccatgcac cgtagccact 850 tcctggcctg cccgctgctg
cgccagctct gccagtacca gcgctgtgtg 900 ccacaggaca ccggcttctc
ctcgctcttc ctgaaggtgc tcctgcagat 950 gctgcagtgg ctggacagcc
ctggcgtgga gggcgggccc ctgcgggcac 1000 agctcaggat gcttgccagc
caggcctcag ccgggcgcag gctcagtgat 1050 gtgcgagggg ggctcctgcg
cctggccgag gccctggcct tccgtcagga 1100 cctggaggtg gtcagctcca
ccgtccgtgc cgtcatcgcc accctgaggt 1150 ctggggagca gtgcagcgtg
gagccggacc tgatcagcaa agtcctccag 1200 gggctgatcg aggtgaggtc
cccccacctg gaggagctgc tgactgcatt 1250 cttctctgcc actgcggatg
ctgcctcccc gtttccagcc tgtaagcccg 1300 ttgtggtggt gagctccctg
ctgctgcagg aggaggagcc cctggctggg 1350 gggaagccgg gtgcggacgg
tggcagcctg gaggccgtgc ggctggggcc 1400 ctcgtcaggc ctcctagtgg
actggctgga aatgctggac cccgaggtgg 1450 tcagcagctg ccccgacctg
cagctcaggc tgctcttctc ccggaggaag 1500 ggcaaaggtc aggcccaggt
gccctcgttc cgtccctacc tcctgaccct 1550 cttcacgcat cagtccagct
ggcccacact gcaccagtgc atccgagtcc 1600 tgctgggcaa gagccgggaa
cagaggttcg acccctctgc ctctctggac 1650 ttcctctggg cctgcatcca
tgttcctcgc atctggcagg ggcgggacca 1700 gcgcaccccg cagaagcggc
gggaggagct ggtgctgcgg gtccagggcc 1750 cggagctcat cagcctggtg
gagctgatcc tggccgaggc ggagacgcgg 1800 agccaggacg gggacacagc
cgcctgcagc ctcatccagg cccggctgcc 1850 cctgctgctc agctgctgct
gtggggacga tgagagtgtc aggaaggtga 1900 cggagcacct gtcaggctgc
atccagcagt ggggagacag cgtgctggga 1950 aggcgctgcc gagaccttct
cctgcagctc tacctacagc ggccggagct 2000 gcgggtgccc gtgcctgagg
tcctactgca cagcgaaggg gctgccagca 2050 gcagcgtctg caagctggac
ggactcatcc accgcttcat cacgctcctt 2100 gcggacacca gcgactcccg
ggcgttggag aaccgagggg cggatgccag 2150 catggcctgc cggaagctgg
cggtggcgca cccgctgctg ctgctcaggc 2200 acctgcccat gatcgcggcg
ctcctgcacg gccgcaccca cctcaacttc 2250 caggagttcc ggcagcagaa
ccacctgagc tgcttcctgc acgtgctggg 2300 cctgctggag ctgctgcagc
cgcacgtgtt ccgcagcgag caccaggggg 2350 cgctgtggga ctgccttctg
tccttcatcc gcctgctgct gaattacagg 2400 aagtcctccc gccatctggc
tgccttcatc aacaagtttg tgcagttcat 2450 ccataagtac attacctaca
atgccccagc agccatctcc ttcctgcaga 2500 agcacgccga cccgctccac
gacctgtcct tcgacaacag tgacctggtg 2550 atgctgaaat ccctccttgc
agggctcagc ctgcccagca gggacgacag 2600 gaccgaccga ggcctggacg
aagagggcga ggaggagagc tcagccggct 2650 ccttgcccct ggtcagcgtc
tccctgttca cccctctgac cgcggccgag 2700 atggccccct acatgaaacg
gctttcccgg ggccaaacgg tggaggatct 2750 gctggaggtt ctgagtgaca
tagacgagat gtcccggcgg agacccgaga 2800 tcctgagctt cttctcgacc
aacctgcagc ggctgatgag ctcggccgag 2850 gagtgttgcc gcaacctcgc
cttcagcctg gccctgcgct ccatgcagaa 2900 cagccccagc attgcagccg
ctttcctgcc cacgttcatg tactgcctgg 2950 gcagccagga ctttgaggtg
gtgcagacgg ccctccggaa cctgcctgag 3000 tacgctctcc tgtgccaaga
gcacgcggct gtgctgctcc accgggcctt 3050 cctggtgggc atgtacggcc
agatggaccc cagcgcgcag atctccgagg 3100 ccctgaggat cctgcatatg
gaggccgtga tgtgagcctg tggcagccga 3150 cccccctcca agccccggcc
cgtcccgtcc ccggggatcc tcgaggcaaa 3200 gcccaggaag cgtgggcgtt
gctggtctgt ccgaggaggt gagggcgccg 3250 agccctgagg ccaggcaggc
ccaggagcaa tactccgagc cctggggtgg 3300 ctccgggccg gccgctggca
tcaggggccg tccagcaagc cctcattcac 3350 cttctgggcc acagccctgc
cgcggagcgg cggatccccc cgggcatggc 3400 ctgggctggt tttgaatgaa
acgacctgaa ctgtcaa 3437 22 1029 PRT Homo sapiens 22 Met His Ile Leu
Val Val His Ala Met Val Ile Leu Leu Thr Leu 1 5 10 15 Gly Pro Pro
Arg Ala Asp Asp Ser Glu Phe Gln Ala Leu Leu Asp 20 25 30 Ile Trp
Phe Pro Glu Glu Lys Pro Leu Pro Thr Ala Phe Leu Val 35 40 45 Asp
Thr Ser Glu Glu Ala Leu Leu Leu Pro Asp Trp Leu Lys Leu 50 55 60
Arg Met Ile Arg Ser Glu Val Leu Arg Leu Val Asp Ala Ala Leu 65 70
75 Gln Asp Leu Glu Pro Gln Gln Leu Leu Leu Phe Val Gln Ser Phe 80
85 90 Gly Ile Pro Val Ser Ser Met Ser Lys Leu Leu Gln Phe Leu Asp
95 100 105 Gln Ala Val Ala His Asp Pro Gln Thr Leu Glu Gln Asn Ile
Met 110 115 120 Asp Lys Asn Tyr Met Ala His Leu Val Glu Val Gln His
Glu Arg 125 130 135 Gly Ala Ser Gly Gly Gln Thr Phe His Ser Leu Leu
Thr Ala Ser 140 145 150 Leu Pro Pro Arg Arg Asp Ser Thr Glu Ala Pro
Lys Pro Lys Ser 155 160 165 Ser Pro Glu Gln Pro Ile Gly Gln Gly Arg
Ile Arg Val Gly Thr 170 175 180 Gln Leu Arg Val Leu Gly Pro Glu Asp
Asp Leu Ala Gly Met Phe 185 190 195 Leu Gln Ile Phe Pro Leu Ser Pro
Asp Pro Arg Trp Gln Ser Ser 200 205 210 Ser Pro Arg Pro Val Ala Leu
Ala Leu Gln Gln Ala Leu Gly Gln 215 220 225 Glu Leu Ala Arg Val Val
Gln Gly Ser Pro Glu Val Pro Gly Ile 230 235 240 Thr Val Arg Val Leu
Gln Ala Leu Ala Thr Leu Leu Ser Ser Pro 245 250 255 His Gly Gly Ala
Leu Val Met Ser Met His Arg Ser His Phe Leu 260 265 270 Ala Cys Pro
Leu Leu Arg Gln Leu Cys Gln Tyr Gln Arg Cys Val 275 280 285 Pro Gln
Asp Thr Gly Phe Ser Ser Leu Phe Leu Lys Val Leu Leu 290 295 300 Gln
Met Leu Gln Trp Leu Asp Ser Pro Gly Val Glu Gly Gly Pro 305 310 315
Leu Arg Ala Gln Leu Arg Met Leu Ala Ser Gln Ala Ser Ala Gly 320 325
330 Arg Arg Leu Ser Asp Val Arg Gly Gly Leu Leu Arg Leu Ala Glu 335
340 345 Ala Leu Ala Phe Arg Gln Asp Leu Glu Val Val Ser Ser Thr Val
350 355 360 Arg Ala Val Ile Ala Thr Leu Arg Ser Gly Glu Gln Cys Ser
Val 365 370 375 Glu Pro Asp Leu Ile Ser Lys Val Leu Gln Gly Leu Ile
Glu Val 380 385 390 Arg Ser Pro His Leu Glu Glu Leu Leu Thr Ala Phe
Phe Ser Ala 395 400 405 Thr Ala Asp Ala Ala Ser Pro Phe Pro Ala Cys
Lys Pro Val Val 410 415 420 Val Val Ser Ser Leu Leu Leu Gln Glu Glu
Glu Pro Leu Ala Gly 425 430 435 Gly Lys Pro Gly Ala Asp Gly Gly Ser
Leu Glu Ala Val Arg Leu 440 445 450 Gly Pro Ser Ser Gly Leu Leu Val
Asp Trp Leu Glu Met Leu Asp 455 460 465 Pro Glu Val Val Ser Ser Cys
Pro Asp Leu Gln Leu Arg Leu Leu 470 475 480 Phe Ser Arg Arg Lys Gly
Lys Gly Gln Ala Gln Val Pro Ser Phe 485 490 495 Arg Pro Tyr Leu Leu
Thr Leu Phe Thr His Gln Ser Ser Trp Pro 500 505 510 Thr Leu His Gln
Cys Ile Arg Val Leu Leu Gly Lys Ser Arg Glu 515 520 525 Gln Arg Phe
Asp Pro Ser Ala Ser Leu Asp Phe Leu Trp Ala Cys 530 535 540 Ile His
Val Pro Arg Ile Trp Gln Gly Arg Asp Gln Arg Thr Pro 545 550 555 Gln
Lys Arg Arg Glu Glu Leu Val Leu Arg Val Gln Gly Pro Glu 560 565 570
Leu Ile Ser Leu Val Glu Leu Ile Leu Ala Glu Ala Glu Thr Arg 575 580
585 Ser Gln Asp Gly Asp Thr Ala Ala Cys Ser Leu Ile Gln Ala Arg 590
595 600 Leu Pro Leu Leu Leu Ser Cys Cys Cys Gly Asp Asp Glu Ser Val
605 610 615 Arg Lys Val Thr Glu His Leu Ser Gly Cys Ile Gln Gln
Trp
Gly 620 625 630 Asp Ser Val Leu Gly Arg Arg Cys Arg Asp Leu Leu Leu
Gln Leu 635 640 645 Tyr Leu Gln Arg Pro Glu Leu Arg Val Pro Val Pro
Glu Val Leu 650 655 660 Leu His Ser Glu Gly Ala Ala Ser Ser Ser Val
Cys Lys Leu Asp 665 670 675 Gly Leu Ile His Arg Phe Ile Thr Leu Leu
Ala Asp Thr Ser Asp 680 685 690 Ser Arg Ala Leu Glu Asn Arg Gly Ala
Asp Ala Ser Met Ala Cys 695 700 705 Arg Lys Leu Ala Val Ala His Pro
Leu Leu Leu Leu Arg His Leu 710 715 720 Pro Met Ile Ala Ala Leu Leu
His Gly Arg Thr His Leu Asn Phe 725 730 735 Gln Glu Phe Arg Gln Gln
Asn His Leu Ser Cys Phe Leu His Val 740 745 750 Leu Gly Leu Leu Glu
Leu Leu Gln Pro His Val Phe Arg Ser Glu 755 760 765 His Gln Gly Ala
Leu Trp Asp Cys Leu Leu Ser Phe Ile Arg Leu 770 775 780 Leu Leu Asn
Tyr Arg Lys Ser Ser Arg His Leu Ala Ala Phe Ile 785 790 795 Asn Lys
Phe Val Gln Phe Ile His Lys Tyr Ile Thr Tyr Asn Ala 800 805 810 Pro
Ala Ala Ile Ser Phe Leu Gln Lys His Ala Asp Pro Leu His 815 820 825
Asp Leu Ser Phe Asp Asn Ser Asp Leu Val Met Leu Lys Ser Leu 830 835
840 Leu Ala Gly Leu Ser Leu Pro Ser Arg Asp Asp Arg Thr Asp Arg 845
850 855 Gly Leu Asp Glu Glu Gly Glu Glu Glu Ser Ser Ala Gly Ser Leu
860 865 870 Pro Leu Val Ser Val Ser Leu Phe Thr Pro Leu Thr Ala Ala
Glu 875 880 885 Met Ala Pro Tyr Met Lys Arg Leu Ser Arg Gly Gln Thr
Val Glu 890 895 900 Asp Leu Leu Glu Val Leu Ser Asp Ile Asp Glu Met
Ser Arg Arg 905 910 915 Arg Pro Glu Ile Leu Ser Phe Phe Ser Thr Asn
Leu Gln Arg Leu 920 925 930 Met Ser Ser Ala Glu Glu Cys Cys Arg Asn
Leu Ala Phe Ser Leu 935 940 945 Ala Leu Arg Ser Met Gln Asn Ser Pro
Ser Ile Ala Ala Ala Phe 950 955 960 Leu Pro Thr Phe Met Tyr Cys Leu
Gly Ser Gln Asp Phe Glu Val 965 970 975 Val Gln Thr Ala Leu Arg Asn
Leu Pro Glu Tyr Ala Leu Leu Cys 980 985 990 Gln Glu His Ala Ala Val
Leu Leu His Arg Ala Phe Leu Val Gly 995 1000 1005 Met Tyr Gly Gln
Met Asp Pro Ser Ala Gln Ile Ser Glu Ala Leu 1010 1015 1020 Arg Ile
Leu His Met Glu Ala Val Met 1025 23 2186 DNA Homo sapiens 23
ccgggccatg cagcctcggc cccgcgggcg cccgccgcgc acccgaggag 50
atgaggctcc gcaatggcac cttcctgacg ctgctgctct tctgcctgtg 100
cgccttcctc tcgctgtcct ggtacgcggc actcagcggc cagaaaggcg 150
acgttgtgga cgtttaccag cgggagttcc tggcgctgcg cgatcggttg 200
cacgcagctg agcaggagag cctcaagcgc tccaaggagc tcaacctggt 250
gctggacgag atcaagaggg ccgtgtcaga aaggcaggcg ctgcgagacg 300
gagacggcaa tcgcacctgg ggccgcctaa cagaggaccc ccgattgaag 350
ccgtggaacg gctcacaccg gcacgtgctg cacctgccca ccgtcttcca 400
tcacctgcca cacctgctgg ccaaggagag cagtctgcag cccgcggtgc 450
gcgtgggcca gggccgcacc ggagtgtcgg tggtgatggg catcccgagc 500
gtgcggcgcg aggtgcactc gtacctgact gacactctgc actcgctcat 550
ctccgagctg agcccgcagg agaaggagga ctcggtcatc gtggtgctga 600
tcgccgagac tgactcacag tacacttcgg cagtgacaga gaacatcaag 650
gccttgttcc ccacggagat ccattctggg ctcctggagg tcatctcacc 700
ctccccccac ttctaccctg acttctcccg cctccgagag tcctttgggg 750
accccaagga gagagtcagg tggaggacca aacagaacct cgattactgc 800
ttcctcatga tgtacgcgca gtccaaaggc atctactacg tgcagctgga 850
ggatgacatc gtggccaagc ccaactacct gagcaccatg aagaactttg 900
cactgcagca gccttcagag gactggatga tcctggagtt ctcccagctg 950
ggcttcattg gtaagatgtt caagtcgctg gacctgagcc tgattgtaga 1000
gttcattctc atgttctacc gggacaagcc catcgactgg ctcctggacc 1050
atattctgtg ggtgaaagtc tgcaaccccg agaaggatgc gaagcactgt 1100
gaccggcaga aagccaacct gcggatccgc ttcaaaccgt ccctcttcca 1150
gcacgtgggc actcactcct cgctggctgg caagatccag aaactgaagg 1200
acaaagactt tggaaagcag gcgctgcgga aggagcatgt gaacccgcca 1250
gcagaggtga gcacgagcct gaagacatac cagcacttca ccctggagaa 1300
agcctacctg cgcgaggact tcttctgggc cttcacccct gccgcggggg 1350
acttcatccg cttccgcttc ttccaacctc taagactgga gcggttcttc 1400
ttccgcagtg ggaacatcga gcacccggag gacaagctct tcaacacgtc 1450
tgtggaggtg ctgcccttcg acaaccctca gtcagacaag gaggccctgc 1500
aggagggccg caccgccacc ctccggtacc ctcggagccc cgacggctac 1550
ctccagatcg gctccttcta caagggagtg gcagagggag aggtggaccc 1600
agccttcggc cctctggaag cactgcgcct ctcgatccag acggactccc 1650
ctgtgtgggt gattctgagc gagatcttcc tgaaaaaggc cgactaagct 1700
gcgggcttct gagggtaccc tgtggccagc cctgaagccc acatttctgg 1750
gggtgtcgtc actgccgtcc ccggagggcc agatacggcc ccgcccaaag 1800
ggttctgcct ggcgtcgggc ttgggccggc ctggggtccg ccgctggccc 1850
ggaggcccta ggagctggtg ctgcccccgc ccgccgggcc gcggaggagg 1900
caggcggccc ccacactgtg cctgaggccc ggaaccgttc gcacccggcc 1950
tgccccagtc aggccgtttt agaagagctt ttacttgggc gcccgccgtc 2000
tctggcgcga acactggaat gcatatacta ctttatgtgc tgtgtttttt 2050
attcttggat acatttgatt ttttcacgta agtccacata tacttctata 2100
agagcgtgac ttgtaataaa gggttaatga agaaaaaaaa aaaaaaaaaa 2150
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2186 24 548 PRT Homo
sapiens 24 Met Arg Leu Arg Asn Gly Thr Phe Leu Thr Leu Leu Leu Phe
Cys 1 5 10 15 Leu Cys Ala Phe Leu Ser Leu Ser Trp Tyr Ala Ala Leu
Ser Gly 20 25 30 Gln Lys Gly Asp Val Val Asp Val Tyr Gln Arg Glu
Phe Leu Ala 35 40 45 Leu Arg Asp Arg Leu His Ala Ala Glu Gln Glu
Ser Leu Lys Arg 50 55 60 Ser Lys Glu Leu Asn Leu Val Leu Asp Glu
Ile Lys Arg Ala Val 65 70 75 Ser Glu Arg Gln Ala Leu Arg Asp Gly
Asp Gly Asn Arg Thr Trp 80 85 90 Gly Arg Leu Thr Glu Asp Pro Arg
Leu Lys Pro Trp Asn Gly Ser 95 100 105 His Arg His Val Leu His Leu
Pro Thr Val Phe His His Leu Pro 110 115 120 His Leu Leu Ala Lys Glu
Ser Ser Leu Gln Pro Ala Val Arg Val 125 130 135 Gly Gln Gly Arg Thr
Gly Val Ser Val Val Met Gly Ile Pro Ser 140 145 150 Val Arg Arg Glu
Val His Ser Tyr Leu Thr Asp Thr Leu His Ser 155 160 165 Leu Ile Ser
Glu Leu Ser Pro Gln Glu Lys Glu Asp Ser Val Ile 170 175 180 Val Val
Leu Ile Ala Glu Thr Asp Ser Gln Tyr Thr Ser Ala Val 185 190 195 Thr
Glu Asn Ile Lys Ala Leu Phe Pro Thr Glu Ile His Ser Gly 200 205 210
Leu Leu Glu Val Ile Ser Pro Ser Pro His Phe Tyr Pro Asp Phe 215 220
225 Ser Arg Leu Arg Glu Ser Phe Gly Asp Pro Lys Glu Arg Val Arg 230
235 240 Trp Arg Thr Lys Gln Asn Leu Asp Tyr Cys Phe Leu Met Met Tyr
245 250 255 Ala Gln Ser Lys Gly Ile Tyr Tyr Val Gln Leu Glu Asp Asp
Ile 260 265 270 Val Ala Lys Pro Asn Tyr Leu Ser Thr Met Lys Asn Phe
Ala Leu 275 280 285 Gln Gln Pro Ser Glu Asp Trp Met Ile Leu Glu Phe
Ser Gln Leu 290 295 300 Gly Phe Ile Gly Lys Met Phe Lys Ser Leu Asp
Leu Ser Leu Ile 305 310 315 Val Glu Phe Ile Leu Met Phe Tyr Arg Asp
Lys Pro Ile Asp Trp 320 325 330 Leu Leu Asp His Ile Leu Trp Val Lys
Val Cys Asn Pro Glu Lys 335 340 345 Asp Ala Lys His Cys Asp Arg Gln
Lys Ala Asn Leu Arg Ile Arg 350 355 360 Phe Lys Pro Ser Leu Phe Gln
His Val Gly Thr His Ser Ser Leu 365 370 375 Ala Gly Lys Ile Gln Lys
Leu Lys Asp Lys Asp Phe Gly Lys Gln 380 385 390 Ala Leu Arg Lys Glu
His Val Asn Pro Pro Ala Glu Val Ser Thr 395 400 405 Ser Leu Lys Thr
Tyr Gln His Phe Thr Leu Glu Lys Ala Tyr Leu 410 415 420 Arg Glu Asp
Phe Phe Trp Ala Phe Thr Pro Ala Ala Gly Asp Phe 425 430 435 Ile Arg
Phe Arg Phe Phe Gln Pro Leu Arg Leu Glu Arg Phe Phe 440 445 450 Phe
Arg Ser Gly Asn Ile Glu His Pro Glu Asp Lys Leu Phe Asn 455 460 465
Thr Ser Val Glu Val Leu Pro Phe Asp Asn Pro Gln Ser Asp Lys 470 475
480 Glu Ala Leu Gln Glu Gly Arg Thr Ala Thr Leu Arg Tyr Pro Arg 485
490 495 Ser Pro Asp Gly Tyr Leu Gln Ile Gly Ser Phe Tyr Lys Gly Val
500 505 510 Ala Glu Gly Glu Val Asp Pro Ala Phe Gly Pro Leu Glu Ala
Leu 515 520 525 Arg Leu Ser Ile Gln Thr Asp Ser Pro Val Trp Val Ile
Leu Ser 530 535 540 Glu Ile Phe Leu Lys Lys Ala Asp 545 25 43 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 25 tgtaaaacga
cggccagtta aatagacctg caattattaa tct 43 26 41 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 26 caggaaacag ctatgaccac
ctgcacacct gcaaatccat t 41 27 19 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 27 actcgggatt cctgctgtt 19 28 23 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 28 aggcctttac
ccaaggccac aac 23 29 19 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 29 ggcctgtcct gtgttctca 19 30 22 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 30 tcccaccact
tacttccatg aa 22 31 25 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 31 ctgtggtacc caattgccgc cttgt 25 32 23 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 32 attgtcctga
gattcgagca aga 23 33 18 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 33 gtccagcaag ccctcatt 18 34 20 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 34 cttctgggcc
acagccctgc 20 35 21 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 35 cagttcaggt cgtttcattc a 21 36 19 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 36 ccagtcaggc
cgttttaga 19 37 21 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 37 cgggcgccca agtaaaagct c 21 38 28 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 38 cataaagtag
tatatgcatt ccagtgtt 28
* * * * *
References