U.S. patent application number 13/317754 was filed with the patent office on 2012-07-05 for novel pro1199 gene disruptions, and methods relating thereto.
Invention is credited to Stephen Jay Anderson, Jane Brennan, Frederic J. de Sauvage, Zhiyong Ding, Joel Edwards, Nelda A. Fikes, Wenhu Huang, Wenjun Ouyang, Carolina Rangel, Mamta Sangha, Zheng-Zheng Shi, Mary Jean Sparks, Joseph Trackey, Melissa Vetter, Ching-Yun Wang, Jessica Woodings.
Application Number | 20120174239 13/317754 |
Document ID | / |
Family ID | 34700085 |
Filed Date | 2012-07-05 |
United States Patent
Application |
20120174239 |
Kind Code |
A1 |
Anderson; Stephen Jay ; et
al. |
July 5, 2012 |
Novel PRO1199 gene disruptions, and methods relating thereto
Abstract
The present invention relates to transgenic animals, as well as
compositions and methods relating to the characterization of gene
function. Specifically, the present invention provides transgenic
mice comprising disruptions in PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 genes. Such in vivo studies
and characterizations may provide valuable identification and
discovery of therapeutics and/or treatments useful in the
prevention, amelioration or correction of diseases or dysfunctions
associated with gene disruptions such as neurological disorders;
cardiovascular, endothelial or angiogenic disorders; eye
abnormalities; immunological disorders; oncological disorders; bone
metabolic abnormalities or disorders; lipid metabolic disorders; or
developmental abnormalities.
Inventors: |
Anderson; Stephen Jay; (The
Woodlands, TX) ; Brennan; Jane; (Edinburgh, GB)
; de Sauvage; Frederic J.; (Foster City, CA) ;
Ding; Zhiyong; (Longmont, CO) ; Edwards; Joel;
(The Woodlands, TX) ; Fikes; Nelda A.; (Spring,
TX) ; Huang; Wenhu; (San Diego, CA) ; Ouyang;
Wenjun; (Foster City, CA) ; Rangel; Carolina;
(Houston, TX) ; Sangha; Mamta; (Flagstaff, AZ)
; Shi; Zheng-Zheng; (The Woodlands, TX) ; Sparks;
Mary Jean; (Magnolia, TX) ; Trackey; Joseph;
(The Woodlands, TX) ; Vetter; Melissa; (Spring,
TX) ; Wang; Ching-Yun; (The Woodlands, TX) ;
Woodings; Jessica; (Longmont, CO) |
Family ID: |
34700085 |
Appl. No.: |
13/317754 |
Filed: |
October 27, 2011 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10583466 |
Sep 5, 2007 |
8067664 |
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PCT/US04/41721 |
Dec 13, 2004 |
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13317754 |
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60530043 |
Dec 16, 2003 |
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Current U.S.
Class: |
800/3 ; 435/7.1;
435/7.92; 506/9 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 13/12 20180101; A61P 35/00 20180101; A01K 2227/105 20130101;
A61P 17/06 20180101; A61P 19/00 20180101; A61P 19/10 20180101; A61P
11/02 20180101; A61P 37/02 20180101; A61P 25/24 20180101; A61P
17/02 20180101; A01K 2217/075 20130101; A01K 67/0276 20130101; A61P
25/28 20180101; A61P 27/02 20180101; A61P 25/04 20180101; A61P 9/00
20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 27/12
20180101; A61P 11/00 20180101; A01K 2267/0306 20130101; A61P 29/00
20180101; A61P 25/18 20180101; A61P 25/20 20180101; A61P 25/22
20180101 |
Class at
Publication: |
800/3 ; 435/7.1;
435/7.92; 506/9 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 23/083 20060101 G01N023/083; C40B 30/04 20060101
C40B030/04; G01N 33/53 20060101 G01N033/53 |
Claims
1-25. (canceled)
26. A method of identifying an agent that modulates a phenotype
associated with a disruption of a gene which encodes for a PRO1199
polypeptide, the method comprising: (a) providing a non-human
transgenic animal whose genome comprises a disruption of the gene
which encodes for the PRO1199 polypeptide; (b) measuring a
physiological characteristic of the non-human transgenic animal of
(a); (c) comparing the measured physiological characteristic of (b)
with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from said gene
disruption in the non-human transgenic animal; (d) administering a
test agent to the non-human transgenic animal of (a); and (e)
determining whether the test agent modulates the identified
phenotype associated with the gene disruption in the non-human
transgenic animal.
27. The method of claim 26, wherein the phenotype associated with
the gene disruption comprises a cardiovascular disorder; an
angiogenic disorder; an immunological disorder; or a lipid
metabolic disorder.
28-41. (canceled)
42. The method of claim 27, wherein the cardiovascular or
angiogenic disorders are arterial diseases, including diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions including acute myocardial infarctions,
cardiac hypertrophy, and heart failure including congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders including thrombophlebitis, lymphangitis,
and lymphedema; peripheral vascular disease; cancer including
vascular tumors, capillary hemangioma and cavernous hemangioma,
glomus tumors, telangiectasia, bacillary angiomatosis,
hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi's
sarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis;
trauma including wounds, burns, and other injured tissue, implant
fixation, scarring; ischemia reperfusion injury; rheumatoid
arthritis; cerebrovascular disease; renal diseases including acute
renal failure, or osteoporosis.
43. The method of claim 27, wherein the immunological disorders are
systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
including scleroderma; idiopathic inflammatory myopathies including
dermatomyositis and polymyositis; Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia including
immune pancytopenia and paroxysmal nocturnal hemoglobinuria;
autoimmune thrombocytopenia including idiopathic thrombocytopenic
purpura and immune-mediated thrombocytopenia; thyroiditis including
Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic
thyroiditis, and atrophic thyroiditis; diabetes mellitus;
immune-mediated renal disease including glomerulonephritis and
tubulointerstitial nephritis; demyelinating diseases of the central
nervous system and of the peripheral nervous system, including
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases including infectious
hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic
viruses), autoimmune chronic active hepatitis, primary biliary
cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;
inflammatory bowel disease including ulcerative colitis and Crohn's
disease; gluten-sensitive enteropathy, and Whipple's disease;
autoimmune or immune-mediated skin diseases including bullous skin
diseases, erythema multiforme and contact dermatitis, psoriasis;
allergic diseases such as asthma, allergic rhinitis, atopic
dermatitis, food hypersensitivity and urticaria; immunologic
diseases of the lung including eosinophilic pneumonias, idiopathic
pulmonary fibrosis and hypersensitivity pneumonitis; or
transplantation associated diseases including graft rejection and
graft-versus-host disease.
44. (canceled)
45. The method of claim 26, wherein the non-human transgenic animal
exhibits at least one of the following physiological characteristic
compared with gender-matched wild-type littermates: an increased
mean serum triglyceride level; an increased total tissue mass, or
an increased lean body mass.
46-49. (canceled)
50. A method of identifying an agent that modulates a physiological
characteristic associated with a disruption of the gene which
encodes for a PRO1199 polypeptide, the method comprising: (a)
providing a non-human transgenic animal whose genome comprises a
disruption of the gene which encodes for a PRO1199 polypeptide; (b)
measuring a physiological characteristic exhibited by the non-human
transgenic animal of (a); (c) comparing the measured physiological
characteristic of (b) with that of a gender matched wild-type
animal, wherein the physiological characteristic exhibited by the
non-human transgenic animal that differs from the physiological
characteristic exhibited by the wild-type animal is identified as a
physiological characteristic associated with said gene disruption;
(d) administering a test agent to the non-human transgenic animal
of (a); and (e) determining whether the physiological
characteristic associated with the gene disruption is
modulated.
51. The method of claim 50, wherein the non-human transgenic animal
exhibits the following physiological characteristic compared with
gender matched wild-type littermates: an increased mean serum
triglyceride level; an increased total tissue mass, or an increased
lean body mass.
52-55. (canceled)
56. A method of identifying an agent which modulates a behavior
associated with a disruption of a gene which encodes for a PRO1199
polypeptide, the method comprising: (a) providing a non-human
transgenic animal whose genome comprises a disruption of a gene
which is an ortholog of a human gene that encodes for a PRO1199
polypeptide; (b) observing the behavior exhibited by the non-human
transgenic animal of (a); (c) comparing the observed behavior of
(b) with that of a gender matched wild-type animal, wherein the
observed behavior exhibited by the non-human transgenic animal that
differs from the observed behavior exhibited by the wild-type
animal is identified as a behavior associated with said gene
disruption; (d) administering a test agent to the non-human
transgenic animal of (a); and (e) determining whether the agent
modulates the behavior associated with the gene disruption.
57-66. (canceled)
67. A method of identifying an agent that ameliorates or modulates
a cardiovascular disorder; an angiogenic disorder; an immunological
disorder; or a lipid metabolic disorder associated with a
disruption of a gene which encodes for a PRO1199 polypeptide, the
method comprising: (a) providing a non-human transgenic animal
whose genome comprises a disruption of the gene which encodes for a
PRO1199 polypeptide; (b) administering a test agent to said
non-human transgenic animal; and (c) determining whether said test
agent ameliorates or modulates a cardiovascular disorder; an
angiogenic disorder; an immunological disorder; or a lipid
metabolic disorder in the non-human transgenic animal.
68-81. (canceled)
82. The method of claim 67, wherein the cardiovascular or
angiogenic disorders are arterial diseases, including diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure including congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders including thrombophlebitis, lymphangitis,
and lymphedema; peripheral vascular disease; cancer including
vascular tumors, including capillary hemangioma and cavernous
hemangioma, glomus tumors, telangiectasia, bacillary angiomatosis,
hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi's
sarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis;
trauma including wounds, burns, and other injured tissue, implant
fixation, and scarring; ischemia reperfusion injury; rheumatoid
arthritis; cerebrovascular disease; renal diseases including acute
renal failure, or osteoporosis.
83. The method of claim 67, wherein the immunological disorders are
systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
including scleroderma; idiopathic inflammatory myopathies including
dermatomyositis; and polymyositis; Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia including
immune pancytopenia and paroxysmal nocturnal hemoglobinuria;
autoimmune thrombocytopenia including idiopathic thrombocytopenic
purpura and immune-mediated thrombocytopenia; thyroiditis including
Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic
thyroiditis, and atrophic thyroiditis; diabetes mellitus;
immune-mediated renal disease including glomerulonephritis and
tubulointerstitial nephritis; demyelinating diseases of the central
nervous system and peripheral nervous system including multiple
sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases including infectious
hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic
viruses), autoimmune chronic active hepatitis, primary biliary
cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;
inflammatory bowel disease including ulcerative colitis and Crohn's
disease; gluten-sensitive enteropathy, and Whipple's disease;
autoimmune or immune-mediated skin diseases including bullous skin
diseases, erythema multiforme and contact dermatitis, psoriasis;
allergic diseases including asthma, allergic rhinitis, atopic
dermatitis, food hypersensitivity and urticaria; immunologic
diseases of the lung including eosinophilic pneumonia, idiopathic
pulmonary fibrosis and hypersensitivity pneumonitis; or
transplantation associated diseases including graft rejection and
graft-versus-host disease.
84. (canceled)
85. The method of claim 67, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
an increased mean serum triglyceride level; an increased total
tissue mass, or an increased lean body mass.
86-95. (canceled)
96. A method of evaluating a therapeutic agent capable of affecting
a condition associated with a disruption of a gene which encodes
for a PRO1199 polypeptide, the method comprising: (a) providing a
non-human transgenic animal whose genome comprises a disruption of
a gene which is an ortholog of a human gene that encodes for the
PRO1199 polypeptide; (b) measuring a physiological characteristic
of the non-human transgenic animal of (a); (c) comparing the
measured physiological characteristic of (b) with that of a gender
matched wild-type animal, wherein the physiological characteristic
of the non-human transgenic animal that differs from the
physiological characteristic of the wild-type animal is identified
as a condition resulting from said gene disruption in the non-human
transgenic animal; (d) administering a test agent to the non-human
transgenic animal of (a); and (e) evaluating the effects of the
test agent on the identified condition associated with the gene
disruption in the non-human transgenic animal.
97. The method of claim 96, wherein the condition is a
cardiovascular disorder; an angiogenic disorder; an immunological
disorder; or a lipid metabolic disorder.
98-149. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of, claims the benefit of,
and claims priority under 35 U.S.C. .sctn.120 from, U.S. patent
application Ser. No. 10/583,466, filed Sep. 5, 2007, which is a
U.S. national stage continuation application claiming the benefit
of, and priority under 35 U.S.C. .sctn.371 from international
application PCT/US2004/041721, filed Dec. 13, 2004, which claims
the benefit of, and priority under 35 U.S.C. .sctn.119 from U.S.
Provisional Patent Application 60/530,043, filed Dec. 16, 2003, all
of which applications are hereby incorporated by reference in their
entireties.
[0002] This disclosure includes a Sequence Listing provided in a
file entitled "Sequence Listing P5201R1" including the sequences
identified as SEQ ID NOs: 1-70, created on Sep. 7, 2006, the byte
size of which is 129 KB, which is hereby incorporated by reference
n its entirety.
[0003] This disclosure includes a Computer Program Listing
Appendix, provided on a single compact disc created on Jan. 11,
2012, which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0004] The present invention relates to compositions, including
transgenic and knockout animals and methods of using such
compositions for the diagnosis and treatment of diseases or
disorders.
BACKGROUND OF THE INVENTION
[0005] 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. 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,
erythropoietin, 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.
93:710817113 (1996); U.S. Pat. No. 5,536,637)].
[0006] 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 adhesion 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.
[0007] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. Receptor immuno-adhesions, 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.
[0008] 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.
[0009] Given the importance of secreted and membrane-bound proteins
in biological and disease processes, in vivo studies and
characterizations may provide valuable identification and discovery
of therapeutics and/or treatments useful in the prevention,
amelioration or correction of diseases or dysfunctions. In this
regard, genetically engineered mice have proven to be invaluable
tools for the functional dissection of biological processes
relevant to human disease, including immunology, cancer,
neuro-biology, cardiovascular biology, obesity and many others.
Gene knockouts can be viewed as modeling the biological mechanism
of drug action by presaging the activity of highly specific
antagonists in vivo. Knockout mice have been shown to model drug
activity; phenotypes of mice deficient for specific pharmaceutical
target proteins can resemble the human clinical phenotype caused by
the corresponding antagonist drug. Gene knockouts enable the
discovery of the mechanism of action of the target, the predominant
physiological role of the target, and mechanism-based side-effects
that might result from inhibition of the target in mammals.
Examples of this type include mice deficient in the angiotensin
converting enzyme (ACE) [Esther, C. R. et al., Lab. Invest.,
74:953-965 (1996)] and cyclooxygenase-1 (COX1) genes [Langenbach,
R. et al., Cell, 83:483-492 (1995)]. Conversely, knocking the gene
out in the mouse can have an opposite phenotypic effect to that
observed in humans after administration of an agonist drug to the
corresponding target. Examples include the erythropoietin knockout
[Wu, C. S. et al., Cell, 83:59-67 (1996)], in which a consequence
of the mutation is deficient red blood cell production, and the
GABA(A)-R-(.beta.3 knockout [DeLorey, T. M., J. Neurosci.,
18:8505-8514 (1998)], in which the mutant mice show hyperactivity
and hyper-responsiveness. Both these phenotypes are opposite to the
effects of erythropoietin and benzodiazepine administration in
humans. A striking example of a target validated using mouse
genetics is the ACC2 gene. Although the human ACC2 gene had been
identified several years ago, interest in ACC2 as a target for drug
development was stimulated only recently after analysis of ACC2
function using a knockout mouse. ACC2 mutant mice eat more than
their wild-type littermates, yet burn more fat and store less fat
in their adipocytes, making this enzyme a probable target for
chemical antagonism in the treatment of obesity [Abu-Elheiga, L. et
al., Science, 291:2613-2616 (2001)].
[0010] In the instant application, mutated gene disruptions have
resulted in phenotypic observations related to various disease
conditions or dysfunctions including: CNS/neurological disturbances
or disorders such as anxiety; eye abnormalities and associated
diseases; cardiovascular, endothelial or angiogenic disorders
including atherosclerosis; abnormal metabolic disorders including
diabetes and dyslipidemias associated with elevated serum
triglycerides and cholesterol levels; immunological and
inflammatory disorders; oncological disorders; bone metabolic
abnormalities or disorders such as arthritis, osteoporosis and
osteopetrosis; or a developmental disease such as embryonic
lethality.
SUMMARY OF THE INVENTION
A. Embodiments
[0011] The invention provides an isolated nucleic acid molecule
comprising a nucleotide sequence that encodes a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide.
[0012] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% nucleic acid
sequence identity, alternatively at least about 81% nucleic acid
sequence identity, alternatively at least about 82% nucleic acid
sequence identity, alternatively at least about 83% nucleic acid
sequence identity, alternatively at least about 84% nucleic acid
sequence identity, alternatively at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid
sequence identity, alternatively at least about 87% nucleic acid
sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid
sequence identity, alternatively at least about 90% nucleic acid
sequence identity, alternatively at least about 91% nucleic acid
sequence identity, alternatively at least about 92% nucleic acid
sequence identity, alternatively at least about 93% nucleic acid
sequence identity, alternatively at least about 94% nucleic acid
sequence identity, alternatively at least about 95% nucleic acid
sequence identity, alternatively at least about 96% nucleic acid
sequence identity, alternatively at least about 97% nucleic acid
sequence identity, alternatively at least about 98% nucleic acid
sequence identity and alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule encoding a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu. TRPM2 or PRO1801
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).
[0013] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81% nucleic
acid sequence identity, alternatively at least about 82% nucleic
acid sequence identity, alternatively at least about 83% nucleic
acid sequence identity, alternatively at least about 84% nucleic
acid sequence identity, alternatively at least about 85% nucleic
acid sequence identity, alternatively at least about 86% nucleic
acid sequence identity, alternatively at least about 87% nucleic
acid sequence identity, alternatively at least about 88% nucleic
acid sequence identity, alternatively at least about 89% nucleic
acid sequence identity, alternatively at least about 90% nucleic
acid sequence identity, alternatively at least about 91% nucleic
acid sequence identity, alternatively at least about 92% nucleic
acid sequence identity, alternatively at least about 93% nucleic
acid sequence identity, alternatively at least about 94% nucleic
acid sequence identity, alternatively at least about 95% nucleic
acid sequence identity, alternatively at least about 96% nucleic
acid sequence identity, alternatively at least about 97% nucleic
acid sequence identity, alternatively at least about 98% nucleic
acid sequence identity and alternatively at least about 99% nucleic
acid sequence identity to (a) a DNA molecule comprising the coding
sequence of a full-length PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide cDNA as
disclosed herein, the coding sequence of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide lacking the signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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).
[0014] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% nucleic acid sequence identity, alternatively at
least about 81% nucleic acid sequence identity, alternatively at
least about 82% nucleic acid sequence identity, alternatively at
least about 83% nucleic acid sequence identity, alternatively at
least about 84% nucleic acid sequence identity, alternatively at
least about 85% nucleic acid sequence identity, alternatively at
least about 86% nucleic acid sequence identity, alternatively at
least about 87% nucleic acid sequence identity, alternatively at
least about 88% nucleic acid sequence identity, alternatively at
least about 89% nucleic acid sequence identity, alternatively at
least about 90% nucleic acid sequence identity, alternatively at
least about 91% nucleic acid sequence identity, alternatively at
least about 92% nucleic acid sequence identity, alternatively at
least about 93% nucleic acid sequence identity, alternatively at
least about 94% nucleic acid sequence identity, alternatively at
least about 95% nucleic acid sequence identity, alternatively at
least about 96% nucleic acid sequence identity, alternatively at
least about 97% nucleic acid sequence identity, alternatively at
least about 98% nucleic acid sequence identity and alternatively at
least about 99% nucleic acid 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).
[0015] Another aspect of the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides are contemplated.
[0016] The invention also provides fragments of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide coding sequence, or the complement thereof, that may
find use as, for example, hybridization probes, for encoding
fragments of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide that may optionally encode
a polypeptide comprising a binding site for an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody or as antisense oligonucleotide
probes. Such nucleic acid fragments usually are or are at least
about 10 nucleotides in length, alternatively are or are at least
about 15 nucleotides in length, alternatively are or are at least
about 20 nucleotides in length, alternatively are or are at least
about 30 nucleotides in length, alternatively are or are at least
about 40 nucleotides in length, alternatively are or are at least
about 50 nucleotides in length, alternatively are or are at least
about 60 nucleotides in length, alternatively are or are at least
about 70 nucleotides in length, alternatively are or are at least
about 80 nucleotides in length, alternatively are or are at least
about 90 nucleotides in length, alternatively are or are at least
about 100 nucleotides in length, alternatively are or are at least
about 110 nucleotides in length, alternatively are or are at least
about 120 nucleotides in length, alternatively are or are at least
about 130 nucleotides in length, alternatively are or are at least
about 140 nucleotides in length, alternatively are or are at least
about 150 nucleotides in length, alternatively are or are at least
about 160 nucleotides in length, alternatively are or are at least
about 170 nucleotides in length, alternatively are or are at least
about 180 nucleotides in length, alternatively are or are at least
about 190 nucleotides in length, alternatively are or are at least
about 200 nucleotides in length, alternatively are or are at least
about 250 nucleotides in length, alternatively are or are at least
about 300 nucleotides in length, alternatively are or are at least
about 350 nucleotides in length, alternatively are or are at least
about 400 nucleotides in length, alternatively are or are at least
about 450 nucleotides in length, alternatively are or are at least
about 500 nucleotides in length, alternatively are or are at least
about 600 nucleotides in length, alternatively are or are at least
about 700 nucleotides in length, alternatively are or are at least
about 800 nucleotides in length, alternatively are or are at least
about 900 nucleotides in length and alternatively are or are 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide-encoding nucleotide
sequence may be determined in a routine manner by aligning the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any of a number of well known sequence
alignment programs and determining which PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide-encoding nucleotide sequence fragment(s) are novel. All
of such PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide-encoding nucleotide sequences are
contemplated herein. Also contemplated are the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide fragments that
comprise a binding site for an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody.
[0017] The invention provides isolated PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides
encoded by any of the isolated nucleic acid sequences hereinabove
identified.
[0018] In a certain aspect, the invention concerns an isolated
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, comprising an amino acid sequence having at
least about 80% amino acid sequence identity, alternatively at
least about 81% amino acid sequence identity, alternatively at
least about 82% amino acid sequence identity, alternatively at
least about 83% amino acid sequence identity, alternatively at
least about 84% amino acid sequence identity, alternatively at
least about 85% amino acid sequence identity, alternatively at
least about 86% amino acid sequence identity, alternatively at
least about 87% amino acid sequence identity, alternatively at
least about 88% amino acid sequence identity, alternatively at
least about 89% amino acid sequence identity, alternatively at
least about 90% amino acid sequence identity, alternatively at
least about 91% amino acid sequence identity, alternatively at
least about 92% amino acid sequence identity, alternatively at
least about 93% amino acid sequence identity, alternatively at
least about 94% amino acid sequence identity, alternatively at
least about 95% amino acid sequence identity, alternatively at
least about 96% amino acid sequence identity, alternatively at
least about 97% amino acid sequence identity, alternatively at
least about 98% amino acid sequence identity and alternatively at
least about 99% amino acid sequence identity to a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
[0019] In a further aspect, the invention concerns an isolated
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide comprising an amino acid sequence having at
least about 80% amino acid sequence identity, alternatively at
least about 81% amino acid sequence identity, alternatively at
least about 82% amino acid sequence identity, alternatively at
least about 83% amino acid sequence identity, alternatively at
least about 84% amino acid sequence identity, alternatively at
least about 85% amino acid sequence identity, alternatively at
least about 86% amino acid sequence identity, alternatively at
least about 87% amino acid sequence identity, alternatively at
least about 88% amino acid sequence identity, alternatively at
least about 89% amino acid sequence identity, alternatively at
least about 90% amino acid sequence identity, alternatively at
least about 91% amino acid sequence identity, alternatively at
least about 92% amino acid sequence identity, alternatively at
least about 93% amino acid sequence identity, alternatively at
least about 94% amino acid sequence identity, alternatively at
least about 95% amino acid sequence identity, alternatively at
least about 96% amino acid sequence identity, alternatively at
least about 97% amino acid sequence identity, alternatively at
least about 98% amino acid sequence identity and alternatively at
least about 99% amino acid sequence identity to an amino acid
sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
[0020] In one aspect, the invention concerns PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polypeptides which are or are at least about 10 amino acids in
length, alternatively are or are at least about 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,
470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,
600 amino acids in length, or more. Optionally, PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polypeptides will have or have no more than one conservative amino
acid substitution as compared to the native PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence, alternatively will have or will have no more
than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid
substitution as compared to the native PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
sequence.
[0021] In a specific aspect, the invention provides an isolated
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide and recovering the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide from the cell
culture.
[0022] Another aspect the invention provides an isolated PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide and recovering
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide from the cell culture.
[0023] The invention provides agonists and antagonists of a native
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide as defined herein. In particular, the agonist
or antagonist is an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody
or a small molecule.
[0024] The invention provides a method of identifying agonists or
antagonists to a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide which comprise
contacting the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide with a candidate molecule
and monitoring a biological activity mediated by said PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. Preferably, the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide is a native
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide.
[0025] The invention provides a composition of matter comprising a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, or an agonist or antagonist of a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide as herein described, or an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody, in combination with a carrier. Optionally, the carrier is
a pharmaceutically acceptable carrier.
[0026] The invention provides the use of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, or an agonist or antagonist thereof as hereinbefore
described, or an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody, for the preparation of a medicament useful in the
treatment of a condition which is responsive to the anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody.
[0027] 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.
[0028] 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.
[0029] The invention provides an antibody which binds, preferably
specifically, to any of the above or below described polypeptides.
Optionally, the antibody is a monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
[0030] The invention provides oligonucleotide probes which may be
useful for isolating genomic and cDNA nucleotide sequences,
measuring or detecting expression of an associated gene or as
antisense probes, wherein those probes may be derived from any of
the above or below described nucleotide sequences. Preferred probe
lengths are described above.
[0031] The invention also provides a method of identifying a
phenotype associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the method comprising:
[0032] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0033] (b) measuring a physiological characteristic of the
non-human transgenic animal; and
[0034] .COPYRGT.) comparing the measured physiological
characteristic with that of a gender matched wild-type animal,
wherein the physiological characteristic of the non-human
transgenic animal that differs from the physiological
characteristic of the wild-type animal is identified as a phenotype
resulting from the gene disruption in the non-human transgenic
animal. In one aspect, the non-human transgenic animal is a mammal.
In another aspect, the mammal is a rodent. In still another aspect,
the mammal is a rat or a mouse. In one aspect, the non-human
transgenic animal is heterozygous for the disruption of a gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In another
aspect, the phenotype exhibited by the non-human transgenic animal
as compared with gender matched wild-type littermates is at least
one of the following: a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality.
[0035] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0036] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0037] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), conical and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia,
incontinentiapigmenti, Batten's disease, mucopolysaccharidoses,
homocystinuria, or mannosidosis.
[0038] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0039] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0040] In still yet another aspect, the cardiovascular, endothelial
or angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0041] In still another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0042] In still another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0043] In another aspect, the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wildtype littermates: a decreased
anxiety-like response during open field activity testing; an
increased anxiety-like response during open field activity testing;
balding, exothalamus observations, and piloerection observations in
functional observation battery (FOB) testing; an increased mean
artery-to-vein ratio associated with retinal degeneration;
developing cataracts; an increased mean serum cholesterol level; an
increased mean serum triglyceride level; a decreased mean serum
insulin level, a decreased mean percentage of B cells in the spleen
and lymph node; a decreased mean serum IgG2a response to an
ovalbumin challenge; decreased mean serum IgA levels; an increased
mean serum IgG2a response to an ovalbumin challenge; increased mean
serum IgM, IgG1, IgG2a and IgG2b levels; increased mean serum IgM,
IgA and IgG3 levels; increased mean serum IgM, IgG1, IgG2a and
IgG2b levels; an increased mean percentage of CD4 cells and a
decreased mean percentage of CD8 cells in spleen and thymus;
mobilization of neutrophils in response to peritoneal inflammation;
an enhanced DDS-induced colitis response; an enhanced ConA-induced
hepatitis response; a decreased skin fibroblast proliferation; a
decreased volumetric bone mineral density, a decreased bone mineral
content index (BMC/LBM), and a decreased mean bone mineral density
in total body, femur and vertebrate; a decreased mean bone mineral
density, a decreased mean trabecular bone volume, decreased
thickness, and decreased connectivity density; a decreased body
weight and length, decreased total tissue mass and lean body mass,
a decreased femoral midshaft cross-sectional area with decreased
alkaline phosphatase levels; growth retardation with decreased body
weight and length, total tissue mass, and lean body mass; a
diaphragmatic hernia; an increased total tissue mass, increased
lean body mass, increased bone mineral content, increased total
body and increased femoral bone mineral density; an enhanced
glucose tolerance; developmental disorders including abnormal
kidney development marked by kidney agenesis; embryonic lethality;
or embryonic lethality wherein heterozygous adults exhibited
decreased serum IgM, IgG1, IgG2a, IgG2b and IgG3 levels.
[0044] The invention also provides an isolated cell derived from a
non-human transgenic animal whose genome comprises a disruption of
the gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In one aspect,
the isolated cell is a murine cell. In yet another aspect, the
murine cell is an embryonic stem cell. In still another aspect, the
isolated cell is derived from a non-human transgenic animal which
exhibits at least one of the following phenotypes compared with
gender matched wild-type littermates: a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality.
[0045] The invention also provides a method of identifying an agent
that modulates a phenotype associated with a disruption of a gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0046] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0047] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0048] .COPYRGT.) comparing the measured physiological
characteristic of (b) with that of a gender matched wild-type
animal, wherein the physiological characteristic of the non-human
transgenic animal that differs from the physiological
characteristic of the wild-type animal is identified as a phenotype
resulting from the gene disruption in the non-human transgenic
animal;
[0049] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0050] (e) determining whether the test agent modulates the
identified phenotype associated with gene disruption in the
non-human transgenic animal.
[0051] In one aspect, the phenotype associated with the gene
disruption comprises a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality.
[0052] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0053] In yet another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0054] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplasia spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0055] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia,
[0056] Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism, or Conradi
syndrome.
[0057] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0058] In still another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0059] In still another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, B and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0060] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0061] In still another aspect, the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
a decreased anxiety-like response during open field activity
testing; an increased anxiety-like response during open field
activity testing; balding, exothalamus observations, and
piloerection observations in functional observation battery (FOB)
testing; an increased mean artery-to-vein ratio associated with
retinal degeneration; developing cataracts; an increased mean serum
cholesterol level; an increased mean serum triglyceride level; a
decreased mean serum insulin level, a decreased mean percentage of
B cells in the spleen and lymph node; a decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean serum IgA
levels; an increased mean serum IgG2a response to an ovalbumin
challenge; increased mean serum IgM, IgG1, IgG2a and IgG2b levels;
increased mean serum IgM, IgA and IgG3 levels; increased mean serum
IgM, IgG1, IgG2a and IgG2b levels; an increased mean percentage of
CD4 cells and a decreased mean percentage of CD8 cells in spleen
and thymus; mobilization of neutrophils in response to peritoneal
inflammation; an enhanced DDS-induced colitis response; an enhanced
ConA-induced hepatitis response; a decreased skin fibroblast
proliferation; a decreased volumetric bone mineral density, a
decreased bone mineral content index (BMC/LBM), and a decreased
mean bone mineral density in total body, femur and vertebrate; a
decreased mean bone mineral density, a decreased mean trabecular
bone volume, decreased thickness, and decreased connectivity
density; a decreased body weight and length, decreased total tissue
mass and lean body mass, a decreased femoral midshaft
cross-sectional area with decreased alkaline phosphatase levels;
growth retardation with decreased body weight and length, total
tissue mass, and lean body mass; a diaphragmatic hernia; an
increased total tissue mass, increased lean body mass, increased
bone mineral content, increased total body and increased femoral
bone mineral density; an enhanced glucose tolerance; developmental
disorders including abnormal kidney development marked by kidney
agenesis; embryonic lethality; or embryonic lethality wherein
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels.
[0062] The invention also provides an agent which modulates the
phenotype associated with gene disruption. In one aspect, the agent
is an agonist or antagonist of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet
another aspect, the agonist agent is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. In still another aspect, the antagonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody.
[0063] The invention also provides a method of identifying an agent
that modulates a physiological characteristic associated with a
disruption of the gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, the method comprising:
[0064] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0065] (b) measuring a physiological characteristic exhibited by
the non-human transgenic animal of (a);
[0066] .COPYRGT.) comparing the measured physiological
characteristic of (b) with that of a gender matched wild-type
animal, wherein the physiological characteristic exhibited by the
non-human transgenic animal that differs from the physiological
characteristic exhibited by the wild-type animal is identified as a
physiological characteristic associated with gene disruption;
[0067] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0068] (e) determining whether the physiological characteristic
associated with gene disruption is modulated.
[0069] In one aspect, the non-human transgenic animal exhibits at
least one of the following physiological characteristics compared
with gender matched wild-type littermates: a decreased anxiety-like
response during open field activity testing; an increased
anxiety-like response during open field activity testing; balding,
exothalamus observations, and piloerection observations in
functional observation battery (FOB) testing; an increased mean
artery-to-vein ratio associated with retinal degeneration;
developing cataracts; an increased mean serum cholesterol level; an
increased mean serum triglyceride level; a decreased mean serum
insulin level, a decreased mean percentage of B cells in the spleen
and lymph node; a decreased mean serum IgG2a response to an
ovalbumin challenge; decreased mean serum IgA levels; an increased
mean serum IgG2a response to an ovalbumin challenge; increased mean
serum IgM, IgG1, IgG2a and IgG2b levels; increased mean serum IgM,
IgA and IgG3 levels; increased mean serum IgM, IgG1, IgG2a and
IgG2b levels; an increased mean percentage of CD4 cells and a
decreased mean percentage of CD8 cells in spleen and thymus;
mobilization of neutrophils in response to peritoneal inflammation;
an enhanced DDS-induced colitis response; an enhanced ConA-induced
hepatitis response; a decreased skin fibroblast proliferation; a
decreased volumetric bone mineral density, a decreased bone mineral
content index (BMC/LBM), and a decreased mean bone mineral density
in total body, femur and vertebrate; a decreased mean bone mineral
density, a decreased mean trabecular bone volume, decreased
thickness, and decreased connectivity density; a decreased body
weight and length, decreased total tissue mass and lean body mass,
a decreased femoral midshaft cross-sectional area with decreased
alkaline phosphatase levels; growth retardation with decreased body
weight and length, total tissue mass, and lean body mass; a
diaphragmatic hernia; an increased total tissue mass, increased
lean body mass, increased bone mineral content, increased total
body and increased femoral bone mineral density; an enhanced
glucose tolerance; developmental disorders including abnormal
kidney development marked by kidney agenesis; embryonic lethality;
or embryonic lethality wherein heterozygous adults exhibited
decreased serum IgM, IgG1, IgG2a, IgG2b and IgG3 levels.
[0070] The invention also provides an agent that modulates a
physiological characteristic which is associated with gene
disruption. In one aspect, the agent is an agonist or antagonist of
the phenotype associated with a disruption of a gene which encodes
for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide. In yet another aspect, the agent is
an agonist or antagonist of a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another
aspect, the agonist agent is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. In still another aspect, the antagonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody.
[0071] The invention also provides a method of identifying an agent
which modulates a behavior associated with a disruption of the gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0072] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0073] (b) observing the behavior exhibited by the non-human
transgenic animal of (a);
[0074] .COPYRGT.) comparing the observed behavior of (b) with that
of a gender matched wild-type animal, wherein the observed behavior
exhibited by the non-human transgenic animal that differs from the
observed behavior exhibited by the wild-type animal is identified
as a behavior associated with gene disruption;
[0075] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0076] (e) determining whether the agent modulates the behavior
associated with gene disruption.
[0077] In one aspect, the observed behavior is an increased
anxiety-like response during open field activity testing. In yet
another aspect, the observed behavior is a decreased anxiety-like
response during open field activity testing. In yet another aspect,
the observed behavior is an abnormal circadian rhythm during
home-cage activity testing. In yet another aspect, the observed
behavior is an enhanced motor coordination during inverted screen
testing. In yet another aspect, the observed behavior is impaired
motor coordination during inverted screen testing. In yet another
aspect, the observed behavior includes depression, generalized
anxiety disorders, attention deficit disorder, sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia and sensory
disorders. Such disorders include the category defined as "anxiety
disorders" which include but are not limited to: mild to moderate
anxiety, anxiety disorder due to a general medical condition,
anxiety disorder not otherwise specified, generalized anxiety
disorder, panic attack, panic disorder with agoraphobia, panic
disorder without agoraphobia, posttraumatic stress disorder, social
phobia, social anxiety, autism, specific phobia, substance-induced
anxiety disorder, acute alcohol withdrawal, obsessive compulsive
disorder, agoraphobia, monopolar disorders, bipolar disorder I or
II, bipolar disorder not otherwise specified, cyclothymic disorder,
depressive disorder, major depressive disorder, mood disorder,
substance-induced mood disorder, enhancement of cognitive function,
loss of cognitive function associated with but not limited to
Alzheimer's disease, stroke, or traumatic injury to the brain,
seizures resulting from disease or injury including but not limited
to epilepsy, learning disorders/disabilities, cerebral palsy. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[0078] The invention also provides an agent that modulates a
behavior which is associated with gene disruption. In one aspect,
the agent is an agonist or antagonist of the phenotype associated
with a disruption of a gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. In yet another aspect, the agent is an agonist or
antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another aspect,
the agonist agent is an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. In still another aspect, the antagonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody.
[0079] The invention also provides a method of identifying an agent
that ameliorates or modulates a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder, an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality associated with a disruption in the
gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0080] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0081] (b) administering a test agent to said non-human transgenic
animal; and
[0082] .COPYRGT.) determining whether the test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality associated with
the gene disruption in the non-human transgenic animal.
[0083] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0084] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0085] In still another aspect, the retinal abnormalities the
retinal abnormalities are consistent with retinal dysplasia,
various retinopathies, including retinopathy of prematurity,
retrolental fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0086] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0087] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0088] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0089] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0090] In still another aspect, the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
a decreased anxiety-like response during open field activity
testing; an increased anxiety-like response during open field
activity testing; balding, exothalamus observations, and
piloerection observations in functional observation battery (FOB)
testing; an increased mean artery-to-vein ratio associated with
retinal degeneration; developing cataracts; an increased mean serum
cholesterol level; an increased mean serum triglyceride level; a
decreased mean serum insulin level, a decreased mean percentage of
B cells in the spleen and lymph node; a decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean serum IgA
levels; an increased mean serum IgG2a response to an ovalbumin
challenge; increased mean serum IgM, IgG1, IgG2a and IgG2b levels;
increased mean serum IgM, IgA and IgG3 levels; increased mean serum
IgM, IgG1, IgG2a and IgG2b levels; an increased mean percentage of
CD4 cells and a decreased mean percentage of CD8 cells in spleen
and thymus; mobilization of neutrophils in response to peritoneal
inflammation; an enhanced DDS-induced colitis response; an enhanced
ConA-induced hepatitis response; a decreased skin fibroblast
proliferation; a decreased volumetric bone mineral density, a
decreased bone mineral content index (BMC/LBM), and a decreased
mean bone mineral density in total body, femur and vertebrate; a
decreased mean bone mineral density, a decreased mean trabecular
bone volume, decreased thickness, and decreased connectivity
density; a decreased body weight and length, decreased total tissue
mass and lean body mass, a decreased femoral midshaft
cross-sectional area with decreased alkaline phosphatase levels;
growth retardation with decreased body weight and length, total
tissue mass, and lean body mass; a diaphragmatic hernia; an
increased total tissue mass, increased lean body mass, increased
bone mineral content, increased total body and increased femoral
bone mineral density; an enhanced glucose tolerance; developmental
disorders including abnormal kidney development marked by kidney
agenesis; embryonic lethality; or embryonic lethality wherein
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels.
[0091] The invention also provides an agent that ameliorates or
modulates a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an eye abnormality; an immunological disorder;
an oncological disorder; a bone metabolic abnormality or disorder;
a lipid metabolic disorder; or a developmental abnormality which is
associated with gene disruption. In one aspect, the agent is an
agonist or antagonist of the phenotype associated with a disruption
of a gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another
aspect, the agent is an agonist or antagonist of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. In still
another aspect, the antagonist agent is an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody.
[0092] The invention also provides a therapeutic agent for the
treatment of a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality.
[0093] The invention also provides a method of identifying an agent
that modulates the expression of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the
method comprising:
[0094] (a) contacting a test agent with a host cell expressing a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide; and
[0095] (b) determining whether the test agent modulates the
expression of the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide by the host
cell.
[0096] The invention also provides an agent that modulates the
expression of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. In one aspect, the agent
is an agonist or antagonist of the phenotype associated with a
disruption of a gene which encodes for a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet
another aspect, the agent is an agonist or antagonist of a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. In still
another aspect, the antagonist agent is an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody.
[0097] The invention also provides a method of evaluating a
therapeutic agent capable of affecting a condition associated with
a disruption of a gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, the method comprising:
[0098] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0099] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0100] .COPYRGT.) comparing the measured physiological
characteristic of (b) with that of a gender matched wild-type
animal, wherein the physiological characteristic of the non-human
transgenic animal that differs from the physiological
characteristic of the wild-type animal is identified as a condition
resulting from the gene disruption in the non-human transgenic
animal;
[0101] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0102] (e) evaluating the effects of the test agent on the
identified condition associated with gene disruption in the
non-human transgenic animal.
[0103] In one aspect, the condition is a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality.
[0104] The invention also provides a therapeutic agent which is
capable of affecting a condition associated with gene disruption.
In one aspect, the agent is an agonist or antagonist of the
phenotype associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide. In yet another aspect, the agent is
an agonist or antagonist of a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another
aspect, the agonist agent is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. In still another aspect, the antagonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody.
[0105] The invention also provides a pharmaceutical composition
comprising a therapeutic agent capable of affecting the condition
associated with gene disruption.
[0106] The invention also provides a method of treating or
preventing or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method comprising
administering to a subject in need of such treatment whom may
already have the disorder, or may be prone to have the disorder or
may be in whom the disorder is to be prevented, a therapeutically
effective amount of a therapeutic agent, or agonists or antagonists
thereof, thereby effectively treating or preventing or ameliorating
said disorder or disease.
[0107] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0108] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0109] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia,
incontinentiapigmenti, Batten's disease, mucopolysaccharidoses,
homocystinuria, or mannosidosis.
[0110] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0111] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0112] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0113] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0114] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0115] In another aspect the therapeutic agent is an agonist or
antagonist of the phenotype associated with a disruption of a gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another
aspect, the agent is an agonist or antagonist of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. In still
another aspect, the antagonist agent is an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody.
[0116] The invention also provides a method of identifying an agent
that ameliorates or modulates a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality associated with a disruption in the
gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0117] (a) providing a non-human transgenic animal cell culture,
each cell of said culture comprising a disruption of the gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide;
[0118] (b) administering a test agent to said cell culture; and
[0119] .COPYRGT.) determining whether the test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in said
culture.
[0120] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0121] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia. In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0122] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0123] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0124] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0125] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0126] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0127] The invention also provides an agent that ameliorates or
modulates a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an eye abnormality; an immunological disorder;
an oncological disorder; a bone metabolic abnormality or disorder;
a lipid metabolic disorder; or a developmental abnormality which is
associated with gene disruption in said culture. In one aspect, the
agent is an agonist or antagonist of the phenotype associated with
a disruption of a gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. In yet another aspect, the agent is an agonist or
antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. In yet another aspect,
the agonist agent is an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. In still another aspect, the antagonist agent is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody.
[0128] The invention also provides a method of modulating a
phenotype associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the method comprising administering
to a subject whom may already have the phenotype, or may be prone
to have the phenotype or may be in whom the phenotype is to be
prevented, an effective amount of an agent identified as modulating
said phenotype, or agonists or antagonists thereof, thereby
effectively modulating the phenotype.
[0129] The invention also provides a method of modulating a
physiological characteristic associated with a disruption of a gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising administering to a subject whom may already exhibit the
physiological characteristic, or may be prone to exhibit the
physiological characteristic or may be in whom the physiological
characteristic is to be prevented, an effective amount of an agent
identified as modulating said physiological characteristic, or
agonists or antagonists thereof, thereby effectively modulating the
physiological characteristic.
[0130] The invention also provides a method of modulating a
behavior associated with a disruption of a gene which encodes for a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, the method comprising administering to a
subject whom may already exhibit the behavior, or may be prone to
exhibit the behavior or may be in whom the exhibited behavior is to
be prevented, an effective amount of an agent identified as
modulating said behavior, or agonists or antagonists thereof,
thereby effectively modulating the behavior.
[0131] The invention also provides a method of modulating the
expression of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method comprising
administering to a host cell expressing said PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, an effective amount of an agent identified as
modulating said expression, or agonists or antagonists thereof,
thereby effectively modulating the expression of said
polypeptide.
[0132] The invention also provides a method of modulating a
condition associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the method comprising administering
to a subject whom may have the condition, or may be prone to have
the condition or may be in whom the condition is to be prevented, a
therapeutically effective amount of a therapeutic agent identified
as modulating said condition, or agonists or antagonists thereof,
thereby effectively modulating the condition.
[0133] The invention also provides a method of treating or
preventing or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method comprising
administering to a non-human transgenic animal cell culture, each
cell of said culture comprising a disruption of the gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or P12.01801 polypeptide, an effective amount of
an agent identified as treating or preventing or ameliorating said
disorder, or agonists or antagonists thereof, thereby effectively
treating or preventing or ameliorating said disorder.
B. Further Embodiments
[0134] In yet further embodiments, the invention is directed to the
following set of potential claims for this application:
1. A method of identifying a phenotype associated with a disruption
of a gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0135] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0136] (b) measuring a physiological characteristic of the
non-human transgenic animal; and
[0137] (c) comparing the measured physiological characteristic with
that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal.
2. The method of claim 1, wherein the non-human transgenic animal
is heterozygous for the disruption of a gene which encodes for a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide. 3. The method of claim 1, wherein the
phenotype exhibited by the non-human transgenic animal as compared
with gender matched wild-type littermates is at least one of the
following: a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality. 4. The method of claim 3, wherein the neurological
disorder is an increased anxiety-like response during open field
activity testing. 5. The method of claim 3, wherein the
neurological disorder is a decreased anxiety-like response during
open field activity testing. 6. The method of claim 3, wherein the
neurological disorder is an abnormal circadian rhythm during
home-cage activity testing. 7. The method of claim 3, wherein the
neurological disorder is an enhanced motor coordination during
inverted screen testing. 8. The method of claim 3, wherein the
neurological disorder is an impaired motor coordination during
inverted screen testing. 9. The method of claim 3, wherein the
neurological disorder is depression, generalized anxiety disorders,
attention deficit disorder, sleep disorder, hyperactivity disorder,
obsessive compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. 10. The method of claim 3,
wherein the eye abnormality is a retinal abnormality. 11. The
method of claim 3, wherein the eye abnormality is consistent with
vision problems or blindness. 12. The method of claim 10, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
13. The method of claim 10, wherein the retinal abnormality is
characterized by retinal degeneration or retinal dysplasia. 14. The
method of claim 10, wherein the retinal abnormality is consistent
with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. 15. The method of claim 3, wherein the eye
abnormality is a cataract. 16. The method of claim 15, wherein the
cataract is consistent with systemic diseases such as human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism or Conradi syndrome.
17. The method of claim 3, wherein the developmental abnormality
comprises embryonic lethality or reduced viability. 18. The method
of claim 3, wherein the cardiovascular, endothelial or angiogenic
disorders are arterial diseases, such as diabetes mellitus;
papilledema; optic atrophy; atherosclerosis; angina; myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as congestive heart failure;
hypertension; inflammatory vasculitides; Reynaud's disease and
Reynaud's phenomenon; aneurysms and arterial restenosis; venous and
lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. 19. The method of claim 3, wherein the immunological
disorders are systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
20. The method of claim 3, wherein the bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis. 21. The
method of claim 1, wherein the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wild-type littermates: a decreased
anxiety-like response during open field activity testing; an
increased anxiety-like response during open field activity testing;
balding, exothalamus observations, and piloerection observations in
functional observation battery (FOB) testing; an increased mean
artery-to-vein ratio associated with retinal degeneration;
developing cataracts; an increased mean serum cholesterol level; an
increased mean serum triglyceride level; a decreased mean serum
insulin level, a decreased mean percentage of B cells in the spleen
and lymph node; a decreased mean serum IgG2a response to an
ovalbumin challenge; decreased mean serum IgA levels; an increased
mean serum IgG2a response to an ovalbumin challenge; increased mean
serum IgM, IgG1, IgG2a and IgG2b levels; increased mean serum IgM,
IgA and IgG3 levels; increased mean serum IgM, IgG1, IgG2a and
IgG2b levels; an increased mean percentage of CD4 cells and a
decreased mean percentage of CD8 cells in spleen and thymus;
mobilization of neutrophils in response to peritoneal inflammation;
an enhanced DDS-induced colitis response; an enhanced ConA-induced
hepatitis response; a decreased skin fibroblast proliferation; a
decreased volumetric bone mineral density, a decreased bone mineral
content index (BMC/LBM), and a decreased mean bone mineral density
in total body, femur and vertebrate; a decreased mean bone mineral
density, a decreased mean trabecular bone volume, decreased
thickness, and decreased connectivity density; a decreased body
weight and length, decreased total tissue mass and lean body mass,
a decreased femoral midshaft cross-sectional area with decreased
alkaline phosphatase levels; growth retardation with decreased body
weight and length, total tissue mass, and lean body mass; a
diaphragmatic hernia; an increased total tissue mass, increased
lean body mass, increased bone mineral content, increased total
body and increased femoral bone mineral density; an enhanced
glucose tolerance; developmental disorders including abnormal
kidney development marked by kidney agenesis; embryonic lethality;
or embryonic lethality wherein heterozygous adults exhibited
decreased serum IgM, IgG1, IgG2a, IgG2b and IgG3 levels. 22. An
isolated cell derived from a non-human transgenic animal whose
genome comprises a disruption of the gene which encodes for a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide. 23. The isolated cell of claim 22 which is a
murine cell. 24. The isolated cell of claim 23, wherein the murine
cell is an embryonic stem cell. 25. The isolated cell of claim 22,
wherein the non-human transgenic animal exhibits at least one of
the following phenotypes compared with gender matched wild-type
littermates: a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality. 26. A method of identifying an agent that modulates a
phenotype associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the method comprising:
[0138] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0139] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0140] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal;
[0141] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0142] (e) determining whether the test agent modulates the
identified phenotype associated with gene disruption in the
non-human transgenic animal.
27. The method of claim 26, wherein the phenotype associated with
the gene disruption comprises a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality. 28. The method of claim 27, wherein
the neurological disorder is an increased anxiety-like response
during open field activity testing. 29. The method of claim 27,
wherein the neurological disorder is a decreased anxiety-like
response during open field activity testing. 30. The method of
claim 27, wherein the neurological disorder is an abnormal
circadian rhythm during home-cage activity testing. 31. The method
of claim 27, wherein the neurological disorder is an enhanced motor
coordination during inverted screen testing. 32. The method of
claim 27, wherein the neurological disorder is an impaired motor
coordination during inverted screen testing. 33. The method of
claim 27, wherein the neurological disorder is depression,
generalized anxiety disorders, attention deficit disorder, sleep
disorder, hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia or sensory
disorders. 34. The method of claim 27, wherein the eye abnormality
is a retinal abnormality. 35. The method of claim 27, wherein the
eye abnormality is consistent with vision problems or blindness.
36. The method of claim 34, wherein the retinal abnormality is
consistent with retinitis pigmentosa. 37. The method of claim 34,
wherein the retinal abnormality is characterized by retinal
degeneration or retinal dysplasia. 38. The method of claim 34,
wherein the retinal abnormality is consistent with retinal
dysplasia, various retinopathies, including retinopathy of
prematurity, retrolental fibroplasia, neovascular glaucoma,
age-related macular degeneration, diabetic macular edema, corneal
neovascularization, corneal graft neovascularization, corneal graft
rejection, retinal/choroidal neovascularization, neovascularization
of the angle (rubeosis), ocular neovascular disease, vascular
restenosis, arteriovenous malformations (AVM), meningioma,
hemangioma, angiofibroma, thyroid hyperplasias (including Grave's
disease), corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. 39. The method of claim 27, wherein the eye
abnormality is a cataract. 40. The method of claim 39, wherein the
cataract is consistent with systemic diseases such as human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism or Conradi syndrome.
41. The method of claim 27, wherein the developmental abnormality
comprises embryonic lethality or reduced viability. 42. The method
of claim 27, wherein the cardiovascular, endothelial or angiogenic
disorders are arterial diseases, such as diabetes mellitus;
papilledema; optic atrophy; atherosclerosis; angina; myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as congestive heart failure;
hypertension; inflammatory vasculitides; Reynaud's disease and
Reynaud's phenomenon; aneurysms and arterial restenosis; venous and
lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. 43. The method of claim 27, wherein the immunological
disorders are systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation-associated
diseases including graft rejection and graft-versus-host disease.
44. The method of claim 27, wherein said bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis. 45. The
method of claim 26, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
a decreased anxiety-like response during open field activity
testing; an increased anxiety-like response during open field
activity testing; balding, exothalamus observations, and
piloerection observations in functional observation battery (FOB)
testing; an increased mean artery-to-vein ratio associated with
retinal degeneration; developing cataracts; an increased mean serum
cholesterol level; an increased mean serum triglyceride level; a
decreased mean serum insulin level, a decreased mean percentage of
B cells in the spleen and lymph node; a decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean serum IgA
levels; an increased mean serum IgG2a response to an ovalbumin
challenge; increased mean serum IgM, IgG1, IgG2a and IgG2b levels;
increased mean serum IgM, IgA and IgG3 levels; increased mean serum
IgM, IgG1, IgG2a and IgG2b levels; an increased mean percentage of
CD4 cells and a decreased mean percentage of CD8 cells in spleen
and thymus; mobilization of neutrophils in response to peritoneal
inflammation; an enhanced DDS-induced colitis response; an enhanced
ConA-induced hepatitis response; a decreased skin fibroblast
proliferation; a decreased volumetric bone mineral density, a
decreased bone mineral content index (BMC/LBM), and a decreased
mean bone mineral density in total body, femur and vertebrate; a
decreased mean bone mineral density, a decreased mean trabecular
bone volume, decreased thickness, and decreased connectivity
density; a decreased body weight and length, decreased total tissue
mass and lean body mass, a decreased femoral midshaft
cross-sectional area with decreased alkaline phosphatase levels;
growth retardation with decreased body weight and length, total
tissue mass, and lean body mass; a diaphragmatic hernia; an
increased total tissue mass, increased lean body mass, increased
bone mineral content, increased total body and increased femoral
bone mineral density; an enhanced glucose tolerance; developmental
disorders including abnormal kidney development marked by kidney
agenesis; embryonic lethality; or embryonic lethality wherein
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels. 46. An agent identified by the method of
claim 26. 47. The agent of claim 46 which is an agonist or
antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. 48. The agent of claim
47, wherein the agonist is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 49. The agent of claim 47, wherein the antagonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 50. A method
of identifying an agent that modulates a physiological
characteristic associated with a disruption of the gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0143] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0144] (b) measuring a physiological characteristic exhibited by
the non-human transgenic animal of (a);
[0145] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic exhibited by the non-human transgenic
animal that differs from the physiological characteristic exhibited
by the wild-type animal is identified as a physiological
characteristic associated with gene disruption;
[0146] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0147] (e) determining whether the physiological characteristic
associated with gene disruption is modulated.
51. The method of claim 50, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
a decreased anxiety-like response during open field activity
testing; an increased anxiety-like response during open field
activity testing; balding, exothalamus observations, and
piloerection observations in functional observation battery (FOB)
testing; an increased mean artery-to-vein ratio associated with
retinal degeneration; developing cataracts; an increased mean serum
cholesterol level; an increased mean serum triglyceride level; a
decreased mean serum insulin level, a decreased mean percentage of
B cells in the spleen and lymph node; a decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean serum IgA
levels; an increased mean serum IgG2a response to an ovalbumin
challenge; increased mean serum IgM, IgG1, IgG2a and IgG2b levels;
increased mean serum IgM, IgA and IgG3 levels; increased mean serum
IgM, IgG1, IgG2a and IgG2b levels; an increased mean percentage of
CD4 cells and a decreased mean percentage of CD8 cells in spleen
and thymus; mobilization of neutrophils in response to peritoneal
inflammation; an enhanced DDS-induced colitis response; an enhanced
ConA-induced hepatitis response; a decreased skin fibroblast
proliferation; a decreased volumetric bone mineral density, a
decreased bone mineral content index (BMC/LBM), and a decreased
mean bone mineral density in total body, femur and vertebrate; a
decreased mean bone mineral density, a decreased mean trabecular
bone volume, decreased thickness, and decreased connectivity
density; a decreased body weight and length, decreased total tissue
mass and lean body mass, a decreased femoral midshaft
cross-sectional area with decreased alkaline phosphatase levels;
growth retardation with decreased body weight and length, total
tissue mass, and lean body mass; a diaphragmatic hernia; an
increased total tissue mass, increased lean body mass, increased
bone mineral content, increased total body and increased femoral
bone mineral density; an enhanced glucose tolerance; developmental
disorders including abnormal kidney development marked by kidney
agenesis; embryonic lethality; or embryonic lethality wherein
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels. 52. An agent identified by the method of
claim 50. 53. The agent of claim 52 which is an agonist or
antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. 54. The agent of claim
53, wherein the agonist is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 55. The agent of claim 53, wherein the antagonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 56. A method
of identifying an agent which modulates a behavior associated with
a disruption of the gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, the method comprising:
[0148] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0149] (b) observing the behavior exhibited by the non-human
transgenic animal of (a);
[0150] (c) comparing the observed behavior of (b) with that of a
gender matched wild-type animal, wherein the observed behavior
exhibited by the non-human transgenic animal that differs from the
observed behavior exhibited by the wild-type animal is identified
as a behavior associated with gene disruption;
[0151] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0152] (e) determining whether the agent modulates the behavior
associated with gene disruption.
57. The method of claim 56, wherein the behavior is an increased
anxiety-like response during open field activity testing. 58. The
method of claim 56, wherein the behavior is a decreased
anxiety-like response during open field activity testing. 59. The
method of claim 56, wherein the behavior is an abnormal circadian
rhythm during home-cage activity testing. 60. The method of claim
56, wherein the behavior is an enhanced motor coordination during
inverted screen testing. 61. The method of claim 56, wherein the
behavior is an impaired motor coordination during inverted screen
testing. 62. The method of claim 56, wherein the behavior is
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. 63. An agent identified by the
method of claim 56. 64. The agent of claim 63 which is an agonist
or antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. 65. The agent
of claim 64, wherein the agonist is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 66. The agent of claim 64, wherein the antagonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 67. A method
of identifying an agent that ameliorates or modulates a
neurological disorder; a cardiovascular, endothelial or angiogenic
disorder; an eye abnormality; an immunological disorder; an
oncological disorder; a bone metabolic abnormality or disorder; a
lipid metabolic disorder; or a developmental abnormality associated
with a disruption in the gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, the method comprising:
[0153] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0154] (b) administering a test agent to said non-human transgenic
animal; and
[0155] (c) determining whether said test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in the non-human
transgenic animal.
68. The method of claim 67, wherein the neurological disorder is an
increased anxiety-like response during open field activity testing.
69. The method of claim 67, wherein the neurological disorder is a
decreased anxiety-like response during open field activity testing.
70. The method of claim 67, wherein the neurological disorder is an
abnormal circadian rhythm during home-cage activity testing. 71.
The method of claim 67, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing. 72. The
method of claim 67, wherein the neurological disorder is an
impaired motor coordination during inverted screen testing. 73. The
method of claim 67, wherein the neurological disorder is
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. 74. The method of claim 67,
wherein the eye abnormality is a retinal abnormality. 75. The
method of claim 67, wherein the eye abnormality is consistent with
vision problems or blindness. 76. The method of claim 74, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
77. The method of claim 74, wherein the retinal abnormality is
characterized by retinal degeneration or retinal dysplasia. 78. The
method of claim 74, wherein the retinal abnormality is consistent
with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. 79. The method of claim 67, wherein the eye
abnormality is a cataract. 80. The method of claim 79, wherein the
cataract is a systemic disease such as human Down's syndrome,
Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfan
syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy, Fabry
disease, hypoparathroidism or Conradi syndrome. 81. The method of
claim 67, wherein the developmental abnormality comprises embryonic
lethality or reduced viability. 82. The method of claim 67, wherein
the cardiovascular, endothelial or angiogenic disorders are
arterial diseases, such as diabetes mellitus; papilledema; optic
atrophy; atherosclerosis; angina; myocardial infarctions such as
acute myocardial infarctions, cardiac hypertrophy, and heart
failure such as congestive heart failure; hypertension;
inflammatory vasculitides; Reynaud's disease and Reynaud's
phenomenon; aneurysms and arterial restenosis; venous and lymphatic
disorders such as thrombophlebitis, lymphangitis, and lymphedema;
peripheral vascular disease; cancer such as vascular tumors, e.g.,
hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. 83. The method of claim 67, wherein the immunological
disorders are systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma; allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
84. The method of claim 67, wherein said bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis. 85. The
method of claim 67, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
a decreased anxiety-like response during open field activity
testing; an increased anxiety-like response during open field
activity testing; balding, exothalamus observations, and
piloerection observations in functional observation battery (FOB)
testing; an increased mean artery-to-vein ratio associated with
retinal degeneration; developing cataracts; an increased mean serum
cholesterol level; an increased mean serum triglyceride level; a
decreased mean serum insulin level, a decreased mean percentage of
B cells in the spleen and lymph node; a decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean serum IgA
levels; an increased mean serum IgG2a response to an ovalbumin
challenge; increased mean serum IgM, IgG1, IgG2a and IgG2b levels;
increased mean serum IgM, IgA and IgG3 levels; increased mean serum
IgM, IgG1, IgG2a and IgG2b levels; an increased mean percentage of
CD4 cells and a decreased mean percentage of CD8 cells in spleen
and thymus; mobilization of neutrophils in response to peritoneal
inflammation; an enhanced DDS-induced colitis response; an enhanced
ConA-induced hepatitis response; a decreased skin fibroblast
proliferation; a decreased volumetric bone mineral density, a
decreased bone mineral content index (BMC/LBM), and a decreased
mean bone mineral density in total body, femur and vertebrate; a
decreased mean bone mineral density, a decreased mean trabecular
bone volume, decreased thickness, and decreased connectivity
density; a decreased body weight and length, decreased total tissue
mass and lean body mass, a decreased femoral midshaft
cross-sectional area with decreased alkaline phosphatase levels;
growth retardation with decreased body weight and length, total
tissue mass, and lean body mass; a diaphragmatic hernia; an
increased total tissue mass, increased lean body mass, increased
bone mineral content, increased total body and increased femoral
bone mineral density; an enhanced glucose tolerance; developmental
disorders including abnormal kidney development marked by kidney
agenesis; embryonic lethality; or embryonic lethality wherein
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels. 86. An agent identified by the method of
claim 67. 87. The agent of claim 86 which is an agonist or
antagonist of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide. 88. The agent of claim
87, wherein the agonist is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 89. The agent of claim 87, wherein the antagonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 90. A
therapeutic agent identified by the method of claim 67. 91. A
method of identifying an agent that modulates the expression of a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, the method comprising:
[0156] (a) contacting a test agent with a host cell expressing a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide; and
[0157] (b) determining whether the test agent modulates the
expression of the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide by the host
cell.
92. An agent identified by the method of claim 91. 93. The agent of
claim 92 which is an agonist or antagonist of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. 94. The agent of claim 93, wherein the agonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 95. The agent
of claim 93, wherein the antagonist is an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody. 96. A method of evaluating a
therapeutic agent capable of affecting a condition associated with
a disruption of a gene which encodes for a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, the method comprising:
[0158] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide;
[0159] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0160] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a condition resulting from the gene
disruption in the non-human transgenic animal;
[0161] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0162] (e) evaluating the effects of the test agent on the
identified condition associated with gene disruption in the
non-human transgenic animal.
97. The method of claim 96, wherein the condition is a neurological
disorder; a cardiovascular, endothelial or angiogenic disorder; an
eye abnormality; an immunological disorder; an oncological
disorder; a bone metabolic abnormality or disorder; a lipid
metabolic disorder; or a developmental abnormality. 98. A
therapeutic agent identified by the method of claim 96. 99. The
therapeutic agent of claim 98 which is an agonist or antagonist of
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide. 100. The therapeutic agent of claim
99, wherein the agonist is an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 101. The therapeutic agent of claim 99, wherein the
antagonist is an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 102. A pharmaceutical composition comprising the
therapeutic agent of claim 98. 103. A method of treating or
preventing or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method comprising
administering to a subject in need of such treatment whom may
already have the disorder, or may be prone to have the disorder or
may be in whom the disorder is to be prevented, a therapeutically
effective amount of the therapeutic agent of claim 94, or agonists
or antagonists thereof, thereby effectively treating or preventing
or ameliorating said disorder. 104. The method of claim 103,
wherein the neurological disorder is an increased anxiety-like
response during open field activity testing. 105. The method of
claim 103, wherein the neurological disorder is a decreased
anxiety-like response during open field activity testing. 106. The
method of claim 103, wherein the neurological disorder is an
abnormal circadian rhythm during home-cage activity testing. 107.
The method of claim 103, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing. 108.
The method of claim 103, wherein the neurological disorder is an
impaired motor coordination during inverted screen testing. 109.
The method of claim 103, wherein the neurological disorder is
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. 110. The method of claim 103,
wherein the eye abnormality is a retinal abnormality. 111. The
method of claim 103, wherein the eye abnormality is consistent with
vision problems or blindness. 112. The method of claim 110, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
113. The method of claim 110, wherein the retinal abnormality is
characterized by retinal degeneration or retinal dysplasia. 114.
The method of claim 110, wherein the retinal abnormality is
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. 115. The method of claim 103, wherein the eye
abnormality is a cataract. 116. The method of claim 115, wherein
the cataract is a systemic disease such as human Down's syndrome,
Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfan
syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy, Fabry
disease, hypoparathroidism or Conradi syndrome. 117. The method of
claim 103, wherein the developmental abnormality comprises
embryonic lethality or reduced viability. 118. The method of claim
103, wherein the cardiovascular, endothelial or angiogenic
disorders are arterial diseases, such as diabetes mellitus;
papilledema; optic atrophy; atherosclerosis; angina; myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as congestive heart failure;
hypertension; inflammatory vasculitides; Reynaud's disease and
Reynaud's phenomenon; aneurysms and arterial restenosis; venous and
lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. 119. The method of claim 103, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
120. The method of claim 103, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. 121. A method of identifying an agent that
ameliorates or modulates a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality associated with a disruption in the gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising:
[0163] (a) providing a non-human transgenic animal cell culture,
each cell of said culture comprising a disruption of the gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide;
[0164] (b) administering a test agent to said cell culture; and
[0165] (c) determining whether said test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in said cell
culture.
122. The method of claim 121, wherein the neurological disorder is
an increased anxiety-like response during open field activity
testing. 123. The method of claim 121, wherein the neurological
disorder is a decreased anxiety-like response during open field
activity testing. 124. The method of claim 121, wherein the
neurological disorder is an abnormal circadian rhythm during
home-cage activity testing. 125. The method of claim 121, wherein
the neurological disorder is an enhanced motor coordination during
inverted screen testing. 126. The method of claim 121, wherein the
neurological disorder is an impaired motor coordination during
inverted screen testing. 127. The method of claim 121, wherein the
neurological disorder is depression, generalized anxiety disorders,
attention deficit disorder, sleep disorder, hyperactivity disorder,
obsessive compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. 128. The method of claim 121,
wherein the eye abnormality is a retinal abnormality. 129. The
method of claim 121, wherein the eye abnormality is consistent with
vision problems or blindness. 130. The method of claim 128, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
131. The method of claim 128, wherein the retinal abnormality is
characterized by retinal degeneration or retinal dysplasia. 132.
The method of claim 128, wherein the retinal abnormality is
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. 133. The method of claim 121, wherein the eye
abnormality is a cataract. 134. The method of claim 133, wherein
the cataract is a systemic disease such as human Down's syndrome,
Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfan
syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy, Fabry
disease, hypoparathroidism or Conradi syndrome. 135. The method of
claim 121, wherein the developmental abnormality comprises
embryonic lethality or reduced viability. 136. The method of claim
121, wherein the cardiovascular, endothelial or angiogenic
disorders are arterial diseases, such as diabetes mellitus;
papilledema; optic atrophy; atherosclerosis; angina; myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as congestive heart failure;
hypertension; inflammatory vasculitides; Reynaud's disease and
Reynaud's phenomenon; aneurysms and arterial restenosis; venous and
lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. 137. The method of claim 121, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, B and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
138. The method of claim 121, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. 139. An agent identified by the method of claim 121.
140. The agent of claim 139 which is an agonist or antagonist of a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide. 141. The agent of claim 140, wherein the
agonist is an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody. 142. The agent of claim 140, wherein the antagonist is an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody. 143. A
therapeutic agent identified by the method of claim 121. 144. A
method of modulating a phenotype associated with a disruption of a
gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising administering to a subject whom may already have the
phenotype, or may be prone to have the phenotype or may be in whom
the phenotype is to be prevented, an effective amount of the agent
of claim 46, or agonists or antagonists thereof, thereby
effectively modulating the phenotype. 145. A method of modulating a
physiological characteristic associated with a disruption of a gene
which encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising administering to a subject whom may already exhibit the
physiological characteristic, or may be prone to exhibit the
physiological characteristic or may be in whom the physiological
characteristic is to be prevented, an effective amount of the agent
of claim 52, or agonists or antagonists thereof, thereby
effectively modulating the physiological characteristic. 146. A
method of modulating a behavior associated with a disruption of a
gene which encodes for a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising administering to a subject whom may already exhibit the
behavior, or may be prone to exhibit the behavior or may be in whom
the exhibited behavior is to be prevented, an effective amount of
the agent of claim 63, or agonists or antagonists thereof, thereby
effectively modulating the behavior. 147. A method of modulating
the expression of a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, the method
comprising administering to a host cell expressing said PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, an effective amount of the agent of claim 92, or
agonists or antagonists thereof, thereby effectively modulating the
expression of said polypeptide. 148. A method of modulating a
condition associated with a disruption of a gene which encodes for
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the method comprising administering
to a subject whom may have the condition, or may be prone to have
the condition or may be in whom the condition is to be prevented, a
therapeutically effective amount of the therapeutic agent of claim
98, or agonists or antagonists thereof, thereby effectively
modulating the condition. 149. A method of treating or preventing
or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, the method comprising
administering to a non-human transgenic animal cell culture, each
cell of said culture comprising a disruption of the gene which
encodes for a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide, a therapeutically
effective amount of the agent of claim 94, or agonists or
antagonists thereof, thereby effectively treating or preventing or
ameliorating said disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0166] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native
sequence PRO224 cDNA, wherein SEQ ID NO:1 is a clone designated
herein as "DNA33221-1133" (UNQ198).
[0167] 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.
[0168] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native
sequence PRO9783 cDNA, wherein SEQ ID NO:3 is a clone designated
herein as "DNA131590-2962" (UNQ2914).
[0169] FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived
from the coding sequence, of SEQ ID NO:3 shown in FIG. 3.
[0170] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native
sequence PRO1108 cDNA, wherein SEQ ID NO:5 is a clone designated
herein as "DNA58848-1472" (UNQ551).
[0171] FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived
from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0172] FIGS. 7A-B show a nucleotide sequence (SEQ ID NO:7) of a
native sequence PRO34000 cDNA, wherein SEQ ID NO:7 is a clone
designated herein as "DNA203528-3014" (UNQ9196).
[0173] FIGS. 8A-B show the amino acid sequence (SEQ ID NO:8)
derived from the coding sequence of SEQ ID NO:7 shown in FIGS.
7A-B.
[0174] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native
sequence PRO240 cDNA, wherein SEQ ID NO:9 is a clone designated
herein as "DNA34387-1138" (UNQ214).
[0175] FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived
from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0176] FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a
native sequence PRO943 cDNA, wherein SEQ ID NO:11 is a clone
designated herein as "DNA52192-1369" (UNQ480).
[0177] FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived
from the coding sequence of SEQ ID NO:11 shown in FIG. 11.
[0178] FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a
native sequence PRO230 cDNA, wherein SEQ ID NO:13 is a clone
designated herein as "DNA33223-1136" (UNQ204).
[0179] FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived
from the coding sequence of SEQ ID NO:13 shown in FIG. 13.
[0180] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a
native sequence PRO178 cDNA, wherein SEQ ID NO:15 is a clone
designated herein as "DNA23339-1130" (UNQ152).
[0181] FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived
from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0182] FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a
native sequence PRO1199 cDNA, wherein SEQ ID NO:17 is a clone
designated herein as "DNA65351-1366-2" (UNQ407).
[0183] FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived
from the coding sequence of SEQ ID NO:17 shown in FIG. 17.
[0184] FIGS. 19A-B show a nucleotide sequence (SEQ ID NO:19) of a
native sequence hu A33 cDNA, wherein SEQ ID NO:19 is a clone
designated herein as "DNA98557" (UNQ1425).
[0185] FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived
from the coding sequence of SEQ ID NO:19 shown in FIGS. 19A-B.
[0186] FIGS. 21A-B show a nucleotide sequence (SEQ ID NO:21) of a
native sequence PRO4333 cDNA, wherein SEQ ID NO:21 is a clone
designated herein as "DNA84210-2576" (UNQ1888).
[0187] FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived
from the coding sequence of SEQ ID NO:21 shown in FIGS. 21A-B.
[0188] FIGS. 23A-B show a nucleotide sequence (SEQ ID NO:23) of a
native sequence PRO1336 cDNA, wherein SEQ ID NO:23 is a clone
designated herein as "DNA65423-1595" (UNQ691).
[0189] FIGS. 24A-B show the amino acid sequence (SEQ ID NO:24)
derived from the coding sequence of SEQ ID NO:23 shown in FIGS.
23A-B.
[0190] FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a
native sequence PRO19598 cDNA, wherein SEQ ID NO:25 is a clone
designated herein as "DNA145887-2849" (UNQ5793).
[0191] FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived
from the coding sequence of SEQ ID NO:25 shown in FIG. 25.
[0192] FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a
native sequence PRO1083 cDNA, wherein SEQ ID NO:27 is a clone
designated herein as "DNA50921-1458" (UNQ540).
[0193] FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:27 shown in FIG. 27.
[0194] FIGS. 29A-C show a nucleotide sequence (SEQ ID NO:29) of a
native sequence hu TRPM2 cDNA, wherein SEQ ID NO:29 is a clone
designated herein as "DNA226659" (UNQ5070).
[0195] FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived
from the coding sequence of SEQ ID NO:29 shown in FIGS. 29A-C.
[0196] FIG. 31 shows a nucleotide sequence (SEQ ID NO:67) of a
native sequence PRO1801 cDNA, wherein SEQ ID NO:67 is a clone
designated herein as "DNA83500-2506" (UNQ852).
[0197] FIG. 32 shows the amino acid sequence (SEQ ID NO:68) derived
from the coding sequence of SEQ ID NO:67 shown in FIG. 31.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0198] 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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. The term "PRO
polypeptide" refers to each individual PRO/number polypeptide
disclosed herein. All disclosures in this specification which refer
to the "PRO polypeptide" refer to each of the polypeptides
individually as well as jointly. For example, descriptions of the
preparation of, purification of, derivation of, formation of
antibodies to or against, administration of, compositions
containing, treatment of a disease with, etc., pertain to each
polypeptide of the invention individually. The term "PRO
polypeptide" also includes variants of the PRO/number polypeptides
disclosed herein.
[0199] A "native sequence PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide" comprises a
polypeptide having the same amino acid sequence as the
corresponding PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide derived from nature. Such
native sequence PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptides can be isolated from
nature or can be produced by recombinant or synthetic means. The
term "native sequence PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide" specifically
encompasses naturally-occurring truncated or secreted forms of the
specific PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the
polypeptide. The invention provides native sequence PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides disclosed herein which 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides.
[0200] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide "extracellular domain" or "ECD"
refers to a form of the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide which is
essentially free of the transmembrane and cytoplasmic domains.
Ordinarily, a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 contemplated by the present invention.
[0201] The approximate location of the "signal peptides" of the
various PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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 mature 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.
[0202] "PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide variant" means a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, preferably an active PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, as
defined herein having at least about 80% amino acid sequence
identity with a full-length native sequence PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence as disclosed herein, a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, with
or without the signal peptide, as disclosed herein or any other
fragment of a full-length PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide sequence as
disclosed herein (such as those encoded by a nucleic acid that
represents only a portion of the complete coding sequence for a
full-length PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide). Such PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
variants include, for instance, PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide variant will have or will
have at least about 80% amino acid sequence identity, alternatively
will have or will have at least about 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
amino acid sequence identity, to a full-length native sequence
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide sequence as disclosed herein, a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, with
or without the signal peptide, as disclosed herein or any other
specifically defined fragment of a full-length PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence as disclosed herein. Ordinarily, PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
variant polypeptides are or are at least about 10 amino acids in
length, alternatively are or are at least about 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,
470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,
600 amino acids in length, or more. Optionally, PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polypeptides will have no more than one conservative amino acid
substitution as compared to the native PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
sequence, alternatively will have or will have no more than 2, 3,
4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as
compared to the native PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide sequence.
[0203] "Percent (%) amino acid sequence identity" with respect to
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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, 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.
[0204] 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. 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.
[0205] "PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 variant polynucleotide" or "PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
nucleic acid sequence" means a nucleic acid molecule which encodes
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, preferably an active PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, as defined herein and which has at least about 80%
nucleic acid sequence identity with a nucleotide acid sequence
encoding a full-length native sequence PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
sequence as disclosed herein, a full-length native sequence PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, with
or without the signal peptide, as disclosed herein or any other
fragment of a full-length PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide sequence as
disclosed herein (such as those encoded by a nucleic acid that
represents only a portion of the complete coding sequence for a
full-length PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide). Ordinarily, a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polynucleotide will have or will have at least about 80% nucleic
acid sequence identity, alternatively will have or will have at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence
identity with a nucleic acid sequence encoding a full-length native
sequence PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide sequence as disclosed herein, a
full-length native sequence PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide sequence lacking
the signal peptide as disclosed herein, an extracellular domain of
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, with or without the signal sequence,
as disclosed herein or any other fragment of a full-length PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide sequence as disclosed herein. Variants do not encompass
the native nucleotide sequence.
[0206] Ordinarily, PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 variant polynucleotides are
or are at least about 5 nucleotides in length, alternatively are or
are at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130,
135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,
200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,
330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,
460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,
590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,
720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,
850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,
980, 990, or 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.
[0207] "Percent (%) nucleic acid sequence identity" with respect to
PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-,
PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-,
PRO1083-, hu TRPM2- or PRO1801-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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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.
[0208] 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. 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.
[0209] The invention also provides PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polynucleotides which are nucleic acid molecules that encode a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide and which are capable of hybridizing,
preferably under stringent hybridization and wash conditions, to
nucleotide sequences encoding a full-length PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide as disclosed herein. PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polypeptides may be those that are encoded by a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 variant
polynucleotide.
[0210] The term "full-length coding region" when used in reference
to a nucleic acid encoding a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide refers to the
sequence of nucleotides which encode the full-length PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide of the invention (which is often shown between start
and stop codons, inclusive thereof, in the accompanying figures).
The term "full-length coding region" when used in reference to an
ATCC deposited nucleic acid refers to the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide-encoding portion of the cDNA that is inserted into the
vector deposited with the ATCC (which is often shown between start
and stop codons, inclusive thereof, in the accompanying
figures).
[0211] "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. The
invention provides that 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0212] An "isolated" PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0213] 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.
[0214] 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.
[0215] "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).
[0216] "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.
[0217] "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.
[0218] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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).
[0219] "Active" or "activity" for the purposes herein refers to
form(s) of a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, wherein "biological" activity refers to a biological
function (either inhibitory or stimulatory) caused by a native or
naturally-occurring PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide other than the
ability to induce the production of an antibody against an
antigenic epitope possessed by a native or naturally-occurring
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide.
[0220] The term "antagonist" is used in the broadest sense [unless
otherwise qualified], and includes any molecule that partially or
fully blocks, inhibits, or neutralizes a biological activity of a
native PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide disclosed herein. In a similar manner,
the term "agonist" is used in the broadest sense [unless otherwise
qualified] and includes any molecule that mimics a biological
activity of a native PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides, peptides, antisense oligonucleotides, small organic
molecules, etc. Methods for identifying agonists or antagonists of
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide may comprise contacting a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide with a candidate agonist or antagonist molecule and
measuring a detectable change in one or more biological activities
normally associated with the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide.
[0221] "Treating" or "treatment" or "alleviation" 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. A subject in need of treatment
may already have the disorder, or may be prone to have the disorder
or may be in whom the disorder is to be prevented.
[0222] "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.
[0223] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, rodents such as rats or
mice, 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.
[0224] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0225] "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..
[0226] 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. 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.
[0227] 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0228] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0229] An "effective amount" of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody, a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
binding oligopeptide, a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 binding organic molecule or
an agonist or antagonist thereof as disclosed herein is an amount
sufficient to carry out a specifically stated purpose. An
"effective amount" may be determined empirically and in a routine
manner, in relation to the stated purpose.
[0230] The term "therapeutically effective amount" refers to an
amount of an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody, a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide, a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 binding
oligopeptide, a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 binding organic molecule or other drug
effective to "treat" a disease or disorder in a subject or mammal.
In the case of cancer, the therapeutically effective amount of the
drug may reduce the number of cancer cells; reduce the tumor size;
inhibit (i.e., slow to some extent and preferably stop) cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some
extent and preferably stop) tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve to some extent one or more of
the symptoms associated with the cancer. See the definition herein
of "treating". To the extent the drug may prevent growth and/or
kill existing cancer cells, it may be cytostatic and/or
cytotoxic.
[0231] The phrases "cardiovascular, endothelial and angiogenic
disorder", "cardiovascular, endothelial and angiogenic
dysfunction", "cardiovascular, endothelial or angiogenic disorder"
and "cardiovascular, endothelial or angiogenic dysfunction" are
used interchangeably and refer in part to systemic disorders that
affect vessels, such as diabetes mellitus, as well as diseases of
the vessels themselves, such as of the arteries, capillaries,
veins, and/or lymphatics. This would include indications that
stimulate angiogenesis and/or cardiovascularization, and those that
inhibit angiogenesis and/or cardiovascularization. Such disorders
include, for example, arterial disease, such as atherosclerosis,
hypertension, inflammatory vasculitides, Reynaud's disease and
Reynaud's phenomenon, aneurysms, and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; and other vascular disorders such as peripheral
vascular disease, cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma, tumor angiogenesis, trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring,
ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular
disease, renal diseases such as acute renal failure, or
osteoporosis. This would also include angina, myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as CHF.
[0232] "Hypertrophy", as used herein, is defined as an increase in
mass of an organ or structure independent of natural growth that
does not involve tumor formation. Hypertrophy of an organ or tissue
is due either to an increase in the mass of the individual cells
(true hypertrophy), or to an increase in the number of cells making
up the tissue (hyperplasia), or both. Certain organs, such as the
heart, lose the ability to divide shortly after birth. Accordingly,
"cardiac hypertrophy" is defined as an increase in mass of the
heart, which, in adults, is characterized by an increase in myocyte
cell size and contractile protein content without concomitant cell
division. The character of the stress responsible for inciting the
hypertrophy, (e.g., increased preload, increased afterload, loss of
myocytes, as in myocardial infarction, or primary depression of
contractility), appears to play a critical role in determining the
nature of the response. The early stage of cardiac hypertrophy is
usually characterized morphologically by increases in the size of
myofibrils and mitochondria, as well as by enlargement of
mitochondria and nuclei. At this stage, while muscle cells are
larger than normal, cellular organization is largely preserved. At
a more advanced stage of cardiac hypertrophy, there are
preferential increases in the size or number of specific
organelles, such as mitochondria, and new contractile elements are
added in localized areas of the cells, in an irregular manner.
Cells subjected to long-standing hypertrophy show more obvious
disruptions in cellular organization, including markedly enlarged
nuclei with highly lobulated membranes, which displace adjacent
myofibrils and cause breakdown of normal Z-band registration. The
phrase "cardiac hypertrophy" is used to include all stages of the
progression of this condition, characterized by various degrees of
structural damage of the heart muscle, regardless of the underlying
cardiac disorder. Hence, the term also includes physiological
conditions instrumental in the development of cardiac hypertrophy,
such as elevated blood pressure, aortic stenosis, or myocardial
infarction.
[0233] "Heart failure" refers to an abnormality of cardiac function
where the heart does not pump blood at the rate needed for the
requirements of metabolizing tissues. The heart failure can be
caused by a number of factors, including ischemic, congenital,
rheumatic, or idiopathic forms.
[0234] "Congestive heart failure" (CHF) is a progressive pathologic
state where the heart is increasingly unable to supply adequate
cardiac output (the volume of blood pumped by the heart over time)
to deliver the oxygenated blood to peripheral tissues. As CHF
progresses, structural and hemodynamic damages occur. While these
damages have a variety of manifestations, one characteristic
symptom is ventricular hypertrophy. CHF is a common end result of a
number of various cardiac disorders.
[0235] "Myocardial infarction" generally results from
atherosclerosis of the coronary arteries, often with superimposed
coronary thrombosis. It may be divided into two major types:
transmural infarcts, in which myocardial necrosis involves the full
thickness of the ventricular wall, and subendocardial
(nontransmural) infarcts, in which the necrosis involves the
subendocardium, the intramural myocardium, or both, without
extending all the way through the ventricular wall to the
epicardium. Myocardial infarction is known to cause both a change
in hemodynamic effects and an alteration in structure in the
damaged and healthy zones of the heart. Thus, for example,
myocardial infarction reduces the maximum cardiac output and the
stroke volume of the heart. Also associated with myocardial
infarction is a stimulation of the DNA synthesis occurring in the
interstice as well as an increase in the formation of collagen in
the areas of the heart not affected.
[0236] As a result of the increased stress or strain placed on the
heart in prolonged hypertension due, for example, to the increased
total peripheral resistance, cardiac hypertrophy has long been
associated with "hypertension". A characteristic of the ventricle
that becomes hypertrophic as a result of chronic pressure overload
is an impaired diastolic performance. Fouad et al., J. Am. Coll.
Cardiol., 4: 1500-1506 (1984); Smith et al., J. Am. Coll. Cardiol.,
5: 869-874 (1985). A prolonged left ventricular relaxation has been
detected in early essential hypertension, in spite of normal or
supranormal systolic function. Hartford et al., Hypertension, 6:
329-338 (1984). However, there is no close parallelism between
blood pressure levels and cardiac hypertrophy. Although improvement
in left ventricular function in response to antihypertensive
therapy has been reported in humans, patients variously treated
with a diuretic (hydrochlorothiazide), a .beta.-blocker
(propranolol), or a calcium channel blocker (diltiazem), have shown
reversal of left ventricular hypertrophy, without improvement in
diastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7
(1984).
[0237] Another complex cardiac disease associated with cardiac
hypertrophy is "hypertrophic cardiomyopathy". This condition is
characterized by a great diversity of morphologic, functional, and
clinical features (Maron et al., N. Engl. J. Med., 316: 780-789
(1987); Spirito et al., N. Engl. J. Med., 320: 749-755 (1989);
Louie and Edwards, Prog. Cardiovasc. Dis., 36: 275-308 (1994);
Wigle et al., Circulation, 92: 1680-1692 (1995)), the heterogeneity
of which is accentuated by the fact that it afflicts patients of
all ages. Spirito et al., N. Engl. J. Med., 336: 775-785 (1997).
The causative factors of hypertrophic cardiomyopathy are also
diverse and little understood. In general, mutations in genes
encoding sarcomeric proteins are associated with hypertrophic
cardiomyopathy. Recent data suggest that .beta.-myosin heavy chain
mutations may account for approximately 30 to 40 percent of cases
of familial hypertrophic cardiomyopathy. Watkins et al., N. Engl.
J. Med., 326: 1108-1114 (1992); Schwartz et al, Circulation, 91:
532-540 (1995); Marian and Roberts, Circulation, 92: 1336-1347
(1995); Thierfelder et al., Cell, 77: 701-712 (1994); Watkins et
al., Nat. Gen., 11: 434-437 (1995). Besides .beta.-myosin heavy
chain, other locations of genetic mutations include cardiac
troponin T, alpha topomyosin, cardiac myosin binding protein C,
essential myosin light chain, and regulatory myosin light chain.
See, Malik and Watkins, Curr. Opin. Cardiol., 12: 295-302
(1997).
[0238] Supravalvular "aortic stenosis" is an inherited vascular
disorder characterized by narrowing of the ascending aorta, but
other arteries, including the pulmonary arteries, may also be
affected. Untreated aortic stenosis may lead to increased
intracardiac pressure resulting in myocardial hypertrophy and
eventually heart failure and death. The pathogenesis of this
disorder is not fully understood, but hypertrophy and possibly
hyperplasia of medial smooth muscle are prominent features of this
disorder. It has been reported that molecular variants of the
elastin gene are involved in the development and pathogenesis of
aortic stenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.
[0239] "Valvular regurgitation" occurs as a result of heart
diseases resulting in disorders of the cardiac valves. Various
diseases, like rheumatic fever, can cause the shrinking or pulling
apart of the valve orifice, while other diseases may result in
endocarditis, an inflammation of the endocardium or lining membrane
of the atrioventricular orifices and operation of the heart.
Defects such as the narrowing of the valve stenosis or the
defective closing of the valve result in an accumulation of blood
in the heart cavity or regurgitation of blood past the valve. If
uncorrected, prolonged valvular stenosis or insufficiency may
result in cardiac hypertrophy and associated damage to the heart
muscle, which may eventually necessitate valve replacement.
[0240] The term "immune related disease" means a disease in which a
component of the immune system of a mammal causes, mediates or
otherwise contributes to a morbidity in the mammal. Also included
are diseases in which stimulation or intervention of the immune
response has an ameliorative effect on progression of the disease.
Included within this term are immune-mediated inflammatory
diseases, non-immune-mediated inflammatory diseases, infectious
diseases, immunodeficiency diseases, neoplasia, etc.
[0241] The term "T cell mediated disease" means a disease in which
T cells directly or indirectly mediate or otherwise contribute to a
morbidity in a mammal. The T cell mediated disease may be
associated with cell mediated effects, lymphokine mediated effects,
etc., and even effects associated with B cells if the B cells are
stimulated, for example, by the lymphokines secreted by T
cells.
[0242] Examples of immune-related and inflammatory diseases, some
of which are immune or T cell mediated, include systemic lupus
erythematosis, rheumatoid arthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's
syndrome, systemic vasculitis, 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 demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis, inflammatory
bowel disease (ulcerative colitis: Crohn's disease),
gluten-sensitive enteropathy, and Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis, allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria, immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis, or transplantation associated
diseases including graft rejection and graft-versus-host-disease.
Infectious diseases including viral diseases such as AIDS (HIV
infection), hepatitis A, B, C, D, and E, herpes, etc., bacterial
infections, fungal infections, protozoal infections and parasitic
infections.
[0243] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and
ankylosing spondylitis), psoriasis, dermatitis including atopic
dermatitis; chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal
necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease (IBD) (Crohn's disease,
ulcerative colitis), and IBD with co-segregate of pyoderma
gangrenosum, erythema nodosum, primary sclerosing cholangitis,
and/or episcleritis), respiratory distress syndrome, including
adult respiratory distress syndrome (ARDS), meningitis,
IgE-mediated diseases such as anaphylaxis and allergic rhinitis,
encephalitis such as Rasmussen's encephalitis, uveitis, colitis
such as microscopic colitis and collagenous colitis,
glomerulonephritis (GN) such as membranous GN, idiopathic
membranous GN, membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
eczema, asthma, conditions involving infiltration of T cells and
chronic inflammatory responses, atherosclerosis, autoimmune
myocarditis, leukocyte adhesion deficiency, systemic lupus
erythematosus (SLE) such as cutaneous SLE, lupus (including
nephritis, cerebritis, pediatric, non-renal, discoid, alopecia),
juvenile onset diabetes, multiple sclerosis (MS) such as
spino-optical MS, allergic encephalomyelitis, immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis, agranulocytosis,
vasculitis (including Large Vessel vasculitis (including
Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis),
Medium Vessel vasculitis (including Kawasaki's Disease and
Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated
vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
immune hemolytic anemia including autoimmune hemolytic anemia
(AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome,
myasthenia gravis, antigen-antibody complex mediated diseases,
anti-glomerular basement membrane disease, anti-phospholipid
antibody syndrome, allergic neuritis, Bechet disease, Castleman's
syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic
Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson
syndrome, solid organ transplant rejection (including pretreatment
for high panel reactive antibody titers, IgA deposit in tissues,
and rejection arising from renal transplantation, liver
transplantation, intestinal transplantation, cardiac
transplantation, etc.), graft versus host disease (GVHD),
pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and
pemphigus mucus-membrane pemphigoid), autoimmune
polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune
complex nephritis, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (ITP), thrombocytopenia (as developed by
myocardial infarction patients, for example), including autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM),
including pediatric IDDM, and Sheehan's syndrome; autoimmune
hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome,
Berger's Disease (IgA nephropathy), primary biliary cirrhosis,
celiac sprue (gluten enteropathy), refractory sprue with
co-segregate dermatitis herpetiformis, cryoglobulinemia,
amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease),
coronary artery disease, autoimmune inner ear disease (AIED),
autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS),
polychondritis such as refractory polychondritis, pulmonary
alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,
monoclonal gammopathy of uncertain/unknown significance (MGUS),
peripheral neuropathy, paraneoplastic syndrome, channelopathies
such as epilepsy, migraine, arrhythmia, muscular disorders,
deafness, blindness, periodic paralysis, and channelopathies of the
CNS; autism, inflammatory myopathy, and focal segmental
glomerulosclerosis (FSGS).
[0244] The phrase "anxiety related disorders" refers to disorders
of anxiety, mood, and substance abuse, including but not limited
to: depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such disorders include the mild
to moderate anxiety, anxiety disorder due to a general medical
condition, anxiety disorder not otherwise specified, generalized
anxiety disorder, panic attack, panic disorder with agoraphobia,
panic disorder without agoraphobia, posttraumatic stress disorder,
social phobia, social anxiety, autism, specific phobia,
substance-induced anxiety disorder, acute alcohol withdrawal,
obsessive compulsive disorder, agoraphobia, monopolar disorders,
bipolar disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder,
enhancement of cognitive function, loss of cognitive function
associated with but not limited to Alzheimer's disease, stroke, or
traumatic injury to the brain, seizures resulting from disease or
injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0245] The term "lipid metabolic disorder" refers to abnormal
clinical chemistry levels of cholesterol and triglycerides, wherein
elevated levels of these lipids is an indication for
atherosclerosis. Additionally, abnormal serum lipid levels may be
an indication of various cardiovascular diseases including
hypertension, stroke, coronary artery diseases, diabetes and/or
obesity.
[0246] The phrase "eye abnormality" refers to such potential
disorders of the eye as they may be related to atherosclerosis or
various ophthalmological abnormalities. Such disorders include but
are nut limited to the following: retinal dysplasia, various
retinopathies, restenosis, retinal artery obstruction or occlusion;
retinal degeneration causing secondary atrophy of the retinal
vasculature, retinitis pigmentosa, macular dystrophies, Stargardt's
disease, congenital stationary night blindness, choroideremia,
gyrate atrophy, Leber's congenital amaurosis, retinoschisis
disorders, Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. Cataracts are also considered an eye abnormality
and are associated with such systemic diseases as: Human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15 condition, Alport syndrome, myotonic
dystrophy, Fabry disease, hypothroidisms, or Conradi syndrome.
Other ocular developmental anomalies include: Aniridia, anterior
segment and dysgenesis syndrome. Cataracts may also occur as a
result of an intraocular infection or inflammation (uveitis).
[0247] A "growth inhibitory amount" of an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody, PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
binding oligopeptide or PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 binding organic molecule is
an amount capable of inhibiting the growth of a cell, especially
tumor, e.g., cancer cell, either in vitro or in vivo. A "growth
inhibitory amount" of an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody, PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide, PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 binding
oligopeptide or PRO224, PRO9783, PRO1108, PRO3400b, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 binding organic molecule for purposes
of inhibiting neoplastic cell growth may be determined empirically
and in a routine manner.
[0248] A "cytotoxic amount" of an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibody, PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide, PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 binding
oligopeptide or PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 binding organic molecule is an amount
capable of causing the destruction of a cell, especially tumor,
e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount"
of an anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody, PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO433, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding oligopeptide or PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 binding
organic molecule for purposes of inhibiting neoplastic cell growth
may be determined empirically and in a routine manner.
[0249] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
monoclonal antibodies (including agonist, antagonist, and
neutralizing antibodies), anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody
compositions with polyepitopic specificity, polyclonal antibodies,
single chain anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibodies, and fragments of anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibodies (see below) as long as they exhibit the desired
biological or immunological activity. The term "immunoglobulin"
(Ig) is used interchangeable with antibody herein.
[0250] 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. The invention provides
that 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.
[0251] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to a H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H 1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0252] The L chain 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. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0253] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[0254] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and
96-101 (L3) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0255] 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. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0256] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of
interest herein include "primatized" antibodies comprising variable
domain antigen-binding sequences derived from a non-human primate
(e.g. Old World Monkey, Ape etc), and human constant region
sequences.
[0257] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H 1, C.sub.H 2 and C.sub.H 3. The constant
domains may be native sequence constant domains (e.g. human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0258] "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 (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0259] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H 1). Each Fab fragment
is monovalent with respect to antigen binding, i.e., it has a
single antigen-binding site. Pepsin treatment of an antibody yields
a single large F(ab').sub.2 fragment which roughly corresponds to
two disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the C.sub.H 1 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.
[0260] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0261] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site.
[0262] This fragment consists of a dimer of one heavy- and one
light-chain variable region domain in tight, non-covalent
association. From the folding of these two domains emanate six
hypervariable loops (3 loops each from the H and L chain) that
contribute the amino acid residues for antigen binding and 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.
[0263] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv 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); Borrebaeck 1995, infra.
[0264] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and V,
domains such that inter-chain but not intra-chain pairing of the V
domains is achieved, resulting in a bivalent fragment, i.e.,
fragment having two antigen-binding sites. Bispecific diabodies are
heterodimers of two "crossover" sFv fragments in which the V.sub.H
and V, domains of the two antibodies are present on different
polypeptide chains. 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).
[0265] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. 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 hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0266] A "species-dependent antibody," e.g., a mammalian anti-human
IgE antibody, is an antibody which has a stronger binding affinity
for an antigen from a first mammalian species than it has for a
homologue of that antigen from a second mammalian species.
Normally, the species-dependent antibody "bind specifically" to a
human antigen (i.e., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second non-human mammalian species which is at
least about 50 fold, or at least about 500 fold, or at least about
1000 fold, weaker than its binding affinity for the human antigen.
The species-dependent antibody can be of any of the various types
of antibodies as defined above, but preferably is a humanized or
human antibody.
[0267] A "PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding oligopeptide" is an oligopeptide that
binds, preferably specifically, to a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide as
described herein. PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 binding oligopeptides may be
chemically synthesized using known oligopeptide synthesis
methodology or may be prepared and purified using recombinant
technology. PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding oligopeptides usually are or are at
least about 5 amino acids in length, alternatively are or are at
least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in
length or more, wherein such oligopeptides that are capable of
binding, preferably specifically, to a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide as
described herein. PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 binding oligopeptides may be
identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening oligopeptide libraries for oligopeptides that are capable
of specifically binding to a polypeptide target are well known in
the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,
4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT
Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol. 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0268] A "PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding organic molecule" is an organic
molecule other than an oligopeptide or antibody as defined herein
that binds, preferably specifically, to a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide as
described herein. PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 binding organic molecules
may be identified and chemically synthesized using known
methodology (see, e.g., PCT Publication Nos. WO00/00823 and
WO00/39585). PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding organic molecules are usually less than
about 2000 daltons in size, alternatively less than about 1500,
750, 500, 250 or 200 daltons in size, wherein such organic
molecules that are capable of binding, preferably specifically, to
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide as described herein may be identified
without undue experimentation using well known techniques. In this
regard, it is noted that techniques for screening organic molecule
libraries for molecules that are capable of binding to a
polypeptide target are well known in the art (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585).
[0269] An antibody, oligopeptide or other organic molecule "which
binds" an antigen of interest, e.g. a tumor-associated polypeptide
antigen target, is one that binds the antigen with sufficient
affinity such that the antibody, oligopeptide or other organic
molecule is preferably useful as a diagnostic and/or therapeutic
agent in targeting a cell or tissue expressing the antigen, and
does not significantly cross-react with other proteins. The extent
of binding of the antibody, oligopeptide or other organic molecule
to a "non-target" protein will be less than about 10% of the
binding of the antibody, oligopeptide or other organic molecule to
its particular target protein as determined by fluorescence
activated cell sorting (FACS) analysis or radioimmunoprecipitation
(RIA). With regard to the binding of an antibody, oligopeptide or
other organic molecule to a target molecule, the term "specific
binding" or "specifically binds to" or is "specific for" a
particular polypeptide or an epitope on a particular polypeptide
target means binding that is measurably different from a
non-specific interaction. Specific binding can be measured, for
example, by determining binding of a molecule compared to binding
of a control molecule, which generally is a molecule of similar
structure that does not have binding activity. For example,
specific binding can be determined by competition with a control
molecule that is similar to the target, for example, an excess of
non-labeled target. In this case, specific binding is indicated if
the binding of the labeled target to a probe is competitively
inhibited by excess unlabeled target. The term "specific binding"
or "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for example, by a molecule having a
Kd for the target of at least about 10.sup.-4M, alternatively at
least about 10.sup.-5 M, alternatively at least about 10.sup.-6 M,
alternatively at least about 10.sup.-7 M, alternatively at least
about 10.sup.-8 M, alternatively at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, alternatively at least
about 10.sup.-11 M, alternatively at least about 10.sup.-12 M, or
greater. The term "specific binding" refers to binding where a
molecule binds to a particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
[0270] An antibody, oligopeptide or other organic molecule that
"inhibits the growth of tumor cells expressing a "PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801" or a
"growth inhibitory" antibody, oligopeptide or other organic
molecule is one which results in measurable growth inhibition of
cancer cells expressing or overexpressing the appropriate PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. The PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide may be a
transmembrane polypeptide expressed on the surface of a cancer cell
or may be a polypeptide that is produced and secreted by a cancer
cell. Preferred growth inhibitory anti-PRO224, anti-PRO9783,
anti-PRO1108, PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibodies, oligopeptides or organic molecules inhibit growth of
PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-,
PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-,
PRO1083-, hu TRPM2- or PRO1801-expressing tumor cells by or by
greater than 20%, preferably from about 20% to about 50%, and even
more preferably, by or by greater than 50% (e.g., from about 50% to
about 100%) as compared to the appropriate control, the control
typically being tumor cells not treated with the antibody,
oligopeptide or other organic molecule being tested. Growth
inhibition can be measured at an antibody concentration of about
0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, where
the growth inhibition is determined 1-10 days after exposure of the
tumor cells to the antibody. Growth inhibition of tumor cells in
vivo can be determined in various ways. The antibody is growth
inhibitory in vivo if administration of the anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody at about 1 .mu.g/kg to about 100
mg/kg body weight results in reduction in tumor size or tumor cell
proliferation within about 5 days to 3 months from the first
administration of the antibody, preferably within about 5 to 30
days.
[0271] An antibody, oligopeptide or other organic molecule which
"induces apoptosis" is one which induces programmed cell death as
determined by binding of annexin V, fragmentation of DNA, cell
shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
and/or formation of membrane vesicles (called apoptotic bodies).
The cell is usually one which overexpresses a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. Preferably the cell is a tumor cell, e.g., a prostate,
breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder
cell. Various methods are available for evaluating the cellular
events associated with apoptosis. For example, phosphatidyl serine
(PS) translocation can be measured by annexin binding; DNA
fragmentation can be evaluated through DNA laddering; and
nuclear/chromatin condensation along with DNA fragmentation can be
evaluated by any increase in hypodiploid cells. Preferably, the
antibody, oligopeptide or other organic molecule which induces
apoptosis is one which results in or in about 2 to 50 fold,
preferably in or in about 5 to 50 fold, and most preferably in or
in about 10 to 50 fold, induction of annexin binding relative to
untreated cell in an annexin binding assay.
[0272] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0273] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No.
5,821,337 may be performed. Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g., in a animal
model such as that disclosed in Clynes et al. Proc. Natl. Acad.
Sci. U.S.A. 95:652-656 (1998).
[0274] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0275] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0276] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0277] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, lung cancer (including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, and squamous
carcinoma of the lung), cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer (including gastrointestinal
cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer, as well as B-cell
lymphoma (including low grade/follicular non-Hodgkin's lymphoma
(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular
NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL;
high grade lymphoblastic NHL; high grade small non-cleaved cell
NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related
lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic
leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell
leukemia; chronic myeloblastic leukemia; and post-transplant
lymphoproliferative disorder (PTLD). Preferably, the cancer
comprises a tumor that expresses an IGF receptor, more preferably
breast cancer, lung cancer, colorectal cancer, or prostate cancer,
and most preferably breast or prostate cancer.
[0278] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, n7 serine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANB.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0279] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARFSTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor;
ABARELIX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0280] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one aspect of the invention, the
cell proliferative disorder is cancer.
[0281] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0282] An antibody, oligopeptide or other organic molecule which
"induces cell death" is one which causes a viable cell to become
nonviable. The cell is one which expresses a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide, preferably a cell that overexpresses a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide as compared to a normal cell of the same tissue type.
The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide may be a transmembrane polypeptide
expressed on the surface of a cancer cell or may be a polypeptide
that is produced and secreted by a cancer cell. Preferably, the
cell is a cancer cell, e.g., a breast, ovarian, stomach,
endometrial, salivary gland, lung, kidney, colon, thyroid,
pancreatic or bladder cell. Cell death in vitro may be determined
in the absence of complement and immune effector cells to
distinguish cell death induced by antibody-dependent cell-mediated
cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
Thus, the assay for cell death may be performed using heat
inactivated serum (i.e., in the absence of complement) and in the
absence of immune effector cells. To determine whether the
antibody, oligopeptide or other organic molecule is able to induce
cell death, loss of membrane integrity as evaluated by uptake of
propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology
17:1-11 (1995)) or 7AAD can be assessed relative to untreated
cells. Preferred cell death-inducing antibodies, oligopeptides or
other organic molecules are those which induce PI uptake in the PI
uptake assay in BT474 cells.
[0283] As used herein, the term "immunoadhesion" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesion") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesions
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 adhesion part of an
immunoadhesion 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
immunoadhesion 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.
[0284] 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.
[0285] "Replication-preventing agent" is an agent wherein
replication, function, and/or growth of the cells is inhibited or
prevented, or cells are destroyed, no matter what the mechanism,
such as by apoptosis, angiostasis, cytosis, tumoricide, mytosis
inhibition, blocking cell cycle progression, arresting cell growth,
binding to tumors, acting as cellular mediators, etc. Such agents
include a chemotherapeutic agent, cytotoxic agent, cytokine,
growth-inhibitory agent, or anti-hormonal agent, e.g., an
anti-estrogen compound such as tamoxifen, an anti-progesterone such
as onapristone (see, EP 616 812); or an anti-androgen such as
flutamide, as well as aromidase inhibitors, or a hormonal agent
such as an androgen.
[0286] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0287] Preferred cytotoxic agents herein for the specific tumor
types to use in combination with the antagonists herein are as
follows:
1. Prostate cancer: androgens, docetaxel, paclitaxel, estramustine,
doxorubicin, mitoxantrone, antibodies to ErbB2 domain(s) such as
2C4 (WO 01/00245; hybridoma ATCC HB-12697), which binds to a region
in the extracellular domain of ErbB2 (e.g., any one or more
residues in the region from about residue 22 to about residue 584
of ErbB2, inclusive), AVASTIN.TM. anti-vascular endothelial growth
factor (VEGF), TARCEVA.TM. OSI-774 (erlotinib) (Genenetech and OSI
Pharmaceuticals), or other epidermal growth factor receptor
tyrosine kinase inhibitors (EGFR TKI's). 2. Stomach cancer:
5-fluorouracil (5FU), XELODA.TM. capecitabine, methotrexate,
etoposide, cisplatin/carboplatin, pacliitaxel, docetaxel,
gemcitabine, doxorubicin, and CPT-11 (camptothcin-11; irinotecan,
USA Brand Name: CAMPTOSAR.RTM.). 3. Pancreatic cancer: gemcitabine,
5FU, XELODA.TM. capecitabine, CPT-11, docetaxel, paclitaxel,
cisplatin, carboplatin, TARCEVA.TM. erlotinib, and other EGFR
TKI's. 4. Colorectal cancer: 5FU, XELODA.TM. capecitabine, CPT-11,
oxaliplatin, AVASTIN.TM. anti-VEGF, TARCEVA.TM. erlotinib and other
EGFR TKI's, and ERBITUXT.TM. (formerly known as IMC-C225)
human:murine-chimerized monoclonal antibody that binds to EGFR and
blocks the ability of EGF to initiate receptor activation and
signaling to the tumor. 5. Renal cancer: IL-2, interferon alpha,
AVASTIN.TM. anti-VEGF, MEGACE.TM. (Megestrol acetate) progestin,
vinblastine, TARCEVA.TM. erlotinib, and other EGFR TKI's.
[0288] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu
A33-, PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-,
PRO1083-, hu TRPM2- or PRO1801-expressing cancer cell, either in
vitro or in vivo. Thus, the growth inhibitory agent may be one
which significantly reduces the percentage of PRO224-, PRO9783-,
PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-, PRO230-, PRO178-,
PRO1199-, PRO4333-, PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or
PRO1801-expressing cells in S phase. Examples of growth inhibitory
agents include agents that block cell cycle progression (at a place
other than S phase), such as agents that induce G1 arrest and
M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. W B Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the
European yew, is a semisynthetic analogue of paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel
promote the assembly of microtubules from tubulin dimers and
stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0289] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0290] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0291] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0292] The term "gene" refers to (a) a gene containing at least one
of the DNA sequences disclosed herein; (b) any DNA sequence that
encodes the amino acid sequence encoded by the DNA sequences
disclosed herein and/or; .COPYRGT.) any DNA sequence that
hybridizes to the complement of the coding sequences disclosed
herein. Preferably, the term includes coding as well as noncoding
regions, and preferably includes all sequences necessary for normal
gene expression.
[0293] The term "gene targeting" refers to a type of homologous
recombination that occurs when a fragment of genomic DNA is
introduced into a mammalian cell and that fragment locates and
recombines with endogenous homologous sequences. Gene targeting by
homologous recombination employs recombinant DNA technologies to
replace specific genomic sequences with exogenous DNA of particular
design.
[0294] The term "homologous recombination" refers to the exchange
of DNA fragments between two DNA molecules or chromatids at the
site of homologous nucleotide sequences.
[0295] The term "target gene" (alternatively referred to as "target
gene sequence" or "target DNA sequence") refers to any nucleic acid
molecule, polynucleotide, or gene to be modified by homologous
recombination. The target sequence includes an intact gene, an exon
or intron, a regulatory sequence or any region between genes. The
target gene my comprise a portion of a particular gene or genetic
locus in the individual's genomic DNA.
[0296] "Disruption" of a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 gene occurs when a fragment
of genomic DNA locates and recombines with an endogenous homologous
sequence wherein the disruption is a deletion of the native gene or
a portion thereof, or a mutation in the native gene or wherein the
disruption is the functional inactivation of the native gene.
Alternatively, sequence disruptions may be generated by nonspecific
insertional inactivation using a gene trap vector (i.e. non-human
transgenic animals containing and expressing a randomly inserted
transgene; see for example U.S. Pat. No. 6,436,707 issued Aug. 20,
2002). These sequence disruptions or modifications may include
insertions, missense, frameshift, deletion, or substitutions, or
replacements of DNA sequence, or any combination thereof.
Insertions include the insertion of entire genes, which may be of
animal, plant, fungal, insect, prokaryotic, or viral origin.
Disruption, for example, can alter the normal gene product by
inhibiting its production partially or completely or by enhancing
the normal gene product's activity. Preferably, the disruption is a
null disruption, wherein there is no significant expression of the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 gene.
[0297] The term "native expression" refers to the expression of the
full-length polypeptide encoded by the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 gene, at expression
levels present in the wild-type mouse. Thus, a disruption in which
there is "no native expression" of the endogenous PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 gene
refers to a partial or complete reduction of the expression of at
least a portion of a polypeptide encoded by an endogenous PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
gene of a single cell, selected cells, or all of the cells of a
mammal.
[0298] The term "knockout" refers to the disruption of a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
gene wherein the disruption results in: the functional inactivation
of the native gene; the deletion of the native gene or a portion
thereof; or a mutation in the native gene.
[0299] The term "knock-in" refers to the replacement of the mouse
ortholog (or other mouse gene) with a human cDNA encoding any of
the specific human PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-,
PRO943-, hu A33-, PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-,
PRO19598-, PRO1083-, hu TRPM2- or PRO1801-encoding genes or
variants thereof (ie. the disruption results in a replacement of a
native mouse gene with a native human gene).
[0300] The term "construct" or "targeting construct" refers to an
artificially assembled DNA segment to be transferred into a target
tissue, cell line or animal. Typically, the targeting construct
will include a gene or a nucleic acid sequence of particular
interest, a marker gene and appropriate control sequences. As
provided herein, the targeting construct comprises a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
targeting construct. A "PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 targeting construct"
includes a DNA sequence homologous to at least one portion of a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 gene and is capable of producing a disruption in a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO-1083, hu TRPM2 or PRO1801
gene in a host cell.
[0301] The term "transgenic cell" refers to a cell containing
within its genome a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 gene that has been
disrupted, modified, altered, or replaced completely or partially
by the method of gene targeting.
[0302] The term "transgenic animal" refers to an animal that
contains within its genome a specific gene that has been disrupted
or otherwise modified or mutated by the methods described herein or
methods otherwise well known in the art. Preferably the non-human
transgenic animal is a mammal. More preferably, the mammal is a
rodent such as a rat or mouse. In addition, a "transgenic animal"
may be a heterozygous animal (i e, one defective allele and one
wild-type allele) or a homozygous animal (i.e., two defective
alleles). An embryo is considered to fall within the definition of
an animal. The provision of an animal includes the provision of an
embryo or foetus in utero, whether by mating or otherwise, and
whether or not the embryo goes to term.
[0303] As used herein, the terms "selective marker" and position
selection marker" refer to a gene encoding a product that enables
only the cells that carry the gene to survive and/or grow under
certain conditions. For example, plant and animal cells that
express the introduced neomycin resistance (Ned) gene are resistant
to the compound G418. Cells that do not carry the Ned gene marker
are killed by G418. Other positive selection markers are known to,
or are within the purview of, those of ordinary skill in the
art.
[0304] The term "modulates" or "modulation" as used herein refers
to the decrease, inhibition, reduction, amelioration, increase or
enhancement of a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 gene function, expression,
activity, or alternatively a phenotype associated with PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
gene.
[0305] The term "ameliorates" or "amelioration" as used herein
refers to a decrease, reduction or elimination of a condition,
disease, disorder, or phenotype, including an abnormality or
symptom.
[0306] The term "abnormality" refers to any disease, disorder,
condition, or phenotype in which PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333,
[0307] PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 is
implicated, including pathological conditions and
behavioral observations.
Table 1
Please See the Computer Program Listing Appendix
TABLE-US-00001 [0308] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15
amino acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids)
Protein % 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%
TABLE-US-00002 TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein %
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%
TABLE-US-00003 TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
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%
TABLE-US-00004 TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison NNNNLLLVV (Length = 9 nucleotides) DNA %
nucleic acid sequence identity = (the number of identically
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
[0309] A. Full-Length PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 Polypeptides
[0310] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides. In particular,
cDNAs encoding various PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0311] 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
[0312] B. PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 Polypeptide Variants
[0313] In addition to the full-length native sequence PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides described herein, it is contemplated that PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
variants can be prepared. PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 variants can be prepared by
introducing appropriate nucleotide changes into the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
DNA, and/or by synthesis of the desired PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. Those
skilled in the art will appreciate that amino acid changes may
alter post-translational processes of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, such
as changing the number or position of glycosylation sites or
altering the membrane anchoring characteristics.
[0314] Variations in the native full-length sequence PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide or in various domains of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide that results in
a change in the amino acid sequence of the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide as compared with the native sequence PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. 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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide 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.
[0315] PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide.
[0316] PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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, PRO224, PRO9783, PRO1108, PRO34000; PRO240,
PRO943, hu A33; PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide fragments share
at least one biological and/or immunological activity with the
native PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide disclosed herein.
[0317] Conservative substitutions of interest are shown in Table 6
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
preferably introduced and the products screened.
TABLE-US-00005 TABLE 6 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg .RTM.)
Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu;
Asn Glu Cys .COPYRGT.) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E)
Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)
Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile;
Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;
Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala
Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y)
Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;
Norleucine
[0318] Substantial modifications in function or immunological
identity of the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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:
Amino acids may be grouped according to similarities in the
properties of their side chains (in A. L. Lehninger, in
Biochemistry, second ed., pp. 73-75, Worth Publishers, New York
(1975)):
TABLE-US-00006 (1) non-polar: Ala (A), Val (V), Leu (L),Ile (I),
Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G),
Ser (S), Thr (T), Cys .COPYRGT.), Tyr (Y), Asn (N), Gln (Q) (3)
acidic: Asp (D), Glu (E) (4) basic: Lys (K), Arg , His(H)
Alternatively, naturally occurring residues may be divided into
groups based on common side-chain properties:
TABLE-US-00007 (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic:
Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence
chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
[0319] 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.
[0320] 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 variant DNA.
[0321] 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.
[0322] C. Modifications of PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801
[0323] Covalent modifications of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides are
included within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues of the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. Derivatization
with bifunctional agents is useful, for instance, for crosslinking
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides to a water-insoluble support matrix or surface
for use in the method for purifying anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
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.
[0324] 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.
[0325] Another type of covalent modification of the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides (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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide. 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.
[0326] Addition of glycosylation sites to the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 (for O-linked glycosylation
sites). The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 amino acid sequence may optionally be altered
through changes at the DNA level, particularly by mutating the DNA
encoding the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide at preselected bases such that
codons are generated that will translate into the desired amino
acids.
[0327] Another means of increasing the number of carbohydrate
moieties on the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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).
[0328] Removal of carbohydrate moieties present on the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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).
[0329] Another type of covalent modification of PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides comprises linking the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0330] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides of the present invention may also
be modified in a way to form a chimeric molecule comprising the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide fused to another, heterologous polypeptide or
amino acid sequence.
[0331] Such a chimeric molecule comprises a fusion of the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. The presence of such epitope-tagged forms of the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide can be detected using an antibody against the
tag polypeptide. Also, provision of the epitope tag enables the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide 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)].
[0332] The chimeric molecule may comprise a fusion of the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide with an immunoglobulin 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide in place of at
least one variable region within an Ig molecule. In a particularly
preferred aspect of the invention, 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.
[0333] D. Preparation of PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 Polypeptides
[0334] The description below relates primarily to production of
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides by culturing cells transformed or transfected
with a vector containing PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 nucleic acid. It is, of
course, contemplated that alternative methods, which are well known
in the art, may be employed to prepare PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides. For
instance, the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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., 82:2149-2154 (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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide may be chemically synthesized separately and combined
using chemical or enzymatic methods to produce the full-length
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide.
[0335] 1. Isolation of DNA Encoding PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 Polypeptides
[0336] DNA encoding PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides may be obtained
from a cDNA library prepared from tissue believed to possess the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 mRNA and to express it at a detectable level. Accordingly,
human PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu
A33-, PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-,
PRO1083-, hu TRPM2- or PRO1801-DNA can be conveniently obtained
from a cDNA library prepared from human tissue, such as described
in the Examples. The PRO224-, PRO9783-, PRO1108-, PRO34000-,
PRO240-, PRO943-, hu A33-, PRO230-, PRO178-, PRO1199-, PRO4333-,
PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or PRO1801-encoding gene
may also be obtained from a genomic library or by known synthetic
procedures (e.g., automated nucleic acid synthesis).
[0337] Libraries can be screened with probes (such as antibodies to
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 is to use
PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR
Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1995)].
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 2. Selection and Transformation of Host Cells
[0342] Host cells are transfected or transformed with expression or
cloning vectors described herein for PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide
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.
[0343] 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 acid
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).
[0344] 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 K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, 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 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoAE15 (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.
[0345] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu
A33-, PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-,
PRO1083-, hu TRPM2- or PRO1801-encoding vectors. Saccharomyces
cerevisiae is a commonly used lower eukaryotic host microorganism.
Others include Schizosaccharomyces pombe (Beach and Nurse, Nature,
290: 140 [1981]; 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; Trichoderma 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, Tolypocladiuin (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).
[0346] Suitable host cells for the expression of glycosylated
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides 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 CCL51). The selection of the
appropriate host cell is deemed to be within the skill in the
art.
[0347] 3. Selection and Use of a Replicable Vector
[0348] The nucleic acid (e.g., cDNA or genomic DNA) encoding
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides 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.
[0349] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide 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 PRO224-,
PRO9783-, PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-, PRO230-,
PRO178-, PRO1199-, PRO4333-, PRO1336-, PRO19598-, PRO1083-, hu
TRPM2- or PRO1801-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.
[0350] 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.
[0351] 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.
[0352] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the PRO224-, PRO9783-, PRO1108-, PRO34000-, PRO240-,
PRO943-, hu A33-, PRO230-, PRO178-, PRO1199-, PRO4333-, PRO1336-,
PRO19598-, PRO1083-, hu TRPM2- or PRO1801-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 plasmid
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)].
[0353] Expression and cloning vectors usually contain a promoter
operably linked to the PRO224-, PRO9783-, PRO1108-, PRO34000-,
PRO240-, PRO943-, hu A33-, PRO230-, PRO178-, PRO1199-, PRO4333-,
PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or PRO1801-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 [Chang et al.,
Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],
alkaline phosphatase, a tryptophan (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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides.
[0354] 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.
[0355] 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, metallothionein,
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.
[0356] PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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.
[0357] Transcription of a DNA encoding the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 coding sequence, but is
preferably located at a site 5' from the promoter.
[0358] 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides.
[0359] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides 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.
[0360] 4. Detecting Gene Amplification/Expression
[0361] 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.
[0362] 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide or against a
synthetic peptide based on the DNA sequences provided herein or
against exogenous sequence fused to PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 DNA and encoding a
specific antibody epitope.
[0363] 5. Purification of Polypeptide
[0364] Forms of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptides 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptides can be disrupted by
various physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption, or cell lysing agents.
[0365] It may be desired to purify PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides 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
metal chelating columns to bind epitope-tagged forms of the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide produced.
[0366] E. Uses for PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 Polypeptides
[0367] Nucleotide sequences (or their complement) encoding PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu. TRPM2 or PRO1801
polypeptides 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.
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 nucleic acid will also be useful for the preparation of
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides by the recombinant techniques described
herein.
[0368] The full-length native sequence PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 cDNA or to isolate still
other cDNAs (for instance, those encoding naturally-occurring
variants of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides or PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides from
other species) which have a desired sequence identity to the native
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801. By way of example, a screening method will
comprise isolating the coding region of the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0369] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0370] Other useful fragments of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 nucleic acids
include antisense or sense oligonucleotides comprising a
singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 mRNA (sense) or PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
DNA (antisense) sequences. Antisense or sense oligonucleotides,
according to the present invention, comprise a fragment of the
coding region of PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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).
[0371] 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801. 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.
[0372] 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 metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0373] 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).
[0374] 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.
[0375] 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.
[0376] 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.
[0377] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 coding sequences.
[0378] Nucleotide sequences encoding a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide can
also be used to construct hybridization probes for mapping the gene
which encodes that PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide 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.
[0379] When the coding sequences for PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 encode a protein
which binds to another protein (for example, where the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
is a receptor), the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide or a receptor for PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides. 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.
[0380] Nucleic acids which encode PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides 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. The
invention provides cDNA encoding a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide which
can be used to clone genomic DNA encoding a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide in accordance with established techniques and the
genomic sequences used to generate transgenic animals that contain
cells which express DNA encoding PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides. Any
technique known in the art may be used to introduce a target gene
transgene into animals to produce the founder lines of transgenic
animals. Such techniques include, but are not limited to pronuclear
microinjection (U.S. Pat. Nos. 4,873,191, 4,736,866 and 4,870,009);
retrovirus mediated gene transfer into germ lines (Van der Putten,
et al., Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); gene
targeting in embryonic stem cells (Thompson, et al., Cell,
56:313-321 (1989)); nonspecific insertional inactivation using a
gene trap vector (U.S. Pat. No. 6,436,707); electroporation of
embryos (Lo, Mol. Cell. Biol., 3:1803-1814 (1983)); and
sperm-mediated gene transfer (Lavitrano, et al., Cell, 57:717-723
(1989)); etc. Typically, particular cells would be targeted for a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides. 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.
[0381] Alternatively, non-human homologues of PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides can be used to construct a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 "knock out" animal
which has a defective or altered gene encoding PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 proteins
as a result of homologous recombination between the endogenous gene
encoding PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides and altered genomic DNA encoding
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides introduced into an embryonic stem cell of the
animal. Preferably the knock out animal is a mammal. More
preferably, the mammal is a rodent such as a rat or mouse. For
example, cDNA encoding PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides can be used to
clone genomic DNA encoding PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides in accordance
with established techniques. A portion of the genomic DNA encoding
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide 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 gene encoding the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide.
[0382] In addition, knockout mice can be highly informative in the
discovery of gene function and pharmaceutical utility for a drug
target, as well as in the determination of the potential on-target
side effects associated with a given target. Gene function and
physiology are so well conserved between mice and humans, since
they are both mammals and contain similar numbers of genes, which
are highly conserved between the species. It has recently been well
documented, for example, that 98% of genes on mouse chromosome 16
have a human ortholog (Mural et al., Science 296:1661-71
(2002)).
[0383] Although gene targeting in embryonic stem (ES) cells has
enabled the construction of mice with null mutations in many genes
associated with human disease, not all genetic diseases are
attributable to null mutations. One can design valuable mouse
models of human diseases by establishing a method for gene
replacement (knock-in) which will disrupt the mouse locus and
introduce a human counterpart with mutation, Subsequently one can
conduct in vivo drug studies targeting the human protein (Kitamoto
et. Al., Biochemical and Biophysical Res. Commun., 222:742-47
(1996)).
[0384] Nucleic acid encoding the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0385] 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 infracellular
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).
[0386] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 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.
[0387] The nucleic acid molecules encoding the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 nucleic acid molecule of the
present invention can be used as a chromosome marker.
[0388] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides and nucleic acid molecules of the
present invention may also be used diagnostically for tissue
typing, wherein the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides of the present
invention may be differentially expressed in one tissue as compared
to another, preferably in a diseased tissue as compared to a normal
tissue of the same tissue type. PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 nucleic acid molecules will
find use for generating probes for PCR, Northern analysis, Southern
analysis and Western analysis.
[0389] The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides described herein may also be
employed as therapeutic agents. The PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides of the
present invention can be formulated according to known methods to
prepare pharmaceutically useful compositions, whereby the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
[0390] 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.
[0391] 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.
[0392] 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.
[0393] 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.
[0394] When in vivo administration of a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0395] Where sustained-release administration of a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide is desired in a formulation with release
characteristics suitable for the treatment of any disease or
disorder requiring administration of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide,
microencapsulation of the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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); Hora et 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.
[0396] 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.
[0397] This invention encompasses methods of screening compounds to
identify those that mimic the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide (agonists) or
prevent the effect of the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide (antagonists).
Agonists that mimic a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide would be
especially valuable therapeutically in those instances where a
negative phenotype is observed based on findings with the non-human
transgenic animal whose genome comprises a disruption of the gene
which encodes for the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. Antagonists
that prevent the effects of a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide would be
especially valuable therapeutically in those instances where a
positive phenotype is observed based upon observations with the
non-human transgenic knockout animal. Screening assays for
antagonist drug candidates are designed to identify compounds that
bind or complex with the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide encoded by the
genes identified herein, or otherwise interfere with the
interaction of the encoded polypeptide 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.
[0398] 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.
[0399] All assays for antagonists are common in that they call for
contacting the drug candidate with a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide encoded
by a nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0400] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide encoded by the gene identified herein
or the drug candidate is immobilized on a solid phase, e.g., on a
microliter plate, by covalent or non-covalent attachments.
Non-covalent attachment generally is accomplished by coating the
solid surface with a solution of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide and
drying. Alternatively, an immobilized antibody, e.g., a monoclonal
antibody, specific for the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0401] If the candidate compound interacts with but does not bind
to a particular PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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 at
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.
[0402] Compounds that interfere with the interaction of a gene
encoding a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 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.
[0403] To assay for antagonists, the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide indicates that the compound is an antagonist to the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide. Alternatively, antagonists may be detected by
combining the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide and a potential antagonist
with membrane-bound PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide receptors or
recombinant receptors under appropriate conditions for a
competitive inhibition assay. The PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide can be
labeled, such as by radioactivity, such that the number of PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. Transfected
cells that are grown on glass slides are exposed to labeled PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. The PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0404] As an alternative approach for receptor identification, the
labeled PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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.
[0405] Another approach in assessing the effect of an antagonist to
a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, would be administering a PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
antagonist to a wild-type mouse in order to mimic a known knockout
phenotype. Thus, one would initially knockout the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 gene of
interest and observe the resultant phenotype as a consequence of
knocking out or disrupting the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 gene. Subsequently, one
could then assess the effectiveness of an antagonist to the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide by administering an antagonist to the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide to a wild-type mouse. An effective antagonist would be
expected to mimic the phenotypic effect that was initially observed
in the knockout animal.
[0406] Likewise, one could assess the effect of an agonist to a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, by administering a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33; PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 agonist to a
non-human transgenic mouse in order to ameliorate a known negative
knockout phenotype. Thus, one would initially knockout the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
gene of interest and observe the resultant phenotype as a
consequence of knocking out or disrupting the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 gene.
Subsequently, one could then assess the effectiveness of an agonist
to the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide by administering an agonist to the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide to the non-human transgenic mouse. An effective
agonist would be expected to ameliorate the negative phenotypic
effect that was initially observed in the knockout animal.
[0407] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with a labeled PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide in the presence of the
candidate compound. The ability of the compound to enhance or block
this interaction could then be measured.
[0408] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide that recognizes the receptor but imparts no effect,
thereby competitively inhibiting the action of the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide.
[0409] Another potential PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
[0410] 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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide,
thereby blocking the normal biological activity of the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
[0411] 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).
[0412] 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.
[0413] 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.
[0414] Diagnostic and therapeutic uses of the herein disclosed
molecules may also be based upon the positive functional assay hits
disclosed and described below.
[0415] F. Anti-PRO224, Anti-PRO9783, Anti-PRO1108, Anti-PRO34000,
Anti-PRO240, Anti-PRO943, Anti-hu A33, Anti-PRO230, Anti-PRO178,
Anti-PRO1199, Anti-PRO4333, Anti-PRO1336, Anti-PRO19598,
Anti-PRO1083, Anti-hu TRPM2 or Anti-PRO1801 Antibodies
[0416] The present invention provides anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibodies which may find use herein as therapeutic and/or
diagnostic agents. Exemplary antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate
antibodies.
[0417] 1. Polyclonal Antibodies
[0418] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl
groups.
[0419] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0420] 2. Monoclonal Antibodies
[0421] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0422] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0423] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0424] Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. 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); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0425] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0426] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980).
[0427] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal e.g., by i.p. injection of the cells
into mice.
[0428] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0429] DNA encoding the monoclonal antibodies is 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 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 E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262
(1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).
[0430] Monoclonal antibodies or antibody fragments can be isolated
from antibody phage libraries generated using the techniques
described in McCafferty et al., Nature, 348:552-554 (1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991) describe the isolation of murine and
human antibodies, respectively, using phage libraries. Subsequent
publications describe the production of high affinity (nM range)
human antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266
(1993)). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
[0431] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0432] 3. Human and Humanized Antibodies
[0433] The anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 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); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0434] 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.
[0435] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0436] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0437] Various forms of a humanized anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody
are contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0438] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and WO 97/17852.
[0439] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 [1990]) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats,
reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J.,
Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0440] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0441] 4. Antibody Fragments
[0442] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors.
[0443] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. The antibody of choice is a single chain Fv
fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S.
Pat. No. 5,587,458. Fv and sFv are the only species with intact
combining sites that are devoid of constant regions; thus, they are
suitable for reduced nonspecific binding during in vivo use. sFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an sFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870 for example. Such linear antibody fragments may be
monospecific or bispecific.
[0444] 5. Bispecific Antibodies
[0445] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 protein as described herein. Other such antibodies may
combine a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 binding site with a binding site for another
protein. Alternatively, an anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 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. CD3), 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 and
localize cellular defense mechanisms to the PRO224-, PRO9783-,
PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-, PRO230-, PRO178-,
PRO1199-, PRO4333-, PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or
PRO1801-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide. These antibodies possess a PRO224-, PRO9783-,
PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-, PRO230-, PRO178-,
PRO1199-, PRO4333-, PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or
PRO1801-binding arm and an arm which binds the cytotoxic agent
(e.g., saporin, anti-interferon-.alpha., vinca alkaloid, ricin A
chain, methotrexate or radioactive isotope hapten). Bispecific
antibodies can be prepared as full length antibodies or antibody
fragments (e.g., F(ab').sub.2 bispecific antibodies).
[0446] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0447] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0448] According to a different approach, antibody variable domains
with the desired binding specificity (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, 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 cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments when unequal ratios
of the three polypeptide chains used in the construction provide
the optimum yield of the desired bispecific antibody. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains into a single expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios have no significant
affect on the yield of the desired chain combination.
[0449] The invention provides bispecific antibodies which are
composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. It was found that this asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details of generating
bispecific antibodies see, for example, Suresh et al., Methods in
Enzymology 121:210 (1986).
[0450] According to another approach described in U.S. Pat. No.
5,731,168, 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 C.sub.H3 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.
[0451] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
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, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0452] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').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.
[0453] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be 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. Various techniques for making and isolating
bispecific antibody fragments directly from recombinant cell
culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers. Kostelny et
al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins were linked to the Fab'
portions of two different antibodies by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a V.sub.H connected to a 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).
[0454] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0455] 6. Heteroconjugate Antibodies
[0456] 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.
[0457] 7. Multivalent Antibodies
[0458] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0459] 8. Effector Function Engineering
[0460] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in 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, B.
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 which 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). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0461] 9. Immunoconjugates
[0462] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a growth inhibitory agent, a 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).
[0463] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPU, 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.131In, .sup.90Y, and
.sup.186Re. 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.
[0464] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0465] The invention provides an anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody
(full length or fragments) which is conjugated to one or more
maytansinoid molecules.
[0466] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
Maytansinoid-Antibody Conjugates
[0467] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansonid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Anti-PRO224, Anti-PRO9783, Anti-PRO1108, Anti-PRO34000,
Anti-PRO240, Anti-PRO943, Anti-hu A33, Anti-PRO230, Anti-PRO178,
Anti-PRO1199, Anti-PRO4333, Anti-PRO1336, Anti-PRO19598,
Anti-PRO1083, Anti-hu TRPM2 or Anti-PRO1801 Antibody-Maytansinoid
Conjugates (Immunoconjugates)
[0468] Anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody-maytansinoid
conjugates are prepared by chemically linking an anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0469] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disufide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0470] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
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 toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0471] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hyrdoxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. The
linkage is formed at the C-3 position of maytansinol or a
maytansinol analogue.
Calicheamicin
[0472] Another immunoconjugate of interest comprises an
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibody conjugated to
one or more calicheamicin molecules. The calicheamicin family of
antibiotics are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. For the preparation of conjugates of
the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296
(all to American Cyanamid Company). Structural analogues of
calicheamicin which may be used include, but are not limited to,
.gamma..sub.1.sup.I, .alpha..sub.2.sup.I, .alpha..sub.3.sup.I,
N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sup.I.sub.1 (Hinman
et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer
Research 58:2925-2928 (1998) and the aforementioned U.S. patents to
American Cyanamid). Another anti-tumor drug that the antibody can
be conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Other Cytotoxic Agents
[0473] Other antitumor agents that can be conjugated to the
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies of the
invention include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively LL-E33288
complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well
as esperamicins (U.S. Pat. No. 5,877,296).
[0474] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0475] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0476] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies. Examples
include At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and
radioactive isotopes of Lu. When the conjugate is used for
diagnosis, it may comprise a radioactive atom for scintigraphic
studies, for example tc.sup.99m or I.sup.123, or a spin label for
nuclear magnetic resonance (NMR) imaging (also known as magnetic
resonance imaging, mri), such as iodine-123 again, iodine-131,
indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,
gadolinium, manganese or iron.
[0477] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0478] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
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. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0479] Alternatively, a fusion protein comprising the anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 antibody and cytotoxic agent may be made,
e.g., by recombinant techniques or peptide synthesis. The length of
DNA may comprise respective regions encoding the two portions of
the conjugate either adjacent one another or separated by a region
encoding a linker peptide which does not destroy the desired
properties of the conjugate.
[0480] The invention provides that the antibody may be conjugated
to a "receptor" (such streptavidin) for utilization in tumor
pre-targeting 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) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0481] 10. Immunoliposomes
[0482] The anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies disclosed
herein may also be formulated as immunoliposomes. A "liposome" is a
small vesicle composed of various types of lipids, phospholipids
and/or surfactant which is useful for delivery of a drug to a
mammal. The components of the liposome are commonly arranged in a
bilayer formation, similar to the lipid arrangement of biological
membranes. 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); Hwang et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0483] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PB). 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 is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0484] 11. Pharmaceutical Compositions of Antibodies
[0485] Antibodies specifically binding a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[0486] If the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 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.
[0487] 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.
[0488] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished, by filtration through
sterile filtration membranes.
[0489] 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.
[0490] G. Uses for Anti-PRO224, Anti-PRO9783, Anti-PRO1108,
Anti-PRO34000, Anti-PRO240, Anti-PRO943, Anti-hu A33, Anti-PRO230,
Anti-PRO178, Anti-PRO1199, Anti-PRO4333 Anti-PRO1336,
Anti-PRO19598, Anti-PRO1083, Anti-hu TRPM2 or Anti-PRO1801
Antibodies
[0491] The anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies of the
invention have various therapeutic and/or diagnostic utilities for
a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an immunological disorder; an oncological
disorder; an embryonic developmental disorder or lethality, or a
metabolic abnormality. For example, anti-PRO224, anti-PRO9783,
anti-PRO1108, anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33,
anti-PRO230, anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
antibodies may be used in diagnostic assays for PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801, e.g.,
detecting its expression (and in some cases, differential
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).
[0492] Anti-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies also are
useful for the affinity purification of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides from
recombinant cell culture or natural sources. In this process, the
antibodies against PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides 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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide, which is bound
to the immobilized antibody. Finally, the support is washed with
another suitable solvent that will release the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide from the antibody.
[0493] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0494] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0495] 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
[0496] 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.).
[0497] 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.
[0498] 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.
[0499] 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 Nod site, linked with blunt
to SalI hemikinased adaptors, cleaved with Nod, 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 pRIC5D that does not contain the Sfd site; see, Holmes et al.,
Science, 253:1278-1280 (1991)) in the unique XhoI and Nod
sites.
Example 2
Isolation of cDNA Clones by Amylase Screening
[0500] 1. Preparation of Oligo dT Primed cDNA Library
[0501] 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.
[0502] 2. Preparation of Random Primed cDNA Library
[0503] 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 Nod 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.
[0504] 3. Transformation and Detection
[0505] DNA from the library described in paragraph 2 above was
chilled on ice to which was added electrocompetent DH10B 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.
[0506] 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.
[0507] 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,
TDJ1p or SSA1p-4-p) or the complex formation of these proteins may
also be preferably employed in combination with the
amylase-expressing yeast.
[0508] 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).
[0509] 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).
[0510] 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).
[0511] Alternatively, instead of multiple small reactions, the
transformation was performed using a single, large scale reaction,
wherein reagent amounts were scaled up accordingly.
[0512] 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.
[0513] 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-Ura agar
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).
[0514] 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.
[0515] 4. Isolation of DNA by PCR Amplification
[0516] 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 distilled water.
The sequence of the forward oligonucleotide 1 was:
TABLE-US-00008 (SEQ ID. NO: 31)
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3'
The sequence of reverse oligonucleotide 2 was:
TABLE-US-00009 (SEQ ID NO: 32)
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3'
PCR was then performed as follows:
TABLE-US-00010 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.
[0517] 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.
[0518] 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 (TEE) 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
[0519] 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.
[0520] Using the techniques described in Examples 1 to 3 above,
numerous full-length cDNA clones were identified as encoding
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083 or PRO1801
polypeptides as disclosed herein. These cDNAs were then deposited
under the terms of the Budapest Treaty with the American Type
Culture Collection, University Blvd., Manassas, Va. 20110-2209, USA
(ATCC) as shown in Table 7 below. In addition, the sequence of
DNA98557, also known as huA33, was identified from GenBank
accession no.: AY112708. The sequence of DNA226659, also known as
hu TRPM2, was identified from GenBank accession no.: AB001535.
TABLE-US-00011 TABLE 7 Material ATCC Dep. No. Deposit Date
DNA33221-1133 209263 Sep. 16, 1997 DNA131590-2962 PTA-2297 Jul. 25,
2000 DNA58848-1472 209955 Jun. 9, 1998 DNA203528-3014 PTA-2780 Sep.
12, 2000 DNA34387-1138 209260 Sep. 16, 1997 DNA52192-1369 203042
Jul. 1, 1998 DNA33223-1136 209264 Sep. 16, 1997 DNA23339-1130
209282 Sep. 18, 1997 DNA65351-1366-2 209856 May 12, 1998
DNA84210-2576 203818 Mar. 2, 1999 DNA65423-1595 203227 Sep. 15,
1998 DNA145887-2849 PTA-1532 Mar. 21, 2000 DNA50921-1458 209859 May
12, 1998 DNA83500-2506 203391 Oct. 29, 1998
[0521] These deposits 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 8860G 638).
[0522] 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.
Example 4
Isolation of cDNA Clones Encoding Human PRO224 Polypeptides
(UNQ198)
[0523] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul 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 sequence. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.).
[0524] A consensus DNA sequence encoding PRO224 was assembled
relative to the other identified EST sequences, wherein the
consensus sequence was designated herein as DNA30845. Based on the
DNA30845 consensus sequence, oligonucleotides were synthesized 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 PRO224.
[0525] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00012 (SEQ ID NO: 33) forward PCR primer
5'-AAGTTCCAGTGCCGCACCAGTGGC-3' (SEQ ID NO: 34) reverse PCR primer
5'-TTGGTTCCACAGCCGAGCTCGTCG-3'
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30845 sequence which had the
following nucleotide sequence
TABLE-US-00013 hybridization probe (SEQ ID NO: 35)
5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAAT GCCCACC-3'
[0526] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO224 gene
using the probe oligonucleotide and one of the PCR primers.
[0527] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. 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.
[0528] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO224 [herein designated as
UNQ198 (DNA33221-1133)] and the derived protein sequence for
PRO224.
[0529] The entire nucleotide sequence of UNQ198 (DNA33221-1133) is
shown in FIG. 1 (SEQ ID NO:1). Clone UNQ198 (DNA33221-1133)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 96-98 [Kozak et al., supra]
and ending at the stop codon at nucleotide positions 942-944 (FIG.
1; SEQ ID NO:1). The start of a transmembrane region begins at
position 777. The predicted polypeptide precursor is 282 amino
acids long (FIG. 2; SEQ ID NO:2). Clone UNQ198 (DNA33221-1133) has
been deposited with ATCC and is assigned ATCC deposit no. 209263 on
Sep. 16, 1997.
[0530] Analysis of the amino acid sequence of the full-length
PRO224 suggests that it has homology to very low-density
lipoprotein receptors, apolipoprotein E receptor and chicken oocyte
receptors P95. Based on BLAST and FastA sequence alignment analysis
of the full-length sequence, PRO224 has amino acid identity to
portions of these proteins in the range from 28% to 45%, and
overall identity with these proteins in the range from 33% to
39%.
Example 5
Isolation of cDNA Clones Encoding Human PRO9783 Polypeptides
(UNQ2914)
[0531] 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, (1) public EST databases
(e.g., GenBank), (2) a proprietary EST database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.), (3) a proprietary EST
database from Genentech. The search was performed using the
computer program BLAST or BLAST2 [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 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 consensus DNA sequences with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.).
[0532] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described above. This consensus sequence
is herein designated DNA52160. In some cases, the consensus
sequence derives from an intermediate consensus DNA sequence which
was extended using repeated cycles of BLAST and phrap to extend
that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed above.
[0533] Based on the DNA52160 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 PRO9783.
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, supra, 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.
[0534] PCR primers (forward and reverse) were synthesized:
TABLE-US-00014 forward PCR primer 5'--3' (SEQ ID NO: 36)
CGAACCTTCCTACTGGGCTCCGGTG reverse PCR primer 5'--3' (SEQ ID NO: 37)
CCAACATCTATGCAGATACCTCAAGCATCTGCAAGACAGCCGTGC
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA52160 sequence which had the
following nucleotide sequence
TABLE-US-00015 hybridization probe (SEQ ID NO: 38) 5'--3'
GCCTGACAGCAAAGATCCGGAAGG
[0535] RNA for construction of the cDNA libraries was isolated from
human placenta tissue. 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 pRK5B or pRK5D;
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.
[0536] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length PRO9783
polypeptide (designated herein as DNA131590-2962 (UNQ2914) [FIG. 3,
SEQ ID NO: 3] and the derived protein sequence for that PRO9783
polypeptide.
[0537] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 237-239 and a stop signal at nucleotide
positions 1728-1730 (FIG. 3, SEQ ID NO:3). The predicted
polypeptide precursor is 497 amino acids long, has a calculated
molecular weight of approximately 55906 daltons and an estimated pI
of approximately 8.43. Analysis of the full-length PRO9783 sequence
shown in FIG. 4 (SEQ ID NO:4) evidences the presence of a variety
of important polypeptide domains as shown in FIG. 4, wherein the
locations given for those important polypeptide domains are
approximate as described above. Clone DNA131590-2962 (UNQ2914) has
been deposited with ATCC on Jul. 25, 2000 and is assigned ATCC
deposit no. PTA-2297.
[0538] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 4 (SEQ ID NO:4), evidenced
sequence identity between the PRO9783 amino acid sequence and the
following Dayhoff sequences: P_Y41738, AF109674.sub.--1,
D45027.sub.--1, P_Y13392, GLIP_HUMAN, CRS3_HUMAN, TPX1_HUMAN,
AF078552.sub.--1, P_Y17828, CRS1_HUMAN.
Example 6
Isolation of cDNA Clones Encoding Human PRO1108 Polypeptides
(UNQ551)
[0539] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul 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 sequence. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.).
[0540] A consensus DNA sequence was assembled relative to other EST
sequences using phrap. This consensus sequence is herein designated
"Consensus". In addition, the "Consensus" DNA sequence was extended
using repeated cycles of BLAST and phrap to extend the sequence as
far as possible using the sources of EST sequences discussed
above.
[0541] In light of the sequence homology between the "Consensus"
sequence and the Incyte EST clone no. 2379881, Incyte EST clone no.
2379881 was purchased and the cDNA insert was obtained and
sequenced. The sequence of this cDNA insert is shown in FIG. 5 and
is herein designated DNA58848-1472.
[0542] The entire nucleotide sequence of UNQ551 (DNA58848-1472) is
shown in FIG. 5 (SEQ ID NO:5). Clone UNQ551 (DNA58848-1472)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 77-79 and ending at the
stop codon at nucleotide positions 1445-1447 (FIG. 5). The
predicted polypeptide precursor is 456 amino acids long (FIG. 6).
The full-length PRO1108 protein shown in FIG. 6 has an estimated
molecular weight of about 52,071 daltons and a pI of about 9.46.
Analysis of the full-length PRO1108 sequence shown in FIG. 6 (SEQ
ID NO:6) evidences the presence of the following: type II
transmembrane domains from about amino acid 22 to about amino acid
42, from about amino acid 156 to about amino acid 176, from about
amino acid 180 to about amino acid 199 and from about amino acid
369 to about amino acid 388, potential N-glycosylaion sites from
about amino acid 247 to about amino acid 250, from about amino acid
327 to about amino acid 330, from about amino acid 328 to about
amino acid 331 and from about amino acid 362 to about amino acid
365 and an amino acid block having homology to ER lumen protein
retaining receptor protein from about amino acid 153 to about amino
acid 190. Clone UNQ551 (DNA58848-1472) has been deposited with ATCC
on Jun. 9, 1998 and is assigned ATCC deposit no. 209955. Analysis
of the amino acid sequence of the full-length PRO1108 polypeptide
suggests that it possesses significant sequence similarity to the
LPAAT protein, thereby indicating that PRO1108 may be a novel LPAAT
homolog. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO1108 amino acid sequence and the following Dayhoff
sequences, AF015811.sub.--1, CER07E3.sub.--2, YL35_CAEEL, S73863,
CEP59F4.sub.--4, P_WO6422, MMU41736.sub.--1, MTV008.sub.--39,
P_R99248 and Y67_BPT7.
Example 7
Isolation of cDNA Clones Encoding Human PRO34000 Polypeptides
(UNQ9196)
[0543] 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
sequence databases. The databases included public databases (e.g.,
GenBank) In this instance, genomic DNA sequence from GenBank was
analyzed using the gene preditiction program GENSCAN, licenced from
Stanford University. GENSCAN analysis predicts gene coding regions,
creating sequences which can be subjected to the ECD search. The
search was performed using the computer program BLAST or BLAST2
[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 sequences. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.) if necessary A consensus DNA sequence was
assembled.
[0544] Based on the consensus sequence as described above,
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
PRO34000. 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, supra, 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.
[0545] A pool of 50 different human cDNA libraries from various
tissues was used in cloning. 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.
[0546] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length PRO34000
polypeptide (designated herein as DNA203528-3014 [FIGS. 7A-B, SEQ
ID NO: 7) and the derived protein sequence for that PRO34000
polypeptide.
[0547] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 584-586 and a stop signal at nucleotide
positions 3203-3205 (FIGS. 7A-B, SEQ ID NO:7). The predicted
polypeptide precursor is 873 amino acids long, has a calculated
molecular weight of approximately 93996 daltons and an estimated pI
of approximately 8.35. Analysis of the full-length PRO34000
sequence shown in FIGS. 8A-B (SEQ ID NO:8) evidences the presence
of a variety of important polypeptide domains as shown in FIGS.
8A-B, wherein the locations given for those important polypeptide
domains are approximate as described above. Clone DNA203528-3014
has been deposited with ATCC on Sep. 12, 2000 and is assigned ATCC
deposit no. PTA-2780.
[0548] An analysis of the protein database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIGS. 8A-B (SEQ ID NO:8), evidenced
sequence identity between the PRO34000 amino acid sequence and the
following sequences:AB019120.sub.--1.
Example 8
Isolation of cDNA Clones Encoding Human PRO240 Polypeptides
(UNQ214)
[0549] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul 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 sequence. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.).
[0550] A consensus DNA sequence encoding PRO240 was assembled
relative to the other identified EST sequences, wherein the
consensus sequence was designated herein as DNA30873. Based on this
consensus sequence, oligonucleotides were synthesized 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 PRO240.
[0551] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00016 forward PCR primer 5'-TCAGCTCCAGACTCTGATACTGCC-3'
(SEQ ID NO: 39) reverse PCR primer 5'-TGCCTTTCTAGGAGGCAGAGCTCC-3'
(SEQ ID NO: 40)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30873 sequence which had the
following nucleotide sequence
TABLE-US-00017 hybridization probe (SEQ ID NO: 41)
5'-GGACCCAGAAATGTGTCCTGAGAATGGATCTTGTGTACCTGATGGTC CAG-3'
[0552] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO240 gene
using the probe oligonucleotide and one of the PCR primers.
[0553] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. 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.
[0554] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO240 [herein designated as
UNQ214 (DNA34387-1138)] and the derived protein sequence for
PRO240.
[0555] The entire nucleotide sequence of UNQ214 (DNA34387-1138) is
shown in FIG. 9 (SEQ ID NO:9). Clone UNQ214 (DNA34387-1138)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 12-14 and ending at the
stop codon at nucleotide positions 699-701 (FIG. 9; SEQ ID NO:9).
The predicted polypeptide precursor is 229 amino acids long (FIG.
10; SEQ ID NO:10). Clone UNQ214 (DNA34387-1138) has been deposited
with ATCC on Sep. 16, 1997 and is assigned ATCC deposit no. 209260.
Analysis of the amino acid sequence of the full-length PRO240
suggests that it possesses 30% and 35% amino acid identity with the
serrate precursor protein from Drospohilia melanogaster and the
C-serrate-1 protein from Gallus gallus.
Example 9
Isolation of cDNA Clones Encoding Human PRO943 Polypeptides
(UNQ480)
[0556] 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 EST databases
(e.g., GenBank) and a proprietary EST database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST2 [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 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 consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.).
[0557] A consensus DNA sequence encoding PRO943 was assembled
relative to other EST sequences using phrap. This consensus
sequence was then extended using repeated cycles of BLAST and phrap
to extend the consensus sequence as far as possible using the
sources of EST sequences discussed above. The extended consensus
sequence is herein designated DNA36360.
[0558] Based on the DNA36360 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 PRO943.
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, supra, 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.
[0559] PCR primers (forward and reverse) were synthesized:
TABLE-US-00018 forward PCR primer (36360.f1)
5'-CGAGATGACGCCGAGCCCCC-3' (SEQ ID NO: 42) reverse PCR primer
(36360.r1) 5'-CGGTTCGACACGCGGCAGGTG-3' (SEQ ID NO: 43)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA36360 sequence which had the
following nucleotide sequence
TABLE-US-00019 hybridization probe (36360.p1) (SEQ ID NO: 44)
5'-TGCTGCTCCTGCTGCCGCCGCTGCTGCTGGGGGCCTTCCCGCCGG- 3'
[0560] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO943 gene
using the probe oligonucleotide and one of the PCR primers.
[0561] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue. The cDNA libraries used to isolated 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 Nod 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 Nod sites.
[0562] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO943 (designated herein as
DNA52192-1369 [FIG. 11, SEQ ID NO: 11]; (UNQ480) and the derived
protein sequence for PRO943.
[0563] The entire nucleotide sequence of UNQ480 (DNA52192-1369) is
shown in FIG. 11 (SEQ ID NO:11). Clone UNQ480 (DNA52192-1369)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 150-152 and ending at the
stop codon at nucleotide positions 1662-1664 (FIG. 11). The
predicted polypeptide precursor is 504 amino acids long (FIG. 12;
SEQ ID NO:12). The full-length PRO943 protein shown in FIG. 12 has
an estimated molecular weight of about 54,537 daltons and a pI of
about 10.04. Analysis of the full-length PRO943 sequence shown in
FIG. 12 (SEQ ID NO:12) evidences the presence of the following: a
signal peptide from about amino acid 1 to about amino acid 17, a
transmembrane domain from about amino acid 376 to about amino acid
396, tyrosine kinase phosphorylation sites from about amino acid
212 to about amino acid 219 and from about amino acid 329 to about
amino acid 336, potential N-glycosylation sites from about amino
acid 111 to about amino acid 114, from about amino acid 231 to
about amino acid 234, from about amino acid 255 to about amino acid
258 and from about amino acid 293 to about amino acid 296 and an
immunoglobulin and MHC protein sequence homology block from about
amino acid 219 to about amino acid 236. Clone UNQ480
(DNA52192-1369) has been deposited with ATCC on Jul. 1, 1998 and is
assigned ATCC deposit no. 203042.
[0564] 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:12), evidenced
significant homology between the PRO943 amino acid sequence and the
following Dayhoff sequences: B49151, A39752, FGR1_XENLA, S38579,
RATHBFGFRB.sub.--1, TVHU2F, FGR2_MOUSE, CEK3_CHICK, P_R21080 and
A27171.sub.--1.
Example 10
Isolation of cDNA Clones Encoding Human PRO230 Polypeptides
(UNQ204)
[0565] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul 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 sequence. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.).
[0566] A consensus DNA sequence encoding PRO230 was assembled
relative to the other identified EST sequences, wherein the
consensus sequence was designated herein as DNA30857. An EST
proprietary to Genentech was employed in the consensus
assembly.
[0567] Based on the DNA30857 consensus sequence, oligonucleotides
were synthesized 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 PRO230.
[0568] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00020 forward PCR primer 5'-TTCGAGGCCTCTGAGAAGTGGCCC-3'
(SEQ ID NO: 45) reverse PCR primer 5'-GGCGGTATCTCTCTGGCCTCCC-3'
(SEQ ID NO: 46)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30857 sequence which had the
following nucleotide sequence
TABLE-US-00021 hybridization probe (SEQ ID NO: 47)
5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCT GGG-3'
[0569] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO230 gene
using the probe oligonucleotide and one of the PCR primers.
[0570] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. 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 Nod 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.
[0571] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO230 (herein designated as
UNQ204 (DNA33223-1136)) and the derived protein sequence for
PRO230.
[0572] The entire nucleotide sequence of UNQ204 (DNA33223-1136) is
shown in FIG. 13 (SEQ ID NO:13). Clone UNQ204 (DNA33223-1136)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 100-103 (Kozak et al.,
supra) and ending at the stop codon at nucleotide positions
1501-1503 (FIG. 13; SEQ ID NO:13). The predicted polypeptide
precursor is 467 amino acids long (FIG. 14; SEQ ID NO:14). Clone
UNQ203 (DNA33223-1136) has been deposited with ATCC on Sep. 16,
1997 and is assigned ATCC deposit no. 209264.
Example 11
Isolation of cDNA Clones Encoding Human PRO178 Polypeptides
(UNQ152)
[0573] An expressed sequence tag (EST) DNA database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST
was identified which showed homology to the human TIE ligand
family.
[0574] RNA for construction of cDNA libraries was then isolated
from human fetal lung tissue. The cDNA libraries used to isolate
the cDNA clones encoding human PRO178 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 Nod 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.
[0575] Oligonucleotides probes based upon the above described EST
sequence were then 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
PRO178. 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 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, supra, 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.
[0576] The oligonucleotide probes employed were as follows:
TABLE-US-00022 NL8.5-1: (SEQ ID NO: 48)
5'-ACGTAGTTCCAGTATGGTGTGAGCAGCAACTGGA-3' NL8.3-1: (SEQ ID NO: 49)
5'-AGTCCAGCCTCCACCCTCCAGTTGCT-3' NL8.3-2: (SEQ ID NO: 50)
5'-CCCCAGTCCTCCAGGAGAACCAGCA-3'
[0577] A full length clone [DNA23339-1130; UNQ152] was identified
that contained a single open reading frame with an apparent
translational initiation site at nucleotide positions 118-120 and a
stop signal at nucleotide positions 1528-1530 (FIG. 15, SEQ ID
NO:15). The predicted polypeptide precursor is 470 amino acids
long, has a calculated molecular weight of approximately 51,694
daltons and an estimated pI of approximately 8.86. Analysis of the
full-length PRO178 sequence shown in FIG. 16 (SEQ ID NO:16)
evidences the presence of a variety of important polypeptide
domains as shown in FIG. 16, wherein the locations given for those
important polypeptide domains are approximate as described above.
Analysis of the full-length PRO178 polypeptide shown in FIG. 16
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 20; N-glycosylation sites
from about amino acid 58 to about amino acid 62, and from about
amino acid 145 to about amino add 149; a cAMP- and cGMP-dependent
protein kinase phosphorylation site from about amino acid 97 to
about amino acid 101; a tyrosine kinase phosphorylation site from
about amino acid 441 to about amino acid 448; N-myristoylation
sites from about amino acid 16 to about amino acid 22; from about
amino acid 23 to about amino acid 29, from about amino acid 87 to
about amino acid 93, from about amino acid 108 to about amino acid
114, from about amino acid 121 to about amino acid 127, from about
amino acid 125 to about amino acid 131, from about amino acid 129
to about amino acid 135, from about amino acid 187 to about amino
acid 193, from about amino acid 293 to about amino acid 299, from
about amino acid 353 to about amino acid 359, from about amino acid
378 to about amino acid 384, from about amino acid 445 to about
amino acid 451, and from about amino acid 453 to about amino acid
459; a cell attachment site from about amino acid 340 to about
amino acid 343; and a fibrinogen beta and gamma chains C-terminal
domain signature from about amino acid 418 to about amino acid 431.
Clone DNA23339-1130 has been deposited with ATCC on Sep. 18, 1997
and is assigned ATCC deposit no. 209282.
[0578] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence shown in FIG. 16 (SEQ ID NO:16), PRO178
(herein designated NL8) shows a 23% amino acid sequence identity to
both ligand 1 and ligand 2 of the TIE2 receptor. Ligand 1 and
ligand 2 of the TIE-2 receptor are 64% identical and 40-43%
identical, respectively, to PRO178. The abbreviation "TIE" is an
acronym which stands for "tyrosine kinase containing Ig and EGF
homology domains" and was coined to designate a new family of
receptor tyrosine kinases.
Example 12
Isolation of cDNA Clones Encoding Human PRO1199 Polypeptides
(UNQ407)
[0579] A public expressed sequence tag (EST) DNA database (GenBank)
was searched with the full-length murine m-FIZZ1 (DNA 53517), and
an EST, designated AA311223 and renamed as DNA53028 was identified,
which showed homology with the m-FIZZ1 DNA.
[0580] Based on the EST sequence, oligonucleotides were synthesized
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 h-PRO1199 (also designated
FIZZ3).
[0581] A pair of PCR primers (forward and reverse) and a probe were
synthesized:
TABLE-US-00023 forward primer (h-FIZZ3.f): (SEQ ID NO: 51)
GGATTTGGTTAGCTGAGCCCACCGAGA reverse primer (h-FIZZ3.r): (SEQ ID NO:
52) GCACTGCGCGCGACCTCAGGGCTGCA probe (h-FIZZ3.p): (SEQ ID NO: 53)
CTTATTGCCCTAAATATTAGGGAGCCGGCGACCTCCTGGATCCTCTCATT
[0582] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO1199
(hFIZZ-3) gene using the probe oligonucleotide and one of the PCR
primers.
[0583] mRNA was isolated from human bone marrow tissue 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 3-4 kb 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.
[0584] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for hFIZZ-3 (DNA65351-1366-2;
UNQ407) and the derived protein sequence for PRO1199. A cDNA clone
was sequenced in entirety. The entire nucleotide sequence of
DNA65351-1366-2 (hFIZZ-3) is shown in FIG. 17 (SEQ ID NO:17). Clone
DNA65351-1366-2 contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
25-27 (FIG. 17; SEQ ID NO:17). The predicted polypeptide precursor
is 108 amino acids long (FIG. 18; SEQ ID NO:18). N-terminal amino
acids 1-18 represent a putative signal peptide, and starting at
position 57 we have identified a cell attachment sequence motif
(RGD). Clone DNA65351-1366-2 has been deposited with ATCC on May
12, 1998 and is assigned ATCC deposit no. 209856.
Example 13
Isolation of cDNA Clones Encoding Human PRO4333 Polypeptides
(UNQ1888)
[0585] An expressed sequence tag (EST) DNA database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched in a manner
similar to that described above under the ECD homology procedure
described above and an EST was identified which showed homology to
lymphotoxin-beta receptor.
[0586] The EST served as the template to create oligonucleotide
primers and probes to screen a human fetal kidney library in a
manner similar to that described above under the ECD homology
procedure. The oligonucleotides created for the above procedure
were the following:
TABLE-US-00024 forward PCR primer: (SEQ ID NO: 54)
5'-GCAAGAATTCAGGGATCGGTCTGG-3' probe: (SEQ ID NO: 55)
5'-CTGTGTTCCCTGCAACCAGTGTGGGCCAGGCATGG AGTTGTCTAAGG-3' reverse:
(SEQ ID NO: 56) 5'-AGATGGCATCACTG GTGGCTGAAC-3' forward: (SEQ ID
NO: 57) 5'-CAGAAGGCAAATTGTTCAGCCACCAG-3' reverse: (SEQ ID NO: 58)
5'-ACAGTTTCCAGACCGATCCCTGAATTC-3'
[0587] The result was the isolation of the full-length DNA sequence
DNA84210-2576 (SEQ ID NO:21, FIGS. 21A-B). The DNA84210-2576 (SEQ
ID NO:21) clone depicted in FIGS. 21A-B contains a single open
reading frame with an apparent translation initiation site at
nucleotide positions 185-187, and a stop codon (TAA) at nucleotide
positions 1436-1438, as indicated by bolded underline. The
predicted PRO4333 polypeptide precursor (i.e., UNQ1888, SEQ ID
NO:22) is 417 amino acids long. The UNQ1888 protein (SEQ ID NO:22)
shown in FIG. 22 has an estimated molecular weight of about 45305
daltons and a pI of about 5.12.
[0588] Analysis of the UNQ1888 polypeptide (SEQ ID NO:22) of FIG.
22 reveals a signal peptide at about amino acid residues 1-25, a
transmembrane domain at about residues 169-192, N-glycosylation
sites about residues 105-109, 214-218, 319-323, 350-354, 368-372,
379-383, cAMP- and cGMP-dependent protein kinase phosphorylation
sites at about residues 200-204 and 238-242, a tyrosine kinase
phosphorylation site at about residues 207-214, an N-myristoylation
site at about residues 55-61, 215-218 and 270-276, a prokaryotic
membrane lipoprotein lipid attachment site at about residues
259-270 and a TNFR/NGFR family cysteine-rich region at about
residues 89-96.
[0589] A cDNA clone containing DNA84210-2576 (SEQ ID NO:21),
designated as DNA84210-2576, has been deposited with ATCC on Mar.
2, 1999 and is assigned ATCC deposit no. 203818.
Example 14
Isolation of cDNA Clones Encoding Human PRO1336 Polypeptides
(UNQ691)
[0590] An EST sequence was identified and entered into a
proprietary Genentech database. The EST was blasted against various
EST databases. The EST databases included public EST databases
(e.g., GenBank), and a proprietary EST database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.), and proprietary ESTs
from Genentech. The search was performed using the computer program
BLAST or BLAST2 [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
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 consensus DNA sequences with the program "phrap" (Phil Green,
University of Washington, Seattle, Wash.).
[0591] A consensus DNA sequence encoding PRO1336 was assembled
relative to other aligned EST sequences (forming an assembly) using
phrap. This consensus sequence is designated herein "DNA43319".
Based on the DNA43319 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 PRO1336.
[0592] PCR primers (forward and reverse) were synthesized:
TABLE-US-00025 forward PCR primer (SEQ ID NO: 59)
5'ATGGAGATTCCTGCCAACTTGCCG3'; and reverse PCR primer (SEQ ID NO:
60) 5'TTGTTGGCATTGAGGAGGAGCAGC3'..
[0593] Additionally, a synthetic, oligonucleotide hybridization
probe was constructed from the consensus DNA43319 sequence which
had the following nucleotide sequence:
TABLE-US-00026 hybridization probe (SEQ ID NO: 61)
5'GAGGGCATCGTCGAAATACGCCTAGAACAGAACTCCATCAAAGCCATC CC3'.
[0594] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO1336 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue.
[0595] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1336 (designated herein as
DNA65423-1595 [FIGS. 23A-B, SEQ ID NO:23]; and the derived protein
sequence for PRO1336.
[0596] The entire coding sequence of PRO1336 is shown in FIGS.
23A-B (SEQ ID NO:23). Clone DNA65423-1595 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 83-85 and an apparent stop codon at nucleotide
positions 4652-4654 of SEQ ID NO:23. The predicted polypeptide
precursor is 1523 amino acids long (FIGS. 24A-B; SEQ ID NO:24). The
approximate locations of the signal peptide (amino acids 1-27),
aspartic acid and asparagine hydroxylation sites, EGF-like domain
cystein pattern signature regions, a leucine zipper pattern region,
a region conserved in immunoglobulins and major histocompatibility
complexes, and N-glycosylation sites are indicated in FIGS. 24A-B.
Clone DNA65423-1595 has been deposited with the ATCC on Sep. 15,
1998 and is assigned ATCC deposit no. 203227. The full-length
PRO1336 protein shown in FIGS. 24A-B has an estimated molecular
weight of about 167,715 daltons and a pI of about 8.06.
[0597] 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 FIGS. 24A-B (SEQ ID NO:24), revealed
sequence identity between the PRO1336 amino acid sequence and the
following Dayhoff sequences (data incorporated herein): SLIT_DROME,
CEF40E10.sub.--1, LCU58977.sub.--1, AF029779.sub.--1, FBPl_STRPU,
NOTC_XENLA, AC004663.sub.--1, XELXDEL.sub.--1, P_WO5835 and
HSU77720.sub.--1.
Example 15
Isolation of cDNA Clones Encoding Human PRO19598 Polypeptides
(UNQ5793)
[0598] 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
sequence databases. The databases included public databases (e.g.,
GenBank) In this instance, genomic DNA sequence from GenBank was
analyzed using the gene preditiction program GENSCAN, licenced from
Stanford University. GENSCAN analysis predicts gene coding regions,
creating sequences which can be subjected to the ECD search. The
search was performed using the computer program BLAST or BLAST2
[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 sequences. 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 consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.).
[0599] A consensus DNA sequence was assembled. This consensus
sequence is herein designated DNA132879. Based on the DNA132879
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 PRO19598. 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, supra, 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.
[0600] PCR primers (forward and reverse) were synthesized:
TABLE-US-00027 forward PCR primer ><H3 ecd.snf1 (SEQ ID NO:
62) 5'CTGGCAACAGCAGTGTCTATTTTGTGC 3' reverse PCR primer ><H3
ecd.snr1 (SEQ ID NO: 63) 5'TAAGTGCCCTCCCAGGCTGCC 3'
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA132879 sequence which had the
following nucleotide sequence
TABLE-US-00028 hybridization probe (SEQ ID NO: 64)
5'TCCTCCAGTCATGAATATAACCCAAGTCAATGGCTCTTTGTTGGTAAT TCTC 3'
[0601] A pool of 50 different human cDNA libraries from various
tissues was used in cloning. 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.
[0602] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length PRO19598
polypeptide (designated herein as DNA145887-2849 [FIG. 25, SEQ ID
NO: 25) and the derived protein sequence for that PRO19598
polypeptide.
[0603] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 241-243 and a stop signal at nucleotide
positions 1027-1030 (FIG. 25, SEQ ID NO:25). The predicted
polypeptide precursor is 262 amino acids long, has a calculated
molecular weight of approximately 30419 daltons and an estimated pI
of approximately 8.44 Analysis of the full-length PRO19598 sequence
shown in FIG. 26 (SEQ ID NO:26) evidences the presence of a variety
of important polypeptide domains as shown in FIG. 26, wherein the
locations given for those important polypeptide domains are
approximate as described above. Clone DNA145887-2849 has been
deposited with ATCC on Mar. 21, 2000 and is assigned ATCC deposit
no. PTA-1532.
[0604] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 26 (SEQ ID NO:26), evidenced
sequence identity between the PRO19598 amino acid sequence and the
following Dayhoff sequence: AF184971.sub.--1.
Example 16
Isolation of cDNA Clones Encoding Human PRO1083 Polypeptides
(UNQ540)
[0605] A cDNA sequence was identified using the amylase screening
technique described in EXAMPLE 2 above. That cDNA sequence was then
compared and aligned with other known EST sequences as described in
Example 1 above to obtain a consensus DNA sequence which is
designated herein as DNA43422. Based on the DNA 43422 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 PRO1083.
[0606] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00029 forward PCR primer 5'-GGCATTGGAGCAGTGCTGGGTG-3';
(SEQ ID NO: 65) reverse PCR primer 5'-TGGAGGCCTAGATGCGGCTGGACG-3'.
(SEQ ID NO: 66)
[0607] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO1083 gene
using the reverse PCR primer. RNA for construction of the cDNA
libraries was isolated from human fetal kidney tissue (LIB227).
[0608] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1083 [herein designated as
UNQ540 (DNA50921-1458)] (SEQ ID NO:27; FIG. 27) and the derived
protein sequence for PRO1083.
[0609] The entire nucleotide sequence of UNQ540 (DNA50921-1458) is
shown in FIG. 27 (SEQ ID NO:27). Clone UNQ540 (DNA50921-1458)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 214-216 and ending at the
stop codon at nucleotide positions 2293-2295 (FIG. 27). The
predicted polypeptide precursor is 693 amino acids long (FIG. 28;
SEQ ID NO:28). The full-length PRO1083 protein shown in FIG. 28 has
an estimated molecular weight of about 77,738 daltons and a pI of
about 8.87. Clone UNQ540 (DNA50921-1458) has been deposited with
the ATCC on May 12, 1998, under ATCC deposit no. 209859. Regarding
the sequence, it is understood that the deposited clone contains
the correct sequence, and the sequences provided herein are based
on known sequencing techniques.
[0610] Still analyzing the amino acid sequence of SEQ ID NO:28, the
putative signal peptide is at about amino acids 1-25 of SEQ ID
NO:28. The transmembrane domains are at about amino acids 382-398,
402-420, 445-468, 473-491, 519-537, 568-590 and 634-657 of SEQ ID
NO:28. A microbodies C-terminal targeting signal is at about amino
acids 691-693 of SEQ ID NO:28. cAMP- and cGMP-dependent protein
kinase phosphorylation sites are at about amino acids 198-201 and
370-373 of SEQ ID NO:28. N-glycosylation sites are at about amino
acids 39-42, 148-151, 171-174, 234-237, 303-306, 324-227 and
341-344 of SEQ ID NO:28. A G-protein coupled receptor family domain
is at about amino acids 475-504 of SEQ ID NO:28. The corresponding
nucleotides can be routinely determined given the sequences
provided herein.
Example 17
Isolation of cDNA Clones Encoding Human PRO1801 Polypeptides
(UNQ852)
[0611] A proprietary expressed sequence tag (EST) DNA database
(LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.) was
searched and an EST was identified which showed homology to the
IL-19 protein. This EST sequence is Incyte EST clone no. 819592 and
is herein designated DNA79293. Based on the DNA79293 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
PRO1801.
[0612] PCR primers (forward and reverse) were synthesized:
TABLE-US-00030 forward PCR primer (SEQ ID NO: 69)
5'-CTCCTGTGGTCTCCAGATTTCAGGCCTA-3' reverse PCR primer (SEQ ID NO:
70) 5'-AGTCCTCCTTAAGATTCTGATGTCAA-3'
[0613] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue. The cDNA libraries used to isolated 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.
[0614] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1801 (designated herein as
DNA83500-2506 [FIG. 31, SEQ ID NO:67]; and the derived protein
sequence for PRO1801.
[0615] The entire nucleotide sequence of DNA83500-2506 is shown in
FIG. 31 (SEQ ID NO:67). Clone DNA83500-2506 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 109-111 and ending at the stop codon at
nucleotide positions 892-894, (FIG. 31). The predicted polypeptide
precursor is 261 amino acids long (FIG. 32). The full-length
PRO1801 protein shown in FIG. 32 has an estimated molecular weight
of about 29,667 daltons and a pI of about 8.76. Analysis of the
full-length PRO1801 sequence shown in FIG. 32 (SEQ ID NO:68)
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 42, cAMP- and cGMP-dependent
protein kinase phosphorylation sites from about amino acid 192 to
about amino acid 195 and from about amino acid 225 to about amino
acid 228 and potential N-myristolation sites from about amino acid
42 to about amino acid 47, from about amino acid 46 to about amino
acid 51 and from about amino acid 136 to about amino acid 141.
Clone DNA83500-2506 has been deposited with ATCC on Oct. 29, 1998
and is assigned ATCC deposit no. 203391.
[0616] 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. 32 (SEQ ID NO:68), evidenced
significant homology between the PRO1801 amino acid sequence and
the following Dayhoff sequences: P_W37935, HGS_B477, P_R32277,
IL10_MACFA, P_W46585, P_R39714, P_R71471, P_R10159, IL10_RAT and
P_W57201.
Example 18
Generation and Analysis of Mice Comprising PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 Gene
Disruptions
[0617] To investigate the role of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 genes were produced
by homologous recombination. Specifically, transgenic mice
comprising disruptions in PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 genes (i.e., knockout mice)
were created by either gene targeting or gene trapping. Mutations
were confirmed by southern blot analysis to confirm correct
targeting on both the 5' and 3' ends. Gene-specific genotyping was
also performed by genomic PCR to confirm the loss of the endogenous
native transcript as demonstrated by RT-PCR using primers that
anneal to exons flanking the site of insertion. Targeting vectors
were electroporated into 129 strain ES cells and targeted clones
were identified. Targeted clones were microinjected into host
blastocysts to produce chimeras. Chimeras were bred with C57
animals to produce F1 heterozygotes. Heterozygotes were
intercrossed to produce F2 wildtype, heterozygote and homozygote
cohorts which were used for phenotypic analysis. Rarely, if not
enough F1 heterozygotes were produced, the F1 hets were bred to
wildtype C57 mice to produce sufficient heterozygotes to breed for
cohorts to be analyzed for a phenotype. All phenotypic analysis was
performed from 12-16 weeks after birth.
Overall Phenotypic Observation Summaries:
[0618] A. Generation and Analysis of Mice Comprising DNA33221-1133
(UNQ198) Gene Disruptions
[0619] In these knockout experiments, the gene encoding PRO224
polypeptides (designated as DNA33221-1133) [UNQ198] was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--019421 or Mus musculus hypothetical protein 425018-1,
protein reference: NP.sub.--062294 or hypothetical protein
425018-1; putative VLDL lipoprotein receptor precursor; DNA
segment, Chr 17, ERATO Doi 716, expressed [Mus musculus]; the human
gene sequence reference: BC007083 or Homo sapiens, 8D6 antigen,
clone MGC:14623 IMAGE:4076237; the human protein sequence
corresponds to reference: NP.sub.--057663 or 8D6 antigen (Homo
sapiens).
[0620] The mutated mouse gene encodes hypothetical protein 425018-1
(LocusLink 54219), which may be orthologus to human 8D6 antigen
(8D6A). Aliases and synonyms include putative VLDL lipoprotein
receptor precursor, DNA segment Chr 17 ERATO Doi 716, L0051293,
cDNA DKFZp56401762, D17Ertd716e and NG29.
[0621] The protein is a member of the family of low-density
lipoprotein (LDL) receptors, class A (Pfam accession number
PF000057) with unknown biological function. LDL receptors play an
important role in cholesterol metabolism, and such motifs are
observed in several extracellular and membrane proteins (Daly et
al., Proc. Natl. Acad. Sci. USA, 92(14):6334-8 (1995)). The
hypothetical protein is 56% similar (over 281 amino acids) to human
8D6 antigen (LocusLink 51293), a follicular dendritic cell signal
molecule that stimulates B cell growth (Li et al., J. Exp. Med.,
191(6):1077-84 (2000)).
[0622] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Retroviral insertion (OST) occurred;
insertion appeared to be in the forward direction prior to the exon
encoding amino acid 45.
TABLE-US-00031 wt het hom Total Observed 22 39 18 79 Expected 19.75
39.5 19.75 79 Chi-Sq. = 0.42 Significance = 0.81151 (hom/n) = 0.23
Avg. Litter Size = 0
[0623] Wild-type expression of the target gene was detected in
embryonic stem (ES) cells and, of the 13 adult tissue samples
tested by RT-PCR, only in kidney, testis, and adipose. RT-PCR
analysis revealed that the transcript was absent in the (-/-) mouse
analyzed.
Phenotypic analysis was performed on mice from this generation as
described below.
[0624] 1. Phenotypic Analysis (for Disrupted Gene: DNA33221-1133
(UNQ198)
[0625] (a) Cardiovascular Phenotypic Analysis:
[0626] In the area of cardiovascular biology, phenotypic testing
was performed to identify potential targets for the treatment of
cardiovascular, endothelial or angiogenic disorders. One such
phenotypic test included optic fundus photography and angiography
to determine the retinal arteriovenous ratio (A/V ratio) in order
to flag various eye abnormalities. An abnormal A/V ratio signals
such systemic diseases or disorders that May be related to the
vascular disease of hypertension (and any disease that causes
hypertension, e.g. atherosclerosis), diabetes or other ocular
diseases corresponding to ophthalmological disorders. Such eye
abnormalities may include but are not limited to the following:
retinal abnormality is retinal dysplasia, various retinopathies,
restenosis, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0627] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Optic fundus photography was
performed on conscious animals using a Kowa Genesis small animal
fundus camera modified according to Hawes and coauthors (Hawes et
al., 1999 Molecular Vision 1999; 5:22). Intra-peritoneal injection
of fluorescein permitted the acquisition of direct light fundus
images and fluorescent angiograms for each examination. In addition
to direct ophthalmological changes, this test can detect retinal
changes associated with systemic diseases such as diabetes and
atherosclerosis or other retinal abnormalities. Pictures were
provided of the optic fundus under normal light. The angiographic
pictures allowed examination of the arteries and veins of the eye.
In addition an artery to vein (A/V) ratio was determined for the
eye.
[0628] Ophthalmology analysis was performed on generated F2 wild
type, heterozygous, and homozygous mutant progeny using the
protocol described above. Specifically, the A/V ratio was measured
and calculated according to the fundus images with Kowa COMIT+
software. This test takes color photographs through a dilated
pupil: the images help in detecting and classifying many diseases.
The artery to vein ratio (A/V) is the ratio of the artery diameter
to the vein diameter (measured before the bifurcation of the
vessels). Many diseases will influence the ratio, i.e., diabetes,
cardiovascular disorders, papilledema, optic atrophy or other eye
abnormalities such as retinal degeneration (known as retinitis
pigmentosa) or retinal dysplasia, vision problems or blindness.
Thus, phenotypic observations which result in an increased
artery-to-vein ratio in homozygous (-/-) and heterozygous (+/-)
mutant progeny compared to wildtype (+/+) littermates would be
indicative of such pathological conditions.
[0629] Results: In this study, the (-/-) and (+/-) mice exhibited
an increased mean artery-to-vein (A/V) ratio when compared with
their (+/+) littermates indicating retinal degeneration. In
summary, by knocking out the gene identified as DNA33221-1133
encoding PRO224 polypeptides, both heterozygous and homozygous
mutant progeny exhibit phenotypes which are associated with retinal
degeneration. Such detected retinal changes are most commonly
associated with cardiovascular systemic diseases or disorders that
may be related to the vascular disease of hypertension (and any
disease that causes hypertension, e.g. atherosclerosis), diabetes
or other ocular diseases corresponding to ophthalmological
disorders such as retinal degeneration. Thus, antagonists of PRO224
encoding genes would lead to similar pathological retinal changes,
whereas agonists would be useful as therapeutic agents in the
treatment of hypertension, atherosclerosis or other opthamological
disorders including retinal degeneration and diseases associated
with this condition (as indicated above).
[0630] B. Generation and Analysis of Mice Comprising DNA131590-2962
(UNQ2914) Gene Disruptions
[0631] In these knockout experiments, the gene encoding PRO9783
polypeptides (designated as DNA131590-2962) [UNQ2914] was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene nucleotide reference corresponds
to: NM.sub.--030209 or Mus musculus RIKEN cDNA 1810049K24 gene
(1810049K24Rik), protein reference: NP.sub.--084485 or RIKEN cDNA
1810049K24 [Mus musculus]; the human nucleotide reference
corresponds to: NM.sub.--031476, protein reference: NP.sub.--113664
or Homo sapiens hypothetical protein DKFZp434B044
(DKFZP434B044).
[0632] The disrupted mouse gene is represented by RIKEN cDNA
1810049K24Rik, ortholog of human hypothetical protein DKFZp434B044.
The putative proteins are members of the SCP-like (Pfam PF00188)
and LCCP (Pfam PF03815) families of extracellular domain containing
proteins. While the individual members of these families have
diverse physiological functions, proteins containing LCCP domains
are suspected to be involved in protein folding.
[0633] The mouse protein has the greatest similarity to mouse
cocoacrisp (cysteine-rich secretory protein or CRISP) and trypsin
inhibitors, which also contain LCCL and SCP-like extracellular
domains. CRISP proteins are typically secreted and expressed in the
male genital tract. They are thought to mediate cell-cell
interactions of male germ cells with other cells during sperm
maturation or during fertilization (Giese et al., Gene,
299(1-2):101-9 (2002)). Overall, this is a large and diverse family
of eukaryotic proteins that includes wasp allergens, plant PR-type
proteins, snail proteases, and even a component of a reptile toxin
(Milne et al., J. Biol. Chem., 278(33):31105-10 (2003)).
[0634] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The wild-type
expression panel resulted in the following observation: expression
of the target gene was detected in embryonic stem (ES) cells and,
among the 13 adult tissue samples tested by RT-PCR, in bone, skin
fibroblast, adipose, and tail. Disruption of the target gene was
confirmed by Southern hybridization analysis. The chimeric mice
were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
TABLE-US-00032 wt het hom Total Observed 22 50 28 100 Expected 25
50 25 100 Chi-Sq. = 0.72 Significance = 0.69768 (hom/n) = 0.28 Avg.
Litter Size = 0
Phenotypic analysis was performed on mice from this generation as
described below.
[0635] 1. Phenotypic Analysis (for Disrupted Gene: DNA131590-2962
(UNQ2914)
[0636] (a) Overall Phenotypic Summary:
[0637] The homozygous mutant mice exhibited numerous immunological
abnormalities when compared with their wild-type littermates and
the historical mean. The male homozygous mutant mice also exhibited
enhanced glucose tolerance at all 3 time intervals tested and
exhibited notably decreased bone-related measurements when compared
with their gender-matched wild-type littermates and the historical
means. Female (-/-) mice showed a decreased skin fibroblast
proliferation rate. Disruption of the target gene was confirmed by
Southern hybridization analysis.
[0638] (b) Phenotypic Analysis: Metabolism--Blood Chemistry
[0639] In the area of metabolism, targets may be identified for the
treatment of various cardiovascular diseases, diabetes and/or
obesity. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases or obesity.
[0640] Procedure: A cohort of 2 wild type and 4 homozygote males
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection.
[0641] Results: These studies indicated that male (-/-) mice
exhibit enhanced glucose tolerance in the presence of normal
fasting glucose at all 3 intervals tested when compared with their
gender-matched (+/+) littermates and the historical means. In
addition, hyperinsulinemia was not apparent in the (-/-) mice.
Thus, knockout mice exhibited the opposite phenotypic pattern of an
impaired glucose homeostasis, and as such antagonists to PRO9783
polypeptides or its encoding gene would be useful in the treatment
of impaired glucose homeostasis and/or various cardiovascular
diseases such as diabetes.
[0642] (c) Immunology Phenotypic Analysis
[0643] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0644] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0645] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0646] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0647] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0648] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0649] The following tests were performed:
[0650] Flourescence-Activated Cell-Sorting (FACS) Analysis
[0651] Procedure: FACS analysis of immune cell composition from
peripheral blood was performed including analysis of CD4, CD8 and T
cell receptors to evaluate T lymphocytes, CD19 for B lymphocytes,
CD45 as a leukocyte marker and pan NK for natural killer cells. The
FACS analysis was carried out on 2 wild type and 6 homozygous mice
and included cells derived from thymus, spleen, bone marrow and
lymph node.
[0652] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+
ratio.
[0653] The mononuclear cell profile was derived by staining a
single sample of lysed peripheral blood from each mouse with a
panel of six lineage-specific antibodies: CD45 PerCP, anti-TCRb
APC, CD4 PE, CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and
PE labeled antibodies stain mutually exclusive cell types. The
samples were analyzed using a Becton Dickinson FACSCalibur flow
cytometer with CellQuest software.
[0654] Results: FACS analysis gave the following results: The male
(-/-) mice exhibited a decreased mean percentage of B cells when
compared with their (+/+) littermates and the historical mean.
[0655] Ovalbumin Challenge
[0656] Procedure: This assay was carried out on 6 wild types and 14
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immunodominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[0657] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Feund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample. [Analyzed wt/het/hom: 6/4/14]
[0658] Results of this challenge: The male (-/-) mice exhibited a
decreased mean serum IgG2a response to the ovalbumin challenge when
compared with their (+/+) littermates. Thus, these knockout mice
exhibited a decreased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen.
[0659] In summary, both FACS analysis of immune cell composition
from peripheral blood and the ovalbumin challenge studies indicate
that knockout mice deficient in the gene encoding PRO9783
polypeptides exhibit immunological abnormalities when compared with
their wild-type littermates. In one instance, the mutant mice
exhibited a decreased ability to elicit an immunological response
when challenged with the T-cell dependent OVA antigen. This coupled
with the immune cell composition analysis of peripheral blood,
suggests that PRO9783 polypeptides or their agonists would be
useful for stimulating the immune system (such as T cell
proliferation) and would find utility in the cases wherein this
effect would be beneficial to the individual such as in the case of
leukemia, and other types of cancer, and in immunocompromised
patients, such as AIDS sufferers. Accordingly, inhibitors
(antagonists) of PRO9783 polypeptides would be useful in inhibiting
the immune response and would be useful candidates for suppressing
harmful immune responses, e.g. in the case of graft rejection or
graft-versus-host diseases.
[0660] (d) Oncology Phenotypic Analysis
[0661] In the area of oncology, targets were identified herein for
the treatment of solid tumors, lymphomas and leukemia.
[0662] Adult Skin Cell Proliferation:
[0663] Procedure: Skin cells were isolated from 16 week old animals
(2 wild type and 4 homozygotes). These were developed into primary
fibroblast cultures and the fibroblast proliferation rates were
measured in a strictly controlled protocol. The ability of this
assay to detect hyper-proliferative and hypo-proliferative
phenotypes has been demonstrated with p53 and Ku80. Proliferation
was measured using Brdu incorporation.
[0664] Specifically, in these studies the skin fibroblast
proliferation assay was used. An increase in the number of cells in
a standardized culture was used as a measure of relative
proliferative capacity. Primary fibroblasts were established from
skin biopsies taken from wild type and mutant mice. Duplicate or
triplicate cultures of 0.05 million cells were plated and allowed
to grow for six days. At the end of the culture period, the number
of cells present in the culture was determined using a electronic
particle counter.
[0665] Results: The female (-/-) mice exhibited a decreased skin
fibroblast proliferation rate when compared with their
gender-matched (+/+) littermates and the historical mean. [Analyzed
wt/het/horn: 2/0/4]
Thus, homozygous mutant mice demonstrated a hypo-proliferative
phenotype. As suggested by these observations, antagonists of a
PRO9783 polypeptide or its encoding gene would be useful in
decreasing abnormal cell proliferation.
[0666] (e) Bone Metabolism: Radiology Phenotypic Analysis
[0667] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included: [0668] DEXA for measurement of bone
mineral density on femur and vertebra [0669] MicroCT for very high
resolution and very high sensitivity measurements of bone mineral
density for both trabecular and cortical bone.
[0670] Dexa Analysis--Test Description:
[0671] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[0672] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PiXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae; and both femurs].
[0673] DEXA Results: The male (-/-) mice exhibited decreased mean
bone mineral content, volumetric bone mineral density, bone mineral
content index (BMC/LBM), and bone mineral density in total body,
femur, and vertebrae when compared with their gender-matched (+/+)
littermates and the historical means.
[0674] Bone microCT Analysis:
[0675] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 veterbra traebecular bone volume, traebecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[0676] Micro-CT Analysis Results: The male (-/-) mice exhibited a
notably decreased mean lumbar 5 vertebral trabecular bone volume,
number, thickness, and connectivity density when compared with
their gender-matched (+/+) littermates and the historical means.
These mutants also exhibited notably decreased mean femoral
midshaft cross-sectional area. [Analyzed wt/het/horn: 4/4/8]
[0677] These results demonstrate that knockout mutant male mice
deficient in the gene encoding PRO9783 polypeptides exhibit
abnormal bone metabolism with significant bone loss characterized
by a decrease in bone mass with decreased density and possibly
fragility leading to bone fractures. No hypercalcemia,
hyperglycemia, or increased alkaline phosphate was detected in
blood chemistry tests to suggest renal, parathyroid, or adrenal
dysfunction that might be related to the decrease in bone mineral
density seen on the DEXA scan. As the bone mineral density defect
was only seen in male knockout mice, this bone abnormality
observation suggests testosterone deficiency. Thus, it appears that
PRO9783 polypeptides or agonists thereof would be useful in
maintaining bone homeostasis mediated by male hormones such as
testosterone. In addition, PRO9783 polypeptides or its encoding
gene would be useful in maintaining bone homeostasis and could be
important in bone healing or for the treatment of arthritis or
osteoporosis; whereas antagonists to PRO9783 polypeptides or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism such as arthritis, osteoporosis, and osteopenia.
[0678] C. Generation and Analysis of Mice Comprising DNA58848-1472
(UNQ551) Gene Disruptions
[0679] In these knockout experiments, the gene encoding PRO1108
polypeptides (designated as DNA58848-1472) [UNQ551] was disrupted.
The gene specific information for these studies is as follows:
mouse nucleotide reference corresponds to: NM.sub.--018743 or Mus
musculus putative lysophosphatidic acid acyltransferase (L0055933),
protein reference: NP.sub.--061213 or putative lysophosphatidic
acid acyltransferase [Mus musculus]; the human nucleotide reference
corresponds to: AF317516 or Homo sapiens putative lysophosphatidic
acid acyltransferase, protein reference: AAG33063 or putative
lysophosphatidic acid acyltransferase [Homo sapiens]. Retroviral
Insertion (OST) appeared to be in the forward direction prior to
the start codon of a predicted protein about 295 amino acids in
length.
[0680] The gene that is mutated in these animals is represented by
NCBI sequence NM.sub.--018743 (expressed sequence AU041707), which
is the ortholog of human sequence AF317516 (DKFZp586M1819). Both
loci encode a hypothetical putative lysophosphatidic acid
acyltransferase. DKFZp586M1819 is likely to be an enzyme that
catalyzes the biosynthesis of glycerolipids or phospholipids.
DKFZp586M1819 contains a phosphate acyltransferase domain, which is
found in enzymes that have glycerolphosphate,
1-acylglycerolphosphate, or 2-acylglycerolphosphoethanolamine
acyltransferase activities (SMART SM00563).
[0681] DKEZp586M1819 has some similarity (33% similar over 270
residues) with 1-acylglycerol-3-phosphate O-acyltransferase 1
(AGPAT1; OMIM 603099). AGPAT1 catalyzes the conversion of
lysophosphatidic acid to phosphatidic acid. Lysophosphatidic acid
and phosphatidic acid are involved in signal transduction and lipid
biosynthesis (Leung, D. W., Front Biosci., 6:D944-53 (2001)).
[0682] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
TABLE-US-00033 wt het hom Total Observed 19 34 0 53 Expected 13.25
26.5 13.25 53 Chi-Sq. = 17.87 Significance = 0.00013 (hom/n) = 0.00
Avg. Litter Size = 0
[0683] In the wild-type animals, expression of the target gene was
detected in embryonic stem (ES) cells and in all 13 adult tissue
samples tested by RT-PCR, except bone and tail. Genetic data
indicate that this retroviral insertion resulted in lethality of
the homozygous mutants. Due to lethality, transcript expression
analysis was not performed. It is not clear when lethality occurs.
Expression of the gene starts at E8.5, continues in all subsequent
stages examined thus far (up to E13). Normal expression is
ubiquitous as shown by in situ hybridization.
[0684] Discussion Related to Embryonic Developmental Abnormality of
Lethality:
[0685] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neurodegenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[0686] In the present example, UNQ 551 is a novel lysophophatidic
acid acyltransferase which catalyses the conversion of
lysophophatidic acid (LPA) to phosphatidic acid (PA). LPA is a
naturally occurring component of phospholipid and is known to have
growth factor like activity in the regulation of numerous cellular
responses through the activation of specific G-protein coupled
receptors. LPA has recently been recognized as diagnostic marker
for ovarian cancer thus indicating that a UNQ 551 mediated pathway
is implicated in cancer progression (Cancer Epidemiol Biomarkers
Prev. 13(7):1185-91 (2004)).
[0687] 1. Phenotypic Analysis (for Disrupted Gene: DNA58848-1472
(UNQ551)
[0688] (a) Overall Phenotypic Summary:
[0689] The heterozygous (-/+) mutant mice exhibited immunological
abnormalities when compared with their wild-type littermates and
the historical mean. Serum IgM, IgG1, IgG2b and IgG3 were decreased
in heterozygous adults.
[0690] (b) Further Embryonic Analysis
[0691] UNQ551 is expressed during the early stages of embryonic
mouse development. A day after gastrulation at 8.5d UNQ551 is
expressed in the newly formed mesoderm and also in the
ectoplacental cone, a structure that will eventually contribute to
the placenta. A day later, UNQ551 expression becomes widespread and
uniform until at least mid-gestation. By 12.5d the placenta is
fully formed and functional. The placenta provides an interface
between mother and embryo to allow exchange of gases, nutrients and
wastes. The placenta is also a source of hormones and growth
factors and is involved in immune protection of the embryo.
Deficiencies in any of these properties can lead to growth
retardation and death. UNQ551 is expressed in the
spongiotrophoblast layer of the placenta.
[0692] Homozygous UNQ551 mutant embryos start dying around 14.5
days. Mutants can first be identified at 12.5d by their small
placentas. Also the amniotic fluid of mutants is yellow and the
yolk sac vasculature is reduced. Both observations are
characteristic of unhealthy embryos that are dying and undergoing
resorption. Examination of placental histology at 12.5d revealed
that projections of the labyrinthine layer into the
spongiotrophoblast layer are reduced in number and extent in the
mutants. Marker gene analysis confirms that the spongiotrophoblast
layer that expresses UNQ551 is reduced in size.
[0693] The labyrinthine layer of the placenta is where the maternal
and fetal blood circulations are juxtaposed for metabolic exchange.
The reduction in branching and therefore reduced surface area in
contact between the two systems in the UNQ551 mutant embryos leads
to insufficient oxygen/nutrient exchange resulting in death of the
embryo. UNQ551 is important in placental development especially in
mediating the invasion of one tissue type into another as seen in
the placenta. A similar role for UNQ551 can occur during
metastasis.
[0694] (c) Immunology Phenotypic Analysis
[0695] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0696] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0697] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0698] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0699] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0700] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0701] The following tests were performed:
[0702] Serum Immunoglobulin Isotyping Assay:
[0703] The Serum Immunoglobulin Isotyping Assay is performed using
a Cytometric Bead Array (CBA) kit. This assay is used to rapidly
identify the heavy and light chain isotypes of a mouse monoclonal
antibody in a single sample. The values expressed are "relative
fluorescence units" and are based on the detection of kappa light
chains. Any value <6 is not significant.
[0704] Results:
[0705] The serum immunoglobulin isotyping assay revealed that
heterozygous adults exhibited decreased serum IgM, IgG1, IgG2a,
IgG2b and IgG3 levels. Thus, heterozygotes showed an abnormally low
serum immunoglobulin levels compared with the (+/+) littermates.
Homozygous mutant mice resulted in embryonic lethality. Thus,
PRO1108 polypeptides or the gene encoding PRO1108 is essential for
embryonic development. In addition, the gene encoding PRO1108 is
essential for making immunoglobulins (or gamma globulins). A
disease associated with the failure to make gamma globulins has
been characterized as agammaglobulinemia linked to a single-gene
defect on the X chromosome and thus occurs mainly in males. This
single-gene defect in antibody production causes the absence of
immunoglobulin isotypes known as IgM, IgA and several subclasses of
IgG. Most inherited immunodeficiency diseases are caused by
recessive gene defects. Recessive defects thus lead to disease or
death only when both chromosomes are defective. In this case, the
heterozygous mutant mice present an excellent model for studying an
immunodeficiency disease such as agammaglobulinemia.
[0706] D. Generation and Analysis of Mice Comprising DNA203528-3014
(UNQ9196) Gene Disruptions
[0707] In these knockout experiments, the gene encoding PRO34000
polypeptides (designated as DNA203528-3014) [UNQ9196] was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene corresponds to nucleotide
reference: BY731963 or BY731963 RIKEN full-length enriched, 8 cells
embryo Mus musculus; the human nucleotide reference corresponds to:
NM.sub.--153835 or homo sapiens G protein-coupled receptor 113
(GPR113), protein reference: NP.sub.--722577 or G-protein coupled
receptor 113 [Homo sapiens].
[0708] The mutated mouse gene is represented by NCBI cDNA BY731963,
which is orthologous to human GPR113 (G-protein coupled receptor
113). Aliases include PGR23 and hGPCR37. A single publication
documents the existence of the human and mouse loci under the name
PGR23 (Vassilatis et al., Proc. Natl. Acad. Sci. USA, 100(8):4903-8
(2003)). The SwissProt record for GPR113 (Q81ZF5) notes that the
sequence was uploaded electronically under the title: New human
G-protein coupled receptors with long N-terminals containing GPS
domains and Ser/Thr rich regions.
[0709] Bioinformatic analysis of the human protein predicts a
region of weak similarity to the hormone-receptor domain (HRM, Pfam
02793) encoded within residues 428-481, and a secretin-type G
protein-coupled receptor motif at the C-terminus (Pfam 01825, Pfam
00002). The N-terminus (amino acids 20-427) is composed of nearly
20% serine or threonine residues.
[0710] GPR113 is not closely related to other human G
protein-coupled receptors. However, it is about 40% similar over
780 residues to GPR116 (KIAA0758 protein), another orphan
receptor.
[0711] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
TABLE-US-00034 wt het hom Total Observed 23 46 31 100 Expected 25
50 25 100 Chi-Sq. = 1.92 Significance = 0.38289 (hom/n) = 0.31 Avg.
Litter Size = 0
Level I phenotypic analysis was performed on mice from this
generation as described below.
[0712] Wild-type expression of the target gene was detected in
embryonic stem (ES) cells and, among the 44 adult and fetal tissue
samples tested by RT-PCR, in thymus, spleen, testis, salivary
gland, skeletal muscle, uterus, adipose, bladder, thyroid, aorta,
eye, placenta, 9.5 day embryo, 12.5 day embryo, fetal brain, and
fetal lung. Disruption of the target gene was confirmed by Southern
hybridization analysis.
[0713] 1. Phenotypic Analysis (for Disrupted Gene: DNA203528-3014
(UNQ9196)
[0714] (a) Overall Phenotypic Summary.
[0715] Mutation of the gene encoding the ortholog of a human
predicted G protein-coupled receptor 113 (GPR113) resulted in
decreased trabecular bone volume, thickness, and connectivity
density in (-/-) mutants. Knockouts also showed an abnormal glucose
tolerance. Gene disruption was confirmed by Southern blot.
[0716] (b) Bone Metabolism: Radiology Phenotypic Analysis
[0717] Procedure:
[0718] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included microCT for very high resolution and very
high sensitivity measurements of bone mineral density for both
trabecular and cortical bone.
[0719] Bone microCT Analysis:
[0720] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 veterbra traebecular bone volume, traebecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[0721] Micro-CT Analysis Results: The (-/-) mice exhibited notably
decreased mean vertebral trabecular bone volume, number, thickness,
and connectivity density when compared with their gender-matched
(+/+) littermates and the historical means. [Analyzed wt/het/hom:
4/0/8]
[0722] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO34000 polypeptides or agonists thereof would be
useful in maintaining bone homeostasis. In addition, PRO34000
polypeptides or its encoding gene would be useful in maintaining
bone homeostasis and would be important in bone healing or for the
treatment of arthritis or osteoporosis; whereas antagonists to
PRO34000 polypeptides or its encoding gene would lead to abnormal
or pathological bone disorders including inflammatory diseases
associated with abnormal bone metabolism including arthritis,
osteoporosis, and osteopenia.
[0723] (c) Phenotypic Analysis: Metabolism--Blood Chemistry
[0724] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
glucose tolerance tests to measure insulin sensitivity and changes
in glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[0725] Procedure: A cohort of 2 wild type and 4 homozygote mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection.
[0726] Results: These studies indicated that (-/-) mice exhibit
enhanced glucose tolerance in the presence of normal fasting
glucose at all 3 intervals tested when compared with their
gender-matched (+/+) littermates and the historical means. In
addition, hyperinsulinemia was not apparent in the (-/-) mice. No
abnormality was seen in the remaining clinical chemistry data.
Thus, knockout mice exhibited the opposite phenotypic pattern of an
impaired glucose homeostasis, and as such antagonists to PRO34000
polypeptides or its encoding gene would be useful in the treatment
of impaired glucose homeostasis and/or diabetes.
[0727] E. Generation and Analysis of Mice Comprising DNA34387-1138
(UNQ214) Gene Disruptions
[0728] In these knockout experiments, the gene encoding PRO240
polypeptides (designated as DNA34387-1138) [UNQ214] was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference: AK002276 or
Mus musculus adult male kidney cDNA, RIKEN full-length enriched
library, clone:0610007C21:homolog to HSPC013, full insert sequence,
protein reference is BAB21981 or data source:SPTR, source
key:Q9Y2R7, evidence:ISS.about.homolog to HSPC013.about.putative
[Mus musculus]. The human gene sequence is as follows: nucleotide
reference:BC021237 or Homo sapiens, clone IMAGE:4303513; protein
reference: AAH11006. Unknown (protein for MGC:13322) [Homo
sapiens].
[0729] The gene that is mutated in these animals is represented by
mouse UniGene cluster Mm.28144, which is the ortholog of human
UniGene cluster Hs.9527 (APR-3). APR-3 encodes apoptosis related
protein APR-3. The protein is also known as p18, HSPC013, and p18
protein.
[0730] SMART (Shultz et al., Nucleic Acids Res., 28(1):231-4
(2000)) analysis predicts an EGF-like domain (Pfam PF00008) and a
cleavable signal peptide in the hypothetical protein, as well as a
possible transmembrane domain. EGF-like domains are a common
feature found in secreted proteins and the extracellular portions
of membrane-bound proteins.
[0731] Little specific information is available concerning APR-3,
but it may be involved in apoptosis and may be involved in
hematopoietic development and differentiation (Zhang et al., Genome
Res., 10(10): 1546-60 (2000)). There are several alternate splice
forms of the mouse and human APR-3 gene.
[0732] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation as described below.
TABLE-US-00035 wt het hom Total Observed 12 31 17 60 Expected 15 30
15 60 Chi-Sq. = 0.90 Significance = 0.63763 (hom/n) = 0.28 Avg.
Litter Size = 0
[0733] Retroviral insertion appears to be in the forward direction
near amino acid 30 out of about 225. Wild-type expression of the
target gene was detected in embryonic stem (ES) cells and in all 13
adult tissues tested by RT-PCR. RT-PCR analysis revealed that the
transcript was absent in the (-/-) mouse analyzed (F-91).
[0734] 1. Phenotypic Analysis (for Disrupted Gene: DNA34387-1138
(UNQ214)
[0735] (a) Cardiovascular Phenotypic Analysis:
[0736] In the area of cardiovascular biology, phenotypic testing
was performed to identify potential targets for the treatment of
cardiovascular, endothelial or angiogenic disorders. One such
phenotypic test included optic fundus photography and angiography
to determine the retinal arteriovenous ratio (A/V ratio) in order
to flag various eye abnormalities. An abnormal A/V ratio signals
such systemic diseases or disorders that may be related to the
vascular disease of hypertension (and any disease that causes
hypertension, e.g. atherosclerosis), diabetes or other ocular
diseases corresponding to ophthalmological disorders. Such eye
abnormalities may include but are not limited to the following:
retinal abnormality is retinal dysplasia, various retinopathies,
restenosis, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0737] In addition, optic fundus photography and angiography may be
used to determine cataract formation. Cataracts are associated with
such systemic diseases as: Human Down's syndrome, Hallerman-Streiff
syndrome, Lowe syndrome, galactosemia, Marfan syndrome, Trismoy
13-15 condition, Alport syndrome, myotonic dystrophy, Fabry
disease, hypothroidisms, or Conradi syndrome. Other ocular
developmental anomalies include: Aniridia, anterior segment and
dysgenesis syndrome: Cataracts may also occur as a result of an
intraocular infection or inflammation (uveitis).
[0738] Procedure:
[0739] A cohort of 4 wild type, 4 heterozygotes and 8 homozygotes
were tested in this assay. Optic fundus photography was performed
on conscious animals using a Kowa Genesis small animal fundus
camera modified according to Hawes and coauthors (Hawes et al.,
1999 Molecular Vision 1999; 5:22). Intra-peritoneal injection of
fluorescein permitted the acquisition of direct light fundus images
and fluorescent angiograms for each examination. In addition to
direct ophthalmological changes, this test can detect retinal
changes associated with systemic diseases such as diabetes and
atherosclerosis. Pictures were provided of the optic fundus under
normal light. The angiographic pictures allowed examination of the
arteries and veins of the eye. In addition an artery to vein (A/V)
ratio was determined for the eye.
[0740] Ophthalmology analysis was performed on generated F2 wild
type, heterozygous, and homozygous mutant progeny using the
protocol described above. Specifically, the A/V ratio was measured
and calculated according to the fundus images with Kowa COMIT+
software. This test takes color photographs through a dilated
pupil: the images help in detecting and classifying many diseases.
The artery to vein ratio (A/V) is the ratio of the artery diameter
to the vein diameter (measured before the bifurcation of the
vessels). Many diseases will influence the ratio, i.e., diabetes,
cardiovascular disorders, papilledema, optic atrophy or other eye
abnormalities such as retinal degeneration (known as retinitis
pigmentosa) or retinal dysplasia, vision problems or blindness.
Thus, phenotypic observations which result in an increased
artery-to-vein ratio in homozygous (-/-) and heterozygous (+/-)
mutant progeny compared to wildtype (+/+) littermates would be
indicative of such pathological conditions.
[0741] Results:
[0742] In this study, developing cataracts were observed in 6 (-/-)
mice (F-97, -115, -117, F-120, -134, and -137) and 1 (+/-) mice
(F-99), being more severe in the (-/-) mice. The male (-/-) mouse
examined (-141) exhibited a cataract in the right eye. A slight
increase in artery to ratio was also seen. In summary, by knocking
out the gene identified as DNA34387-1138 (UNQ214) which encodes
PRO240 polypeptides, both heterozygous and homozygous mutant
progeny exhibit phenotypes which are associated with cataract
formation and/or other opthalmological disorders. Such detected
ophthalmology changes are most commonly associated with
cardiovascular systemic diseases. In particular, cataract formation
may be indicative of a cardiovascular complication related to
disturbances in the blood coagulation cascade. Cataracts are also
associated with such systemic diseases as: Human Down's syndrome,
Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfan
syndrome, Trismoy 13-15 condition, Alport syndrome, myotonic
dystrophy, Fabry disease, hypothroidisms, Conradi syndrome. Thus,
antagonists of PRO240 polypeptides or its encoding genes would lead
to similar pathological changes, whereas agonists would be useful
as therapeutic agents in the prevention of cataract formation
and/or the underlying cardiovascular disease or opthalmological
disorder.
[0743] F. Generation and Analysis of Mice Comprising DNA52192-1369
(UNQ480) Gene Disruptions
[0744] In these knockout experiments, the gene encoding PRO943
polypeptides (designated as DNA52192-1369) [UNQ480] was disrupted.
The gene specific information for these studies is as follows: the
mouse nucleotide reference corresponds to: NM.sub.--054071 or Mus
musculus fibroblast growth factor receptor-like 1 (Fgfrl1); protein
reference: NP.sub.--473412 or fibroblast growth factor
receptor-like 1; fibroblast growth factor receptor 5 [Mus
musculus]; the human nucleotide reference corresponds to:
NM.sub.--021923 or Homo sapiens fibroblast growth factor
receptor-like 1 (FGFRL1), protein reference: NP.sub.--068742 or
fibroblast growth factor receptor-like 1 precursor [Homo
sapiens].
[0745] The gene that is mutated in these animals is represented by
mouse UniGene cluster Mm.35691, which is the ortholog of human
UniGene cluster Hs.193326 (FGFRL1). FGFRL1 encodes fibroblast
growth factor, receptor-like 1. The protein is also known as FGFR5,
FGFR5beta, FGFR5gamma, and fibroblast growth factor receptor 5.
[0746] FGFRL1, a member of the fibroblast growth factor receptor
family, contains a signal peptide, three extracellular Ig-like
modules, six cysteines, an acidic box with no HAV motif, a
transmembrane segment, and a short intracellular domain lacking the
usual tyrosine kinase domain required for signal transduction by
transphosphorylation (Wiedemann, M., and Trued, B., Genomics,
69(2):275-9 (2000); Kim et al., Biocim Biophys Acta,
1518(1-2):152-6 (2001); Wiedemann, M. and Trued, B., Biochim
Biophys Acta, 1520(3):247-50 (2001), Sleeman et al., Gene,
271(2):171-82 (2001)) further classified FGFRL1 as a member of the
I-set subgroup of the Ig-superfamily, consistent with the known
FGFRs.
[0747] Although no precise function has been attributed to FGFRL1,
Wiedemann, M. and Trued, B. suggested that FGFRL1 may play a role
in modulating FGF receptor activity. FGFRL1 is expressed at high
levels in the pancreas, suggesting that FGFRL1 may regulate
pancreatic function (Kim et al, 2001 supra). In addition to kidney,
FGFRL1 expression is high in brain and lung (Sleeman et al, 2001
supra).
[0748] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
TABLE-US-00036 wt het hom Total Observed 30 68 1 99 Expected 24.75
49.5 24.75 99 Chi-Sq. = 30.82 Significance = 0.00000 (hom/n) = 0.01
Avg. Litter Size = 0
[0749] In the wild-type animals, expression of the target gene was
detected in embryonic stem (ES) cells and in all 13 adult tissue
samples tested by RT-PCR. Disruption of the target gene was
confirmed by Southern hybridization analysis. Genetic data indicate
that this mutation resulted in lethality of the homozygous mutants.
No notable phenotype was observed for the heterozygous mice. Due to
lethality, transcript expression analysis was not performed.
[0750] 1. Phenotypic Analysis (for Disrupted Gene: DNA52192-1369
(UNQ480)
[0751] Mutation of the target gene (UNQ480) resulted in embryonic
lethality. Heterozygous male (+/-) mice exhibited an increased mean
femoral midshaft cross-sectional area when compared with their
gender-matched littermates and the historical mean.
Discussion Related to Developmental Abnormality of Embryonic
Lethality for UNQ480 and the Negative Phenotype Associated with
Heterozygous Male Mutant Mice:
[0752] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neurodegenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[0753] With regards to UNQ480, the disease conditions more likely
to be associated with the embryonic lethality are as follows:
chondrodyplasia osteoarthritis pancreatic diseases vascular
development multiple sclerosis
[0754] During embryonic development UNQ480 is preferentially
expressed in the cartilage of skeletal tissues indicating
involvement in the control of proliferation and differentiation of
chondrocytes. Mutations in cartilage proteins can cause severe
congenital disorders, including various forms of chondrodyplasia.
In the adult, degeneration of the cartilage can lead to
osteoarthritis.
[0755] UNQ480 is also expressed at high levels in the developing
foregut endoderm which will give rise to the pancreatic analage. In
adult human tissues UNQ480 is preferentially expressed in the
pancreas. Several FGFs and their receptors are expressed in human
pancreatic cancer cell lines and are overexpressed in human
pancreatic cancers or in the pancreas of chronic pancreatitis.
Therefore, overexpression of UNQ480 is involved in the pathobiology
of pancreatic diseases.
[0756] UNQ480 also plays a role in vascular development as it binds
FGF2 which is known to be involved in this process. FGF2 has also
been shown to play a role in demyelination and thus can be involved
in demyelination diseases such as multiple sclerosis; by inference
so might UNQ480.
[0757] (a) Bone Metabolism: Radiology Phenotypic Analysis
[0758] Procedure:
[0759] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included microCT for very high resolution and very
high sensitivity measurements of bone mineral density for both
trabecular and cortical bone.
[0760] Bone microCT Analysis:
[0761] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 heterozygous mice. Measurements were
taken of lumbar 5 veterbra traebecular bone volume, traebecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[0762] Micro-CT Analysis Results: The male (+/+) mice exhibited an
increased mean femoral midshaft cross-sectional area when compared
with their gender-matched (+/+) littermates and the historical
mean.
[0763] (b) Further Pathology Observations
[0764] In addition to the above discussion, UNQ480 knockout mice
were further investigated to determine what factors were involved
which contributed to the embryonic lethality phenotype for the
homozygous mice. The results of these studies show that UNQ480
(designated as FGFRL1) is essential for kidney development and can
play a positive role in FGF signaling.
[0765] FGFRL1 is expressed during embryonic development in the
intermediate mesoderm that gives rise to the two precursor cell
populations of the kidney; the metenephric mesenchyme and the
Wolffian duct. Kidney development starts at embryonic day 10.5 when
the metenephric mesenchyme signals to the adjacent epithelial
Wolffian duct to induce ureteric bud formation. FGFRL1 is expressed
in the metenephric mesenchyme at this stage. The ureteric bud grows
and branches in response to signals from the metenephric mesenchyme
to form the collecting duct system of the mature kidney. Reciprocal
signals from the ureteric bud cause the metenephric mesenchyme to
undergo a mesenchymal to epithelial transition to form the
excretory portion of the kidney, the nephron.
[0766] UNQ480 homozygous mutants die around birth and exhibit
kidney agenesis. The initial ureteric bud forms normally in the
UNQ480 mutants as illustrated by the normal expression pattern of
c-ret at embryonic day 10.5. However, a day later the ureteric bud
fails to branch and form the characteristic T-shape structure.
Wnt-11 is expressed in the ureteric bud prior to branching but
little to no Wnt-11 is expressed in the UNQ480 mutants. Pax2 is
expressed in mesenchyme condensing around the tips of the ureteric
bud, although Pax2 is expressed in the UNQ480 mutants, the numbers
of cells and levels of expression is greatly reduced. These
observations suggest that FGFRL1 signals from the metenephric
mesenchyme to the ureteric bud to undergo branching morphogenesis.
By 13.5d the ureteric bud has normally branched many times as
illustrated by c-ret expression. Similarly Wnt-11 marks the branch
termini at these stages. Both these genes are either missing or
greatly down regulated in the UNQ480 mutants.
[0767] Little detail is known about the role of FGF signaling
during the early stages of kidney development. However, over
expression of a dominant negative form of FGFR2 blocks ureteric
branching providing compelling evidence that FGF signaling plays a
key role. The endogenous receptor affected in the dominant negative
model is unknown because the FGFR2 knock out develops kidneys
normally. However, FGFRL1 plays a role in this process.
[0768] In summary we have identified UNQ480 (FGFRL1) as a key
regulator during the early stages of kidney development. This data
suggests that FGFRL1 plays a positive role in FGF signaling.
[0769] G. Generation and Analysis of Mice Comprising DNA98557
(UNQ1425) Gene Disruptions
[0770] In these knockout experiments, the gene encoding hu A33
polypeptides (designated as DNA98557) [UNQ1425] was disrupted. The
gene specific information for these studies is as follows: the
mouse nucleotide reference corresponds to: NM.sub.--021610 or Mus
musculus glycoprotein A33 (transmembrane) (Gpa33), the protein
reference: NP.sub.--067623 or glycoprotein A33 (transmembrane); A33
antigen [Mus musculus]; the human nucleotide reference corresponds
to: NM.sub.--005814 or Homo sapiens glycoprotein A33
(transmembrane) (GPA33), protein reference: NP.sub.--005805 or
transmembrane glycoprotein A33 precursor [Homo sapiens]. Retroviral
insertion disrupted the gene prior to the exon encoding amino acid
3 in a protein of 315 amino acids (mouse NCBI accession number
NP.sub.--067623).
[0771] The disrupted mouse gene is glycoprotein A33 (GPA33),
ortholog of human GPA33. Aliases include cell surface antigen A33;
A33, A33 antigen, and cell surface A33 antigen.
[0772] GPA33 is a transmembrane protein containing two
immunoglobulin domains (Pfam PF00047) and is expressed almost
exclusively in intestinal epithelial cells (Abud et al., Mech.
Dev., 98(1-2):111-4 (2000)).
[0773] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation
[0774] Wild-type expression of the target gene was detected in
embryonic stem (ES) cells and, among the 13 adult tissue samples
tested by RT-PCR, in thymus, testis, and small intestine and colon.
RT-PCR analysis revealed that the transcript was absent in the
(-/-) mouse analyzed (F-99).
TABLE-US-00037 wt het hom Total Observed 29 47 24 100 Expected 25
50 25 100 Chi-Sq. = 0.86 Significance = 0.65051 (hom/n) = 0.24 Avg.
Litter Size = 0
[0775] 1. Phenotypic Analysis (for Disrupted Gene: DNA98557
(UNQ1425)
[0776] (a) Overall Phenotypic Summary:
[0777] The homozygous mutant mice exhibited an increased mean
percentage of CD4 cells and a decreased mean percentage of CD8
cells in the spleen and thymus when compared with their wild-type
littermates. In addition, the mutants exhibited a decreased mean
percentage of CD21hiCD23med B cells in the lymph node. RT-PCR
analysis revealed that the transcript was absent in the homozygous
mutant mice.
[0778] The male (-/-) mice also exhibited an increased mean serum
cholesterol level when compared with their gender-matched (+/+)
littermates and the historical mean.
[0779] (b) Phenotypic Analysis: Cardiology
[0780] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), cancer and/or obesity.
[0781] The phenotypic tests included the measurement of serum
cholesterol and triglycerides. In addition, inflammation assays
were performed to identify potential targets for the inflammatory
component of atherosclerosis.
[0782] Blood Lipids
[0783] Procedure:
[0784] A cohort of 5 wild type, 4 heterozygotes and 8 homozygotes
were tested in this assay. High cholesterol levels are recognized
risk factors in the development of cardiovascular disease.
Measuring blood lipids allowed finding of the biological switches
that regulate blood lipid levels and that upon inhibition would
lead to a reduction in the risk for cardiovascular disease.
Cholesterol measurements were recorded. The COBAS Integra 400 (mfr:
Roche) was used for running blood chemistry tests on mice.
[0785] Results:
[0786] As summarized above, the (-/-) mice exhibited a 35%
increased mean serum cholesterol level when compared with their
gender-matched (+/+) littermates and the historical mean. No change
in triglycerides was observed. (Analyzed wt/het/horn: 5/4/8)
[0787] Thus, mutant mice deficient in the A33 gene can serve as a
model for cardiovascular disease. A33 polypeptides or its encoding
gene would be useful in regulating blood lipids and in particular
maintaining normal cholesterol metabolism. Thus A33 polypeptides
would be useful for the treatment of such cardiovascular diseases
as: hypertension, atherosclerosis, heart failure, stroke, various
coronary artery diseases or diabetes.
[0788] (c) Immunology Phenotypic Analysis
[0789] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0790] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0791] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0792] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0793] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc.
[0794] In the area of immunology, targets were identified herein
for the treatment of inflammation and inflammatory disorders.
Immune related diseases could be treated by suppressing the immune
response. Using neutralizing antibodies that inhibit molecules
having immune stimulatory activity would be beneficial in the
treatment of immune-mediated and inflammatory diseases. Molecules
which inhibit the immune response can be utilized (proteins
directly or via the use of antibody agonists) to inhibit the immune
response and thus ameliorate immune related disease.
The following tests were performed:
[0795] Flourescence-Activated Cell-Sorting (FACS) Analysis
[0796] Procedure:
[0797] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[0798] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+
ratio.
[0799] The mononuclear cell profile was derived by staining a
single sample of lysed peripheral blood from each mouse with a
panel of six lineage-specific antibodies: CD45 PerCP, anti-TCRb
APC, CD4 PE, CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and
PE labeled antibodies stain mutually exclusive cell types. The
samples were analyzed using a Becton Dickinson FACSCalibur flow
cytometer with CellQuest software.
[0800] Results:
[0801] The homozygous mutant mice exhibited an increased mean
percentage of CD4+/CD8- cells and a decreased mean percentage of
CD4-/CD8+ cells in the spleen and thymus when compared with their
wild-type littermates. In addition, tissue-specific FACS analysis
resulted in the following observations: The (-/-) mice exhibited a
decreased mean percentage of CD21hiCD23med B cells in the lymph
node when compared with their (+/+) littermates; the (-/-) mice
also showed decreased mean percentages of B220dim CD43dim and
B220Med IgD-cells in bone marrow.
[0802] In summary, FACS analysis of immune cell composition from
peripheral blood indicates that knockout mice exhibit immunological
differences with respect to CD4 and CD8 cells when compared with
their wild-type littermates. Thus, antagonists of A33 polypeptides
or its encoding gene would be expected to mimic these effects. The
co-receptor CD4 molecule cooperates with the T-cell receptor which
differentially recognizes MHC class II molecules in the antigen
recognition process. An increased mean percentage of CD4 cells
plays a central role in B cell activation.
[0803] H. Generation and Analysis of Mice Comprising DNA33223-1136
(UNQ204) Gene Disruptions
[0804] In these knockout experiments, the gene encoding PRO230
polypeptides (designated as DNA33223-1136) [UNQ204] was disrupted.
The gene specific information for these studies is as follows:
mouse nucleotide reference: NM.sub.--023476 or Mus musculus
lipocalin 7 (Lcn7); mouse protein reference: NP.sub.--075965 or
lipocalin 7; androgen-regulated gene 1 [Mus musculus]; human gene
nucleotide reference: NM.sub.--022164 or Homo sapiens lipocalin 7
(LCN7), protein reference: NP.sub.--071447 or P3ECSL;
glucocorticoid-inducible protein; oxidized-LDL responsive gene 2;
tubulointerstitial nephritis antigen-related protein precursor;
likely ortholog of lipocalin 7; androgen-regulated gene 1 [Homo
sapiens].
[0805] The targeted mouse gene is lipocalin 7 (Lcn7), ortholog of
human lipocalin 7 (LCN7). Aliases include androgen-regulated gene
1, Arg1, P3ECSL, oxidized-LDL responsive gene 2;
glucocorticoid-inducible protein, and tubulointerstitial nephritis
antigen-related protein precursor.
[0806] LCN7 is a secreted protein with weak homology to
cathepsin-like proteases. The protein of about 460 amino acids
contains a signal peptide, epidermal growth factor-like repeats,
and a proteolytically inactive cathepsin B-related domain (Mukai et
al., J. Biol. Chem., 278(19):17084-92 (2003)). LCN7 also contains a
lipocalin signature, which typically binds with small hydrophobic
molecules (Kobayashi et al., J. Steroid Biochem. Mol. Biol.,
77(2-3):109-15 (2001)). The molecular function of LCN7 is not
known; it does not have proteolytic activity though it has features
of a protease.
[0807] LCN7 expression is induced by androgens in
androgen-dependent mouse mammary Shionogi carcinoma SC-3 cells and
in prostate. LCN7 is also detected in kidney, heart, lung, spleen,
and liver (Kobayashi et al, 2001 supra). Expression of the protein
may be restricted to vascular smooth muscle cells as well as
skeletal muscle, cardiac muscle, and kidney cells (Wex et al.,
Biochemistry, 40(5):1350-7 (2001)). LCN7 may be involved in zonal
differentiation in the adrenal cortex (Mukai et al, 2003 supra) or
in endosomal trafficking (Bromme et al., Biochem. Biophys. Res.
Commun., 271(2):474-80 (2000)). It may also be a structural or
regulatory protein.
[0808] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation as shown below.
TABLE-US-00038 wt het hom Total Observed 27 51 22 100 Expected 25
50 25 100 Chi-Sq. = 0.54 Significance = 0.76338 (hom/n) = 0.22 Avg.
Litter Size = 0
[0809] Wild-type expression of the target gene was detected in all
13 adult tissue samples tested by RT-PCR, except brain, skeletal
muscle, bone, stomach, small intestine, and colon, and adipose.
Disruption of the target gene was confirmed by Southern
hybridization analysis.
[0810] 1. Phenotypic Analysis (for Disrupted Gene: DNA33223-1136
(UNQ204)
[0811] (a) Overall Phenotypic Summary:
[0812] The female homozygous mutant mice exhibited a decreased
anxiety-like response during open field activity testing when
compared with their gender-matched wild-type littermates and the
historical mean. The homozygous mutant mice also showed an
increased response to the ovalbumin challenge. Disruption of the
target gene was confirmed by Southern hybridization analysis.
[0813] (b) Phenotypic Analysis: CNS/Neurology
[0814] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders. Neurological disorders
include but are not limited to: depression, generalized anxiety
disorders, attention deficit disorder; sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia and sensory
disorders, mild to moderate anxiety, anxiety disorder due to a
general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0815] Procedure:
[0816] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[0817] Open Field Test:
[0818] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning.
First, the field (40.times.40 cm) was selected to be relatively
large for a mouse, thus designed to pick up changes in locomotor
activity associated with exploration. In addition, there were 4
holes in the floor to allow for nose-poking, an activity
specifically related to exploration. Several factors were also
designed to heighten the affective state associated with this test.
The open-field test is the first experimental procedure in which
the mice are tested, and the measurements that were taken were the
subjects' first experience with the chamber. In addition, the
open-field was brightly lit. All these factors will heighten the
natural anxiety associated with novel and open spaces. The pattern
and extent of exploratory activity, and especially the
center-to-total distance traveled ratio, may then be able to
discern changes related to susceptibility to anxiety or depression.
A large arena (40 cm.times.40 cm, VersaMax animal activity
monitoring system from AccuScan Instruments) with infrared beams at
three different levels was used to record rearing, hole poke, and
locomotor activity. The animal was placed in the center and its
activity was measured for 20 minutes. Data from this test was
analyzed in five, 4-minute intervals. The total distance traveled
(cm), vertical movement number (rearing), number of hole pokes, and
the center to total distance ratio were recorded.
[0819] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value.
[0820] Results: A notable difference was observed during open field
activity testing. The female (-/-) mice exhibited an increased
median sum time in the center area when compared with their
gender-matched (+/+) littermates, which is indicative of a
decreased anxiety-like response in the mutants. Thus, knockout mice
demonstrated a phenotype consistent with depression, generalized
anxiety disorders, attention deficit disorder, sleep disorder,
cognitive disorders, hyperalgesia and sensory disorders depressive
disorders. Thus, PRO230 polypeptides and agonists thereof would be
useful for the treatment or amelioration of the symptoms associated
with such depressive disorders.
[0821] (c) Immunology Phenotypic Analysis
[0822] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0823] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0824] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0825] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0826] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0827] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0828] The following test was performed:
[0829] Ovalbumin Challenge
[0830] Procedure: This assay was carried out on 6 wild types and 14
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immunodominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[0831] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Feund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample. [Analyzed wt/het/hom: 6/4/14]
[0832] Results of this challenge: The (-/-) mice exhibited a trend
towards an increased mean serum IgG2a response to the ovalbumin
challenge when compared with their (+/+) littermates. Thus, these
knockout mice exhibited an increased ability to elicit an
OVA-specific antibody response to the T-cell dependent OVA antigen.
Inhibitors (antagonists) of PRO230 polypeptides would be expected
to also stimulate the immune system and would find utility in the
cases wherein this effect would be beneficial to the individual
such as in the case of leukemia, and other types of cancer, and in
immunocompromised patients, such as AIDS sufferers. Accordingly,
PRO230 polypeptides or agonists thereof would be useful in
inhibiting the immune response and would be useful candidates for
suppressing harmful immune responses, e.g. in the case of graft
rejection or graft-versus-host diseases.
[0833] I. Generation and Analysis of Mice Comprising DNA23339-1130
(UNQ152) Gene Disruptions
[0834] In these knockout experiments, the gene encoding PRO178
polypeptides (designated as DNA23339-1130) [UNQ152] was disrupted.
The gene specific information for these studies is as follows:
mouse gene sequence nucleotide reference: NM.sub.--145154 or
accession: NM.sub.--145154 NID: gi 21699061 ref NM.sub.--145154.1,
Mus musculus RIKEN cDNA 6330404E11 gene (6330404E11Rik), protein
reference: Q9CU50 or accession: Q9CU50 NID: Mus musculus (Mouse) or
6330404E11RIK protein (fragment) or MOUSESPTRNRDB; human gene
sequence nucleotide reference: NM.sub.--031917 or accession:
NM.sub.--031917 NID: 13994284 Homo sapiens [Homo sapiens
angiopoietin-related protein 5 (ARP5)], protein reference: Q9BZZO
or accession: Q9BZZO NID: Homo sapiens (Human) or
ANGIOPOMTIN-RELATED PROTEIN 5. HITMANSPTRNRDB. Insertion appears to
be in forward direction near amino acid 180 out of about 460.
[0835] The gene that is mutated in these animals is represented by
mouse UniGene cluster Mm.34019, which is the ortholog of human
UniGene cluster Hs.306971 (ARP5). ARP5 encodes angiopoietin-related
protein 5. The protein is also known as ARP3 or ANGPTL5.
[0836] Angiopoietin-related proteins 1, 3 and 5 suffer from
nomenclature problems and ensuing confusion. All three have been
called ARP3 at one time or another (in mouse or human).
Additionally, other proteins have an b ARPb prefix (e.g.,
actin-related proteins).
[0837] ARP5 is predicted to encode a domain known as b C-terminal
globular domain of fibrinogen beta and gamma chainsb (Pfam 00147;
FBG). This domain is up to 270 residues in length and contains 4
conserved cysteines that participate in 2 disulfide bonds. The FBG
motif is found in the C-terminus of the beta and gamma chains of
fibrinogen, but not the alpha chain.
[0838] Examination of SwissProt records shows that the motif is
found in various, typically extracellular or surface, human
proteins including angiopoietin-1 and b 2 (Q15389, O15123),
angiopoietin-related protein 2 (Q9UKU9), neurexin 4 (P78357),
ficolin 2 (Q15485), and microfibril-associated glycoprotein 4
(P55083). Additionally, the FBG domain is present in 2
fibrinogen-like proteins of sea urchin (Xu, X. and Doolittle, R.
F., Proc. Natl. Acad. Sci. USA, 87(6):2097-101 (1990)) and the
Drosophila scabrous protein (P21520).
[0839] Because a signal peptide is predicted and membrane-anchoring
mechanisms were not, and also because related proteins are
secreted, the protein may be secreted.
[0840] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation as shown below.
TABLE-US-00039 wt het hom Total Observed 31 29 22 82 Expected 20.5
41 20.5 82 Chi-Sq. = 9.00 Significance = 0.01111 (hom/n) = 0.27
Avg. Litter Size = 0
[0841] Wild-type expression of the target gene was detected in
embryonic stem (ES) cells and, among the 13 adult tissue samples
tested by RT-PCR, in brain, thymus, liver, and skin fibroblast.
RT-PCR analysis revealed that the transcript was absent in the
(-/-) mouse analyzed (M-68).
[0842] 1. Phenotypic Analysis (for Disrupted Gene: DNA23339-1130
(UNQ152)
[0843] (a) Overall Phenotypic Summary
[0844] The male homozygous mutant mice exhibited notably increased
mean serum triglyceride and cholesterol levels when compared with
their gender-matched wild-type littermates and the historical
means. No other notable phenotype was observed for the homozygous
mutant mice. RT-PCR analysis revealed that the transcript was
absent in the homozygous mutant mice.
[0845] (b) Phenotypic Analysis: Cardiology
[0846] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[0847] Blood Lipids
[0848] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels. Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche).
[0849] Results: As summarized above, the male (-/-) mice exhibited
notably increased triglyceride and cholesterol levels when compared
with their gender-matched (+/+) littermates and the historical
means for the male (+/+) mice. Thus, mutant mice deficient in the
PRO178 gene can serve as a model for cardiovascular disease. PRO178
polypeptides or its encoding gene would be useful in regulating
blood lipids such as cholesterol and triglycerides. Thus, PRO178
polypeptides or agonists thereof would be useful in the treatment
of such cardiovascular diseases as hypertension, atherosclerosis,
heart failure, stroke, various coronary diseases,
hypercholesterolemia, hypertriglyceridemia, diabetes and/or
obesity.
[0850] J. Generation and Analysis of Mice Comprising
DNA65351-1366-2 (UNQ407) Gene Disruptions
[0851] In these knockout experiments, the gene encoding PRO1199
polypeptides (designated as DNA65351-1366-2) [UNQ407] was
disrupted. The gene specific information for these studies is as
follows: mouse gene sequence nucleotide reference: NM.sub.--022984
or Mus musculus resistin (Retn), protein reference: NP.sub.--075360
or resistin; found in inflammatory zone 3; adipocyte-specific
secretory factor; Fizz3-pending [Mus musculus]; human gene sequence
nucleotide reference: NM.sub.--020415 or Homo sapiens resistin
(RETN), protein reference: NP.sub.--065148 or resistin [Homo
sapiens].
[0852] The mutated gene is resistin (Retn), ortholog of human
resistin (RETN). Aliases include ADSF, RSTN, FIZZ3, RETN1, found in
inflammatory zone 3, C/EBP-epsilon regulated myeloid-specific
secreted cysteine-rich protein, cysteine-rich secreted protein
A12-alpha-like 2, cysteine-rich secreted protein FIZZ3, and
adipocyte-specific secretory factor.
[0853] RETN contains an N-terminal signal peptide and a
cysteine-rich C-terminus. The spacing of the C-terminal cysteines
is highly conserved among FIZZ family members (Holcomb et al., EMBO
J., 19(15):4046-55 (2000); Steppan et al., Nature, 409:307-312
(2001); OMIM 605565). RETN belongs to the RESISTIN/FIZZ family of
proteins, which are involved in inflammation.
[0854] RETN is a hormone expressed mainly in white adipose tissue
that suppresses insulin-stimulated glucose uptake into fat cells
and insulin action in peripheral tissues. In obese individuals,
RETN is reported to be overexpressed in adipose tissue; thus, RETN
may contribute to insulin resistance (Steppan et al, 2001 supra;
Pizzuti et al., J. Clin. Endocr. Metab., 87:4403-4406 (2002)).
However, other studies failed to find RETN overexpression in
adipose tissues (Beltowski, J., Med. Sci. Monit., 9(2):RA55-61
(2003)).
[0855] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were crossed to hybrid 129SvEv.sup.Brd/C57
Cre homozygous mice to generate mice carrying both the mutant and
Cre alleles (compound heterozygous mice). The male compound
heterozygous mice were then crossed to hybrid 129SvEv.sup.Brd/C57
F1 mice, derived from crossing 129SvEv.sup.Brd mice to C57BL/6J
mice, to generate heterozygous Cre-excised animals. Finally, these
progeny were intercrossed to generate wild-type, Cre-excised
heterozygous, and Cre-excised homozygous mice. Level I phenotypic
analysis was performed on mice from this generation as shown
below.
[0856] Wild-type expression of the target gene was detected in all
13 adult tissue samples tested by RT-PCR, except brain, liver,
testis, stomach, and skin fibroblast. Disruption of the target gene
was confirmed by Southern hybridization analysis.
TABLE-US-00040 wt het hom Total Observed 29 48 23 100 Expected 25
50 25 100 Chi-Sq. = 0.88 Significance = 0.64404 (hom/n) = 0.23 Avg.
Litter Size = 0
[0857] 1. Phenotypic Analysis (for Disrupted Gene: DNA65351-1366-2
(UNQ407)
[0858] (a) Overall Phenotypic Summary
[0859] The homozygous mutant mice exhibited an increased response
to an inflammatory stimulus when compared with their wild-type
littermate and the historical range. In addition, the female
homozygous mutant mice exhibited an increased mean total tissue
mass and lean body mass and an increased mean serum triglyceride
level.
[0860] (b) Immunology Phenotypic Analysis
[0861] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0862] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0863] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0864] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0865] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc.
[0866] In the area of immunology, targets were identified herein
for the treatment of inflammation and inflammatory disorders.
Immune related diseases could be treated by suppressing the immune
response. Using neutralizing antibodies that inhibit molecules
having immune stimulatory activity would be beneficial in the
treatment of immune-mediated and inflammatory diseases. Molecules
which inhibit the immune response can be utilized (proteins
directly or via the use of antibody agonists) to inhibit the immune
response and thus ameliorate immune related disease.
The following tests were performed:
[0867] Zymosan Challenge Assay--Peritoneal Inflamnzation:
[0868] Procedure: A cohort of 1 wild type and 2 homozygous mutants
were used in this assay. Peritoneal leukocyte recruitment assays
were used to identify targets that may regulate the inflammatory
component of atherosclerosis. These assays detect abnormalities in
immune cell recruitment to a site of inflammation. Zymosan (an
agent which induces inflammation) was injected into the
intraperitoneal cavity and fluid was later removed and measurements
were taken of total WBC counts, neutrophil/monocyte ratio and
percent granulocytes, monocytes, lymphocytes and eosinophils in the
ip fluid.
[0869] Results: The (-/-) mice exhibited a notably increased total
white blood cell count after zymosan challenge when compared with
their (+/+) littermate and the historical range, suggestive of an
increased response to an inflammatory stimulus in these
mutants.
[0870] In summary, the zymosan challenge studies indicate that
knockout mice deficient in the gene encoding PRO1199 polypeptides
exhibit a proinflammatory response when compared with their
wild-type littermates. Thus, antagonists of PRO1199 polypeptides
would be important agents which would stimulate the immune system
(such as T cell proliferation) and would find utility in the cases
wherein this effect would be beneficial to the individual such as
in the case of leukemia, and other types of cancer, and in
immunocompromised patients, such as AIDS sufferers. Accordingly,
PRO1199 polypeptides or agonists thereto would be useful in
inhibiting the immune response and would be useful candidates for
suppressing harmful immune responses, e.g. in the case of graft
rejection or graft-versus-host diseases.
[0871] (c) Phenotypic Analysis: Cardiology
[0872] In the area of cardiovascular biology, targets were
identified for the treatment of hypertension, atherosclerosis,
heart failure, stroke, coronary artery diseases, and dislipidemias
such as high cholesterol and triglyceride serum levels. The
phenotypic tests included the measurement of serum cholesterol and
triglycerides.
[0873] Blood Lipids
[0874] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High serum trigylcerides
levels are recognized risk factors in the development of
cardiovascular disease. In addition, dislipidemia can be associated
with obesity and diabetes. Measuring blood lipids facilitates the
finding of biological switches that regulate blood lipid levels.
Inhibition of factors which elevate blood lipid levels may be
useful for reducing the risk for cardiovascular disease.
[0875] As summarized above, the female homozygous mutant mice
exhibited an increased mean total tissue mass and lean body mass
and an increased mean serum triglyceride level in homozygous
knockout mice (167% of wild-type littermates). Thus, mutant mice
deficient in the PRO1199 gene can serve as a model for
cardiovascular disease and/or dislipidemia associated with obesity
and diabetes. PRO1199 or its encoding gene would be useful in
regulating blood lipids and in particular may play a role in
maintaining normal lipid metabolism.
[0876] (d) Dexa Analysis--Test Description:
[0877] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[0878] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[0879] DEXA Results: As summarized above, the female homozygous
mutant mice exhibited slightly increased mean total tissue mass and
lean body mass beginning at 8 weeks; at 18 weeks, the female
knockout mice are 18% heavier than their gender-matched (+/+)
littermates and the historical means.
[0880] These studies suggest that mutant (-/-) non-human transgenic
animals exhibit a negative phenotype that can be associated with
obesity. Thus, PRO1199 polypeptides or agonists thereof would be
useful in the treatment of obesity.
[0881] K. Generation and Analysis of Mice Comprising DNA84210-2576
(UNQ1888) Gene Disruptions
[0882] In these knockout experiments, the gene encoding PRO4333
polypeptides (designated as DNA84210-2576 (UNQ1888) was disrupted.
The gene specific information for these studies is as follows:
mouse gene sequence nucleotide reference: NM.sub.--013869 or Mus
musculus tumor necrosis factor receptor superfamily, member 19
(Tnfrsf19), protein reference: NP.sub.--038897 or tumor necrosis
factor receptor superfamily, member 19; tumor necrosis factor
receptor superfamily, member 20 [Mus musculus]; human gene sequence
nucleotide reference: NM.sub.--018647 or Homo sapiens tumor
necrosis factor receptor superfamily, member 19 (TNFRSF19), protein
reference: NP.sub.--061117 or tumor necrosis factor receptor
superfamily, member 19 [Homo sapiens].
[0883] The disrupted mouse gene is tumor necrosis factor receptor
superfamily, member 19 (Tnfrsf19), ortholog of human TNFRSF19.
Aliases and synonyms include TM; Troy; TM-ALPHA; Tnfrsf20; tumor
necrosis factor receptor superfamily, member 20; TRADE; and
toxicity and JNK inducer.
[0884] TNFRSF19 consists of an extracellular ligand-binding domain,
a transmembrane domain, and a cytoplasmic domain that contains a
tumor necrosis factor receptor-associated factor (TRAF) 2-binding
consensus sequence but no death domain. TNFRSF19 recruits binding
of TRAFs, and activates nuclear factor kappa B (NF-kB) and c-Jun
N-terminal kinase (Eby et al., J. Biol. Chem., 275(20):15336-42
(2000); Kojima et al., J. Biol. Chem., 275(27):20742-7 (2000); Hu
et al., Genomics, 62(1):103-7 (1999)).
[0885] In the developing mouse embryo, TNFRSF19 is expressed at
high levels in skin, whereas expression is high in brain and
moderate in heart, lung, liver and hair follicles in post natal
mice. Activation of TNFRSF19 also induces caspace-independent cell
death. The function of TNFRSF19 is likely to be pleiotropic and
important in embryonic development and skin and hair follicle
development (OMIM 606122).
[0886] In mouse, alternative splicing gives rise to 2 additional
variants; one may function as a soluble receptor and the other may
function as a decoy receptor.
[0887] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to 129SvEv.sup.Brd mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant mice. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd mice to yield
additional heterozygous animals for the intercross to generate the
F2 mice. Expression of the target gene was detected in embryonic
stem (ES) cells and, of 13 adult tissues samples tested by RT-PCR,
only in brain. RT-PCR analysis revealed that the transcript was
absent in the (-/-) mouse analyzed (M-66). Level I phenotypic
analysis was performed on mice from this generation.
TABLE-US-00041 wt het hom Total Observed 25 46 15 86 Expected 21.5
43 21.5 86 Chi-Sq. = 2.74 Significance = 0.25358 (hom/n) = 0.17
Avg. Litter Size = 0
[0888] 1. Phenotypic Analysis for Disrupted Gene: DNA84210-2576
(UNQ1888)
[0889] (a) Overall Phenotypic Summary
[0890] The homozygous mutant mice exhibited a decreased exploratory
response and hypoactivity when compared with their wild-type
littermates. The (-/-) mice exhibited decreased serum IgA levels.
In addition, these mutants exhibited enhanced glucose tolerance at
all 3 intervals tested. The (-/-) mutant mice also exhibited a
decreased mean total tissue mass and lean body mass. The male (-/-)
mutant mice exhibited a notably decreased femoral midshaft
cross-sectional area when compared with the historical mean.
[0891] (b) Phenotypic Analysis: CNS/Neurology
[0892] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders. Neurological disorders
include but are not limited to: depression, generalized anxiety
disorders, attention deficit disorder, sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia and sensory
disorders, mild to moderate anxiety, anxiety disorder due to a
general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0893] Procedure:
[0894] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[0895] Open Field Test:
[0896] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning.
First, the field (40.times.40 cm) was selected to be relatively
large for a mouse, thus designed to pick up changes in locomotor
activity associated with exploration. In addition, there were 4
holes in the floor to allow for nose-poking, an activity
specifically related to exploration. Several factors were also
designed to heighten the affective state associated with this test.
The open-field test is the first experimental procedure in which
the mice are tested, and the measurements that were taken were the
subjects' first experience with the chamber. In addition, the
open-field was brightly lit. All these factors will heighten the
natural anxiety associated with novel and open spaces. The pattern
and extent of exploratory activity, and especially the
center-to-total distance traveled ratio, may then be able to
discern changes related to susceptibility to anxiety or depression.
A large arena (40 cm.times.40 cm, VersaMax animal activity
monitoring system from AccuScan Instruments) with infrared beams at
three different levels was used to record rearing, hole poke, and
locomotor activity. The animal was placed in the center and its
activity was measured for 20 minutes. Data from this test was
analyzed in five, 4-minute intervals. The total distance traveled
(cm), vertical movement number (rearing), number of hole pokes, and
the center to total distance ratio were recorded.
[0897] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value.
[0898] Results:
[0899] As indicated in the phenotypic analysis summary, notable
differences were observed during open field activity testing. The
(-/-) mice demonstrated a decrease in locomotor activity, rearing,
and hole poke activity over all intervals when compared with the
(+/+) mice, suggesting a hypoactive and decreased exploratory
response. Thus, knockout mice demonstrated a phenotype consistent
with various neural degenerative diseases which may include
consistent with depression, generalized anxiety disorders,
attention deficit disorder, sleep disorder, cognitive disorders,
hyperalgesia and sensory disorders, schizophrenia and/or bipolar
disorders. Thus, PRO4333 polypeptides and agonists thereof would be
useful for the treatment or amelioration of the symptoms associated
with such depressive disorders.
[0900] (c) Bone Metabolism: Radiology Phenotypic Analysis
[0901] Procedure:
[0902] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included microCT for very high resolution and very
high sensitivity measurements of bone mineral density for both
trabecular and cortical bone.
[0903] DEXA Analysis--Test Description:
[0904] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[0905] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[0906] DEXA Results: As summarized above, The male (-/-) mice
exhibited decreased mean total tissue mass and lean body mass when
compared with their gender-matched (+/-) littermates and the
historical means.
[0907] Bone microCT Analysis:
[0908] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. The .mu.CT40 scans dissected bones and
provides detailed information on bone mass and architecture. One
vertebra and 1 femur were taken from a cohort of 4 wild type and 8
homozygous mice. Measurements were taken of lumbar 5 veterbra
traebecular bone volume, traebecular thickness, connectivity
density and midshaft femur total bone area and cortical thickness.
The .mu.CT40 scans provided detailed information on bone mass and
architecture. Multiple bones were placed into sample holders and
scanned automatically. Instrument software was used to select
regions of interest for analysis. Trabecular bone parameters were
analyzed in the fifth lumbar vertebrae (LV5) at 16 micrometer
resolution and cortical bone parameters were analyzed in the femur
midshaft at a resolution of 20 micrometers.
[0909] Micro-CT Analysis Results: The male (-/-) mutant exhibited a
notably decreased femoral midshaft cross-sectional area when
compared with gender-matched (+/+) littermates and the historical
means.
[0910] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Blood
chemistry tests also showed an increased alkaline phosphatase
activity in (-/-) mutant mice which is commonly associated with
osteoporosis. Thus, it appears that PRO4333 polypeptides or its
encoding gene would be useful in maintaining bone homeostasis or
would be useful for bone healing or for the treatment of arthritis
or osteoporosis, whereas antagonists would lead to abnormal or
pathological bone disorders similar to osteoporosis. In addition to
these studies, (-/-) mutant mice showed signs of growth
retardation. Such growth disorders may be associated with the
phenotype or physiological condition associated with tissue wasting
diseases such as diabetes or cachexia. Thus, PRO4333 polypeptides
or agonists thereof would be useful for treating diabetes or
cachexia.
[0911] (d) Phenotypic Analysis: Metabolism--Blood Chemistry
[0912] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
glucose tolerance tests to measure insulin sensitivity and changes
in glucose metabolism. Abnormal glucose tolerance test results may
indicate the following disorders or conditions: cachexia, Diabetes
Type 1 and Type 2, Syndrome X, various cardiovascular diseases.
[0913] Procedure: A cohort of 4 wild type, 4 heterozygote and 8
homozygote mice were used in this assay. The glucose tolerance test
is the standard for defining impaired glucose homeostasis in
mammals. The intraperitoneal glucose tolerance test (IPGTT)
measures glucose levels following administration of a glucose
challenge. Glucose tolerance tests were performed using a Lifescan
glucometer. Animals were injected IP at 2 g/kg with D-glucose
delivered as a 20% solution and blood glucose levels were measured
at 0, 30, 60 and 90 minutes after injection.
[0914] Results: These studies indicated that male (-/-) mice
exhibit enhanced glucose tolerance in the presence of normal
fasting glucose at all 3 intervals tested when compared with their
gender-matched (+/+) littermates and the historical means. In
addition, hyperinsulinemia was not apparent in the (-/-) mice.
[0915] (e) Immunology Phenotypic Analysis
[0916] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0917] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0918] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0919] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0920] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0921] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulator),
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0922] The following tests were performed:
[0923] Serum Immunoglobulin Isotyping Assay:
[0924] The Serum Immunoglobulin Isotyping Assay is performed using
a Cytometric Bead Array (CBA) kit. This assay is used to rapidly
identify the heavy and light chain isotypes of a mouse monoclonal
antibody in a single sample. The values expressed are "relative
fluorescence units" and are based on the detection of kappa light
chains. Any value <6 is not significant.
[0925] Results:
[0926] The serum immunoglobulin isotyping assay revealed that
homozygous adults exhibited decreased serum IgA levels compared
with the (+/+) littermates. These results suggest that the
phenotype associated with these knockout mice is immunoglobulin
deficiency in IgA. The most common inherited form of immunoglobulin
deficiency is selective IgA deficiency, which is seen in about one
person in 800. Although no obvious disease susceptibility is
associated with selective IgA defects, they are commoner in people
with chronic lung disease than in the general population. This
suggests that lack of IgA may result in a predisposition to lung
infections with various pathogens and is consistent with the role
of IgA in defense at the body surfaces. Thus PRO4333 polypeptides
or agonists thereof, would be useful for natural immunity
protection against skin infections and more importantly could
prevent susceptibility to lung infections.
[0927] L. Generation and Analysis of Mice Comprising DNA65423-1595
(UNQ691) Gene Disruptions
[0928] In these knockout experiments, the gene encoding PRO1336
polypeptides (designated as DNA65423-1595 (UNQ691) was disrupted.
The gene specific information for these studies is as follows:
mouse gene sequence nucleotide reference: AF144629 or Mus musculus
SLIT3 (Slit3), protein reference: AAD44760 or SLIT3 [Mus musculus];
human gene sequence nucleotide reference: NM.sub.--003062 or Homo
sapiens slit homolog 3 (Drosophila) (SLIT3), protein reference:
NP.sub.--003053 or slit homolog 3 (Drosophila); slit (Drosophila)
homolog 3; slit (Drosophila) homolog 2; slit2 [Homo sapiens].
[0929] The gene that is mutated in these animals is represented by
mouse UniGene cluster Mm.28509, which is the ortholog of human
UniGene cluster Hs.57929 (SLIT3). SLIT3 encodes slit homolog 3
(Drosophila). The protein is also known as MEGF5, SLIL2, SLIT1,
SLIT-3, slit2, slit (Drosophila) homolog 2, slit (Drosophila)
homolog 3 and multiple epidermal growth factor-like domains 5. The
Drosophila and vertebrate SLIT proteins are large extracellular
glycoproteins that have been characterized as secreted
chemo-repellents recognized by the ROBO receptor proteins that
function principally for the guidance of neuronal axons and
neurons. Drosophila SLIT3 expression is restricted to the future
chondrogenic core of the limb bud in early development, and in
later stages, it is expressed in proliferating chondrocytes during
cartilage development (Holmes et al., Dev. Dyn., 222(2): 301-7
(2001)).
[0930] The SLIT3 protein is targeted to and localized at two
distinct sites within mouse kidney epithelial cells: the
mitochondria, and then, in more confluent cells, the cell surface.
The investigators concluded that their results indicated that SLIT3
has potentially unique functions not shared by other SLIT proteins
(Little et al., Am J Physiol Cell Physiol, 281(2):C486-95 (August
2001)).
[0931] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were crossed to hybrid 129SvEv.sup.Brd/C57
Cre homozygous mice to generate mice carrying both the mutant and
Cre alleles (compound heterozygous mice). The male compound
heterozygous mice were then crossed to hybrid 129SvEv.sup.Brd/C57
F1 mice, derived from crossing 129SvEv.sup.Brd mice to C57BL/6J
mice, to generate heterozygous Cre-excised animals. Finally, these
progeny were intercrossed to generate wild-type, Cre-excised
heterozygous, and Cre-excised homozygous mice.
[0932] Wild-type expression of the target gene was detected in
embryonic stem (ES) cells and in all 13 adult tissue samples tested
by RT-PCR. RT-PCR analysis revealed that the transcript was absent
in kidney and barely detectable in lung in the (-/-) mouse analyzed
(M-107). Gene expression of the neo transcript was detected in
stomach, adrenal gland cortex, kidney collecting tubule, small
intestine renal tubule, heart parenchyma, and brain hippocampus.
[Analyzed wt/het/horn: 0/1/5]. RT-PCR analysis revealed that the
transcript was absent in the homozygous mutant mice. Level I
phenotypic analysis was performed on mice from this generation as
shown below.
TABLE-US-00042 wt het hom Total Observed 22 39 22 83 Expected 20.75
41.5 20.75 83 Chi-Sq. = 0.30 Significance = 0.86019 (hom/n) = 0.27
Avg. Litter Size = 0
[0933] 1. Phenotypic Analysis (for Disrupted Gene: DNA65423-1595
(UNQ691)
[0934] (a) Overall Phenotypic Summary
[0935] At 2 weeks of age, the homozygous mutant neonates exhibited
balding; however, their hair grew in and appeared normal at
adulthood. Half of the homozygous mutant mice exhibited signs of
growth retardation at 16 weeks of age, namely decreased body weight
and length, total tissue mass, and lean body mass, when compared
with their wild-type littermates. The homozygous mutant mice also
exhibited a diaphragmatic hernia and decreased exploratory activity
and a decreased anxiety-related response during open field
testing.
[0936] (b) Phenotypic Analysis: CNS/Neurology
[0937] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders. Neurological disorders
include but are not limited to: depression, generalized anxiety
disorders, attention deficit disorder, sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia and sensory
disorders, mild to moderate anxiety, anxiety disorder due to a
general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0938] Procedure:
[0939] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[0940] Open Field Test:
[0941] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning.
First, the field (40.times.40 cm) was selected to be relatively
large for a mouse, thus designed to pick up changes in locomotor
activity associated with exploration. In addition, there were 4
holes in the floor to allow for nose-poking, an activity
specifically related to exploration. Several factors were also
designed to heighten the affective state associated with this test.
The open-field test is the first experimental procedure in which
the mice are tested, and the measurements that were taken were the
subjects' first experience with the chamber. In addition, the
open-field was brightly lit. All these factors will heighten the
natural anxiety associated with novel and open spaces. The pattern
and extent of exploratory activity, and especially the
center-to-total distance traveled ratio, may then be able to
discern changes related to susceptibility to anxiety or depression.
A large arena (40 cm.times.40 cm, VersaMax animal activity
monitoring system from AccuScan Instruments) with infrared beams at
three different levels was used to record rearing, hole poke, and
locomotor activity. The animal was placed in the center and its
activity was measured for 20 minutes. Data from this test was
analyzed in five, 4-minute intervals. The total distance traveled
(cm), vertical movement number (rearing), number of hole pokes, and
the center to total distance ratio were recorded.
[0942] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value.
[0943] Results:
[0944] With regards to general and exploratory activity/anxiety
behavior, notable differences were observed during open field
activity testing. The (-/-) mice demonstrated a decrease in rearing
and hole poke activity when compared to the (4+) mice, suggesting a
decreased exploratory response in the (-/-) mice. The (-/-) mice
also demonstrated a decrease in center-to-total distance traveled
ratio at intervals 2, 3, and 5 when compared to the (+/+) mice,
suggesting a decreased anxiety-like response in the (-/-) mice.
Thus, knockout mice demonstrated a phenotype consistent with
various neural degenerative diseases which may include consistent
with depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, cognitive disorders, hyperalgesia and
sensory disorders, schizophrenia and/or bipolar disorders. Thus,
PRO1336 polypeptides and agonists thereof would be useful for the
treatment or amelioration of the symptoms associated with such
depressive disorders.
[0945] Functional Observational Battery (FOB) Test
[0946] The FOB is a series of situations applied to the animal to
determine gross sensory and motor deficits. A subset of tests from
the Irwin neurological screen that evaluates gross neurological
function is used. In general, short-duration, tactile, olfactory,
and visual stimuli are applied to the animal to determine their
ability to detect and respond normally. These simple tests take
approximately 10 minutes and the mouse is returned to its home cage
at the end of testing.
[0947] Results:
[0948] As summarized above, a notable difference was observed
during the functional observational battery. Male heterozygous
(-/+) mice exhibited moderate exophthalmus (an abnormal protrusion
of the eyeball); whereas female homozygous (-/-) mutant mice
exhibited severe exophthalmus. Both male and female (-/-) mice also
exhibited piloerection. Piloerection is an involuntary erection or
bristling of hairs due to a sympathetic reflex usually triggered by
cold, shock, or fright or due to a sympatho-mimetic agent. All of
the (-/-) mice exhibited balding at 2 weeks of age with bald hair
patches around the snout. However, their hair grew in and appeared
normal at adulthood. Thus, functional observational battery testing
showed that the mutant mice exhibited a decreased exploratory
activity phenotype. Neurological disorders associated with this
type of behavior may involve such anxiety disorders of panic or a
panic disorder associated with agoraphobia, agoraphobia or
cyclothymic disorder. Cyclothymic disorders are characterized by
the alteration of depressed moods with elevated, expansive, or
irritable moods without psychotic features such as hallucinations
or delusions.
[0949] (c) Phenotype Analysis: Body Mass:
[0950] Dexa Analysis--Test Description:
[0951] Procedure: A cohort of 4 wild type, 4 heterozygotes and 6
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[0952] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[0953] Results:
[0954] The (-/-) mice analyzed by DEXA exhibited notably decreased
total tissue mass and lean body mass when compared with their (+/+)
littermates, suggestive of growth retardation in these mutants.
These mutants also exhibited a notably increased bone mineral
content index (BMC/LBM). Blood chemistry analysis indicated that
male (-/-) mice showed a decreased mean insulin level when compared
with their gender-matched (+/+) littermates and the historical
mean. This in conjunction with the observations of a decreased mean
body weight and decreased mean body length suggests a tissue
wasting condition such as cachexia, diabetes type 1 and type 2.
Thus, PRO1336 polypeptides or agonists thereof would be useful in
the treatment or prevention of cachexia and/or diabetes.
[0955] CAT-Scan Protocol:
[0956] Mice were injected with a CT contrast agent, Omnipaque 300
(Nycomed Amershan, 300 mg of iodine per ml, 0.25 ml per animal, or
2.50-3.75 g iodine/kg of body weight) intraperitoneally. After
resting in the cage for .about.10 minutes, the mouse was then
sedated by intraperitoneal injection of Avertin (1.25%
2,2,2,-tribromoethanol, 20 ml/kg body weight). A CAT-scan was
performed using a MicroCAT scanner (ImTek, Inc.) with the
anesthetized animal lying prone on the test bed. Three dimensional
images were reconstructed by the Feldkamp algorithm in a cluster of
workstations using an ImTek 3D RECON software.
[0957] Results:
[0958] Of the five (-/-) mutant mice that were examined, four
exhibited a developmental defect of the diaphragm (diaphragmatic
hernia). In addition, the (-/-) mice exhibited an enlarged right
atrium. Microscopic observations were as follows: Histological
analysis of these mutants revealed a variety of lesions, including
hepatic atrophy, congestion, periportal fibrosis, aortic
atherosclerosis, and gastric inflammation. However, all of these
lesions were considered to be secondary to the diaphragmatic
hernia, rather than being primary gene-related lesions.
[0959] M. Generation and Analysis of Mice Disruptions
[0960] In these knockout experiments, the gene encoding PRO19598
polypeptides (designated as DNA145887-2849 (UNQ5793) was disrupted.
The gene specific information for these studies is as follows:
mouse gene sequence nucleotide reference: XM.sub.--136951 or Mus
musculus similar to interleukin-22 binding protein (LOC237310),
protein reference: XP.sub.--136951 similar to interleukin-22
binding protein [Mus musculus]; human gene sequence nucleotide
reference: NM.sub.--052962 or Homo sapiens class II cytokine
receptor (IL22RA2), protein reference: NP 443194 or class II
cytokine receptor; interleukin 22-binding protein [Homo
sapiens].
[0961] The gene that is mutated in these animals is represented by
mouse nucleotide sequence XM.sub.--136951, which is the ortholog of
human UniGene cluster Hs. 126891 (IL22RA2). IL22RA2 encodes
interleukin 22 receptor alpha-2. The protein is also known as
interleukin 22-binding protein (IL22BP) and class II cytokine
receptor, IL22BP, CRF2-S1, and IL-22BP.
[0962] IL22RA2, containing 2 cytokine-binding fibronectin type III
domains, is a secreted glycoprotein resembling the extracellular
domain of cytokine receptors IL20RA and IL22R (33% identity)
(Dumoutier et al, J. Immunol., 166(12):7090-5 (2001)); Xu et al,
Proc. Natl. Acad. Sci. USA, 98(17):9511-6 (2001)). IL22RA2 binds
with IL22, a cytokine produced by T cells, and was shown to inhibit
11.22 signaling in numerous cell lines. Thus, IL22RA2 is likely to
be a negative regulator of inflammatory responses induced by IL22
(Dumoutier et al, 2001 supra; Xu et al, 2001 supra; Kotenko et al,
J. Immunol., 166(12):7096-103 (2001)). IL22RA2 was detected in
breast, lung, colon, skin, placenta, spleen, mononuclear cells, and
to a lesser extent in several other tissues (Dumoutier et al, 2001
supra; Xu et al, 2001 supra; Kotenko et al, 2001 supra). Highest
IL22RA2 expression occurred in mononuclear cells, plasma cells, and
epithelium in inflammatory infiltration sites. IL22RA2 expression
was also detected in certain types of tumors (see U.S. Patent
Application Publication No. US-2002-0137909-A1 published Sep. 26,
2002).
[0963] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Disruption of the target gene was confirmed
by Southern hybridization analysis. Level I phenotypic analysis was
performed on mice from this generation.
TABLE-US-00043 wt het hom Total Observed 22 52 23 97 Expected 24.25
48.5 24.25 97 Chi-Sq. = 0.53 Significance = 0.76883 (hom/n) = 0.24
Avg. Litter Size = 0
[0964] 1. Phenotypic Analysis (for Disrupted Gene: DNA145887-2849
(UNQ5793)
[0965] (a) Overall Phenotypic Summary
[0966] Mutant (-/-) mice exhibited elevation of serum
immunoglobulins compared to their gender-matched (+/+) littermates.
In addition, (-/-) mice exhibited elevated levels of alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) levels
when the mice after inducing a hepatitis phenotype with ConA
compared to the controls and wild-type littermates.
[0967] (b) Immunology Phenotypic Analysis
[0968] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0969] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0970] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines; which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0971] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0972] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc.
[0973] In the area of immunology, targets were identified herein
for the treatment of inflammation and inflammatory disorders.
Immune related diseases could be treated by suppressing the immune
response. Using neutralizing antibodies that inhibit molecules
having immune stimulatory activity would be beneficial in the
treatment of immune-mediated and inflammatory diseases. Molecules
which inhibit the immune response can be utilized (proteins
directly or via the use of antibody agonists) to inhibit the immune
response and thus ameliorate immune related disease.
[0974] Serum Immunoglobulin Isotyping Assay:
[0975] The Serum Immunoglobulin Isotyping Assay was performed using
a Cytometric Bead Array (CBA) kit. This assay was used to rapidly
identify the heavy and light chain isotypes of a mouse monoclonal
antibody in a single sample. The values expressed are "relative
fluorescence units" and are based on the detection of kappa light
chains. Any value <6 is not significant.
[0976] Results:
[0977] Mutant (-/-) mice exhibited elevation of IgM, IgG1, IgG2a
and IgG2b serum immunoglobulins compared to their gender-matched
(+/+) littermates. IgM immunoglobulins are the first to be produced
in a humoral immune response for neutralization of bacterial toxins
and are particularly important in activating the complement system.
Likewise, IgG immunoglobulins have neutralization effects and to a
lesser extent are important for activation of the complement
system. The observed phenotype suggests that the PRO19598
polypeptide is a negative regulator of inflammatory responses.
These immunological abnormalities suggest that inhibitors
(antagonists) of PRO19598 polypeptides would be useful in
stimulating the immune system (such as T cell proliferation) and
would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, PRO9783 polypeptides or agonists
thereof would be useful in inhibiting the immune response and would
be useful candidates for suppressing harmful immune responses, e.g.
in the case of graft rejection or graft-versus-host diseases.
[0978] ConA-Induced Hepatitis
[0979] Studies were performed to see the effects of ConA-induced
hepatitis on homozygous and heterozygous mutant mice in comparison
to their wild-type gender-matched littermates.
[0980] Procedure:
[0981] The effect of ConA-induced hepatitis was studied in UNQ5793
knockout mice. Groups used for this study include wild-type (+/+)
mice, heterozygous (+/-) mice, UNQ5793 homozygous (-/-) mice, and
untreated C57B16 mice. All mice except for the C57B16 control group
were injected with 10 mg/kg of ConA in 100 ul saline by intravenous
injection. Sixteen hours later, mice were sacrificed and serum was
collected for analysis of alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) levels. Serum was diluted 1:3 in
water and placed on a Roche reagent cassette and read using a
Chemistry analyzer (Integra 400). Livers were also collected at 16
hours. The liver samples were fixed and stained with hematoxylin
and eosin in order to determine the extent of liver damage.
[0982] Results:
[0983] Both the homozygous (-/-) and heterozygous (+/-) mutant mice
exhibited significantly elevated levels of serum alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) 16
hours after injection with ConA compared with untreated C57B16
controls and their wild-type (+/+) gender-matched littermates. In
addition, histological findings showed significant liver damage in
the mutant (+/-) and (-/-) mice compared with the untreated
controls and wild-type littermates. The knockout (-/-) mice showed
marked to severe multifocal bridging acute hepatocellular necrosis.
Likewise, the heterozygotes (+/-) showed marked to moderate
multifocal acute hepatocellular necrosis indicative of extensive
liver damage. Thus, the mutant knockout mice appeared to have an
exacerbated liver damage when hepatitis was induced with ConA,
suggestive of an enhanced inflammatory response. These studies
indicate that PRO9783 polypeptides or agonists thereof would be
useful in inhibiting or suppressing harmful immune responses and in
particular would be useful in the treatment of hepatitis.
[0984] N. Generation and Analysis of Mice Comprising DNA50921-1458
(UNQ540) Gene Disruptions
[0985] In these knockout experiments, the gene encoding PRO1083
polypeptides (designated as DNA50921-1458 (UNQ540) was disrupted.
The gene specific information for these studies is as follows:
mouse gene sequence nucleotide reference: NM.sub.--018882 or Mus
musculus G protein-coupled receptor 56 (Gpr56), protein reference:
NP.sub.--061370 or serpentine receptor; secretin receptor [Mus
musculus]; human gene sequence nucleotide reference:
NM.sub.--005682 or Homo sapiens G protein-coupled receptor 56
(GPR56), protein reference: NP.sub.--005673 or G protein-coupled
receptor 56; EGF-TM7-like [Homo sapiens].
[0986] The gene that is mutated in these animals is represented by
mouse UniGene cluster Mm.13799, which is the ortholog of human
UniGene cluster Hs.6527 (GPR56). GPR56 encodes G protein-coupled
receptor 56. The protein is also known as Cyt28, TM7LN4, TM7XN1,
secretin receptor, serpentine receptor, and EGF-TM7-like. GPR56 is
strongly expressed in hippocampus and hypothalamic nuclei (Liu et
al., Genomics, 55(3):296-305 (1999)) and was strongly downregulated
in highly metastatic cell lines (Zendman et al, FEBS Lett.,
446(2-3):292-8 (Mar. 12, 1999)). The presence of an extracellular
mucin-like domain suggests a possible role for the protein in
cell-cell interactions.
[0987] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation. Wild-type expression of the target gene
was detected in embryonic stem (ES) cells and in all 13 adult
tissue samples tested by RT-PCR, except adipose. Disruption of the
target gene was confirmed by Southern hybridization analysis.
TABLE-US-00044 wt het hom Total Observed 14 43 16 73 Expected 18.25
36.5 18.25 73 Chi-Sq. = 2.42 Significance = 0.29750 (hom/n) = 0.22
Avg. Litter Size = 0
[0988] 1. Phenotypic Analysis (for Disrupted Gene: DNA50921-1458
(UNQ540)
[0989] (a) Overall Phenotypic Summary
[0990] The (-/-) mice exhibited an increased mean serum IgM level
and notably increased mean serum IgA and IgG3 levels when compared
with their (+/+) littermates and the historical means. The female
(-/-) mice exhibited slightly increased mean total tissue mass,
lean body mass, bone mineral content, and total body and femoral
bone mineral density when compared with their gender-matched (+/+)
littermates.
[0991] (b) Immunology Phenotypic Analysis
[0992] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0993] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0994] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0995] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0996] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc.
[0997] In the area of immunology, targets were identified herein
for the treatment of inflammation and inflammatory disorders.
Immune related diseases could be treated by suppressing the immune
response. Using neutralizing antibodies that inhibit molecules
having immune stimulatory activity would be beneficial in the
treatment of immune-mediated and inflammatory diseases. Molecules
which inhibit the immune response can be utilized (proteins
directly or via the use of antibody agonists) to inhibit the immune
response and thus ameliorate immune related disease.
[0998] Serum Immunoglobulin Isotyping Assay:
[0999] The Serum Immunoglobulin Isotyping Assay was performed using
a Cytometric Bead Array (CBA) kit. This assay was used to rapidly
identify the heavy and light chain isotypes of a mouse monoclonal
antibody in a single sample. The values expressed are "relative
fluorescence units" and are based on the detection of kappa light
chains. Any value <6 is not significant.
[1000] Results:
[1001] Mutant (-/-) mice exhibited elevation of IgM, IgA, and IgG3
serum immunoglobulins compared to their gender-matched (+/+)
littermates. IgM immunoglobulins are the first to be produced in a
humoral immune response for neutralization of bacterial toxins and
are particularly important in activating the complement system.
Likewise, IgG immunoglobulins have neutralization effects and to a
lesser extent are important for activation of the complement
system. IgA mainly functions as an epithelial cell protector which
can neutralize bacterial toxins and viruses. Although no obvious
disease susceptibility is associated with selective IgA defects,
they are commoner in people with chronic lung disease than in the
general population. This suggests that lack of IgA may result in a
predisposition to lung infections with various pathogens and is
consistent with the role of IgA in defense at the body surfaces. In
this case, the phenotype observed for knockout mice resulted in an
increase in IgA serum levels suggesting that inhibitors
(antagonists) of PRO1083 polypeptides would be useful for natural
immunity protection against skin infections and more importantly
would prevent susceptibility to lung infections. The observed
phenotype suggests that the PRO1083 polypeptide is a negative
regulator of inflammatory responses. These immunological
abnormalities suggest that inhibitors (antagonists) of PRO1083
polypeptides would be useful as important agents which could
stimulate the immune system (such as T cell proliferation) and
would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, PRO1083 polypeptides or agonists
thereof would be useful in inhibiting the immune response and would
be useful candidates for suppressing harmful immune responses, e.g.
in the case of graft rejection or graft-versus-host diseases.
[1002] (c) Bone Metabolism: Radiology Phenotypic Analysis
[1003] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included: [1004] DEXA for measurement of bone
mineral density on femur and vertebra [1005] MicroCT for very high
resolution and very high sensitivity measurements of bone mineral
density for both trabecular and cortical bone.
[1006] Dexa Analysis--Test Description:
[1007] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1008] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1009] DEXA Results:
[1010] The female (-/-) mice exhibited increased mean total tissue
mass, lean body mass, bone mineral content, and total body and
femoral bone mineral density when compared with their
gender-matched (+/+) littermates. These results indicate that the
knockout mutant phenotype may be associated with such bone
abnormalities as osteopetrosis. Osteopetrosis is a condition
characterized by abnormal thickening and hardening of bone and
abnormal fragility of the bones. As such, PRO1083 polypeptides or
agonists thereof would be useful for the treatment of
osteopetrosis. A phenotype associated with an increased bone
mineral content, and total body and femoral bone mineral density
suggests that agents which mimic these effects (e.g. antagonists of
PRO1083 polypeptides) would be useful in bone healing.
[1011] O. Generation and Analysis of Mice Comprising DNA226659
(UNQ5070) Gene Disruptions
[1012] In these knockout experiments, the gene encoding hu TRPM2
polypeptides (designated as DNA226659 (UNQ5070) was disrupted. The
gene specific information for these studies is as follows: mouse
gene sequence nucleotide reference: NM.sub.--138301 or Mus musculus
transient receptor potential cation channel, subfamily M, member 2
(Trpm2), protein reference: NP.sub.--612174 or transient receptor
potential cation channel, subfamily M, member 2; transient receptor
potential channel 7; transient receptor protein 7 [Mus musculus];
human gene sequence nucleotide reference: NM.sub.--003307 or Homo
sapiens transient receptor potential cation channel, subfamily M,
member 2 (TRPM2), protein reference: NP.sub.--003298 or transient
receptor potential cation channel, subfamily M, member 2; transient
receptor potential-related channel 7, a novel putative Ca2+ channel
protein; transient receptor potential channel 7 [Homo sapiens].
[1013] The disrupted mouse gene is transient receptor potential
cation channel, subfamily M, member 2 (Trpm2), ortholog of human
transient receptor potential cation channel, subfamily M, member 2
(TRPM2). Aliases include TRPC7; Trrp7; LTRPC2; transient receptor
protein 7; transient receptor potential channel 7; KNP3; and
transient receptor potential-related channel 7, a novel putative
Ca2+ channel protein.
[1014] TRPM2, a member of the short subfamily of transient receptor
potential channels, functions as a Ca2+-permeable nonselective
cation channel highly expressed in brain and immunocytes (OMIM
603749). The protein contains an ion transport protein domain (Pfam
PF00520) and a NUDIX domain (Pfam PF00293), which functions as a
specific ADP-ribose pyrophosphatase.
[1015] ADP-ribose, which is a breakdown product of the
calcium-release second messenger cyclic ADP-ribose and NAD
hydrolysis, activates TRPM2 by binding to the C-terminal Nudix
domain. ADP-ribose-activated TRPM2 is likely to play an important
role in triggering immune responses in certain types of immune
cells (Sano et al., Science, 293(5533):1327-30 (Aug. 17, 2001);
Perraud et al, Nature, 411(6837):595-9 (May 31, 2001)).
[1016] A specific TRPM2 variant lacking the C-terminal Nudix domain
is activated by hydrogen peroxide but not ADP-ribose and is thus
activated by oxidative stress. This variant is expressed in
neutrophil granulocytes and is likely to play a role in respiratory
burst, which generates high concentrations of superoxide anions and
free radicals (Wehage et al, J. Biol. Chem., 277(26):23150-6
(2002)). Moreover, the TRPM2 variant with the Nudix domain is also
activated by hydrogen peroxide. Since it is widely expressed, this
TRPM2 variant may play a more general role in necrosis and
apoptosis in response to oxidative stress (Hara et al, Mol. Cell.,
2(11:163-73 (2002)).
[1017] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells: The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation. Wild-type expression of the target gene
was detected in embryonic stem (ES) cells and, among the 19 adult
tissue samples tested by RT-PCR, in spleen, liver, bone, stomach,
small intestine and colon, skin fibroblast, prostate, heart, and
aortic tree. Disruption of the target gene was confirmed by
Southern hybridization analysis.
TABLE-US-00045 wt het hom Total Observed 22 43 22 87 Expected 21.75
43.5 21.75 87 Chi-Sq. = 0.01 Significance = 0.99427 (hom/n) = 0.25
Avg. Litter Size = 0
[1018] 1. Phenotypic Analysis (for Disrupted Gene: DNA226659
(UNQ5070)
[1019] (a) Overall Phenotypic Summary
[1020] The male (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, thickness, and connectivity density and
decreased mean femoral midshaft cortical thickness and
cross-sectional area when compared with their gender-matched (+/+)
littermates and the historical means.
[1021] (b) Bone Metabolism: Radiology Phenotypic Analysis
[1022] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included: [1023] DEXA for measurement of bone
mineral density on femur and vertebra [1024] MicroCT for very high
resolution and very high sensitivity measurements of bone mineral
density for both trabecular and cortical bone.
[1025] Dexa Analysis--Test Description:
[1026] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1027] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1028] Bone micro CT Analysis:
[1029] Procedure:
[1030] MicroCT was also used to get very sensitive measurements of
BMD. One vertebra and 1 femur were taken from a cohort of 4 wild
type and 8 homozygous mice. Measurements were taken of lumbar 5
veterbra traebecular bone volume, traebecular thickness,
connectivity density and midshaft femur total bone area and
cortical thickness. The 4CT40 scans provided detailed information
on bone mass and architecture. Multiple bones were placed into
sample holders and scanned automatically. Instrument software was
used to select regions of interest for analysis. Trabecular bone
parameters were analyzed in the fifth lumbar vertebrae (LV5) at 16
micrometer resolution and cortical bone parameters were analyzed in
the femur midshaft at a resolution of 20 micrometers.
[1031] Micro-CT Analysis Results: The male (-/-) mice exhibited
notably decreased mean lumbar 5 vertebral trabecular bone volume,
number, thickness, and connectivity density when compared with
their gender-matched (+/+) littermates and the historical means.
These mutants also exhibited notably decreased mean femoral
midshaft cortical thickness and cross-sectional area when compared
with their gender-matched (+/+) littermates and the historical
mean. [Analyzed wt/het/horn: 5/4/8]
[1032] These results demonstrate that knockout mutant male mice
deficient in the gene encoding hu TRPM2 polypeptides exhibit
abnormal bone metabolism with significant bone loss characterized
by a decrease in bone mass with decreased density and possibly
fragility leading to bone fractures. Thus, it appears that hu TRPM2
or its encoding gene would be useful in maintaining bone
homeostasis and would be important in bone healing or for the
treatment of arthritis or osteoporosis; whereas antagonists to hu
TRPM2 or its encoding gene would lead to abnormal or pathological
bone disorders including inflammatory diseases associated with
abnormal bone metabolism including arthritis, osteoporosis, and
osteopenia.
[1033] P. Generation and Analysis of Mice Comprising DNA83500-2506
(UNQ852) Gene Disruptions
[1034] In these knockout experiments, the gene encoding PRO1801
polypeptides (designated as DNA83500-2506) (UNQ852) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--021380 or Mus musculus interleukin 20 (1120); protein
reference: NP.sub.--067355 or interleukin 20; four helix bundle
cytokine 10 [Mus musculus]; the human gene sequence reference:
NM.sub.--018724 or Homo sapiens interleukin 20 (IL20); the human
protein sequence corresponds to reference: NP.sub.--061194 or
interleukin 20 [Homo sapiens]
[1035] The gene that is mutated in these animals is interleukin 20
(1120), which is the ortholog of human IL20. Aliases include IL-20,
IL10D, ZCYTO10, and four alpha helix cytokine.
[1036] IL20 is most closely related by sequence to several members
of the IL-10 family: IL-19, MDA7, and IL10 (OMIM 605619). Northern
blot studies demonstrate that the gene is expressed in skin, and
trachea. Overexpression of the gene in mouse caused neonatal death
as well as skin abnormalities. An IL20 receptor molecule is also
expressed in skin.
[1037] IL20 is reported to share receptor complexes with IL19 and
IL24, however the biological consequences of ligand-receptor
interactions are very different for the three cytokines. IL20 is
reported to enhance hematopoiesis, and has been proposed as a
target for inflammatory skin disease treatments.
[1038] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice. Level I phenotypic analysis was performed on
mice from this generation. Wild-type expression of the target gene
was detected in embryonic stem (ES) cells and, among the 19 adult
tissue samples tested by RT-PCR, in spleen, liver, bone, stomach,
small intestine and colon, skin fibroblast, prostate, heart, and
aortic tree. Disruption of the target gene was confirmed by
Southern hybridization analysis.
TABLE-US-00046 wt het hom Total Observed 18 34 18 70 Expected 17.5
35 17.5 70 Chi-Sq. = 0.06 Significance = 0.97183 (hom/n) = 0.26
Avg. Litter Size = 8
[1039] 41.1.1. Phenotypic Analysis (for Disrupted Gene:
DNA83500-2506) (UNQ852)
[1040] (a) Overall Phenotypic Summary:
[1041] Mutation of the gene encoding the ortholog of human
interleukin 20 (IL-20) resulted in a notably increased inflammatory
response in the (-/-) mice. Gene disruption was confirmed by
Southern blot.
[1042] (b) Immunology Phenotypic Analysis
[1043] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1044] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1045] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1046] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histologic examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1047] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc.
[1048] In the area of immunology, targets were identified herein
for the treatment of inflammation and inflammatory disorders.
Immune related diseases could be treated by suppressing the immune
response. Using neutralizing antibodies that inhibit molecules
having immune stimulatory activity would be beneficial in the
treatment of immune-mediated and inflammatory diseases. Molecules
which inhibit the immune response can be utilized (proteins
directly or via the use of antibody agonists) to inhibit the immune
response and thus ameliorate immune related disease.
[1049] DSS-Induced Colitis:
[1050] Both male wildtype and homozygous (-/-) mutant mice were
given 3% DSS (dextran sulfate sodium) in their drinking water from
day 0 to day 7. On day 7, the drinking water was replaced with
regular water. The mice were weighed and colons were analyzed by
histology on day 7 and day 14. The knockout (-/-) mice showed a
markedly decreased survival rate on Day 7 (60% survival) compared
to their wild-type littermate controls (100% survival). The
knockout (-/-) mice were much more susceptible to the DSS challenge
(inducing a colitis condition) and they exhibited an enhanced
inflammatory response compared to their gender-matched littermate
controls. Thus, PRO1801 polypeptides or agonists thereof would be
useful in the healing process of inflammatory conditions of the
colon such as occurs in colitis or irritable bowel disorders.
Example 19
Use of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 as a hybridization probe
[1051] The following method describes use of a nucleotide sequence
encoding a PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide as a hybridization probe.
[1052] DNA comprising the coding sequence of full-length or mature
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptides as disclosed herein is employed as a probe to
screen for homologous DNAs (such as those encoding
naturally-occurring variants of PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides) in human
tissue cDNA libraries or human tissue genomic libraries.
[1053] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO224-, PRO9783-,
PRO1108-, PRO34000-, PRO240-, PRO943-, hu A33-, PRO230-, PRO178-,
PRO1199-, PRO4333-, PRO1336-, PRO19598-, PRO1083-, hu TRPM2- or
PRO1801-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.
[1054] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides can
then be identified using standard techniques known in the art.
Example 20
Expression of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 in E. coli
[1055] This example illustrates preparation of an unglycosylated
form of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptides by recombinant expression in E.
coli.
[1056] The DNA sequence encoding a PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
coding region, lambda transcriptional terminator, and an argU
gene.
[1057] 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.
[1058] 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.
[1059] 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 protein can then be purified
using a metal chelating column under conditions that allow tight
binding of the protein.
[1060] PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 may be expressed in E. coli in a poly-His tagged
form, using the following procedure. The DNA encoding PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 metal 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) Ion galE rpoHts(htpRts) clpP(lacIq).
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
citrate.2H.sub.2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g
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.
[1061] 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 0.02 M, 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 metal 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 metal
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.
[1062] 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 50 to 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.
[1063] Fractions containing the desired folded PRO224, PRO9783,
PRO1.108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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.
Example 21
Expression of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 in Mammalian Cells
[1064] This example illustrates preparation of a potentially
glycosylated form of a PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide by recombinant
expression in mammalian cells.
[1065] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
DNA is ligated into pRK5 with selected restriction enzymes to allow
insertion of the PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 DNA using ligation methods
such as described in Sambrook et al., supra. The resulting vector
is called pRIC5-PRO224, pRK5-PRO9783, pRK5-PRO1108, pRIC5-PRO34000,
pRIC5-PRO240, pRK5-PRO943, pRK5-hu A33, pRIC5-PRO230, pRIC5-PRO178,
pRK5-PRO1199, pRK5-PRO4333, pRK5-PRO1336, pRK5-PRO19598,
pRK5-PRO1083, pRK5-hu TRPM2 or pRK5-PRO1801.
[1066] 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 fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-PRO224, pRK5-PRO9783, pRIC5-PRO1108, pRK5-PRO34000,
pRK5-PRO240, pRK5-PRO943, pRK5-hu A33, pRK5-PRO230, pRK5-PRO178,
pRK5-PRO1199, pRK5-PRO4333, pRK5-PRO1336, pRK5-PRO19598,
pRIC5-PRO1083, pRK5-hu TRPM2 or pRK5-PRO1801 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.3. To this mixture is added,
dropwise, 500 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.
[1067] 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.Ci/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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptides. The cultures containing
transfected cells may undergo further incubation (in serum free
medium) and the medium is tested in selected bioassays.
[1068] In an alternative technique, PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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-PRO224, pRK5-PRO9783, pRIC5-PRO1108,
pRK5-PRO34000, pRK5-PRO240, pRK5-PRO943, pRK5-hu A33, pRK5-PRO230,
pRK5-PRO178, pRK5-PRO1199, pRIC5-PRO4333, pRK5-PRO1336,
pRIC5-PRO19598, pRIC5-PRO1083, pRK5-hu TRPM2 or pRIC5-PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 can then be concentrated and
purified by any selected method, such as dialysis and/or column
chromatography.
[1069] In another embodiment, PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 can be expressed in CHO
cells. The pRK5-PRO224, pRK5-PRO9783, pRIC5-PRO1108, pRK5-PRO34000,
pRK5-PRO240, pRK5-PRO943, pRK5-hu A33, pRK5-PRO230, pRK5-PRO178,
pRK5-PRO1199, pRK5-PRO4333, pRK5-PRO1336, pRK5-PRO19598,
pRK5-PRO1083, pRK5-hu TRPM2 or pRK5-PRO1801 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 S-methionine. After determining the presence of
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 can then be concentrated and
purified by any selected method.
[1070] Epitope-tagged PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 may also be expressed in
host CHO cells. The PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 can then be concentrated and purified
by any selected method, such as by Ni.sup.2+-chelate affinity
chromatography.
[1071] PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[1072] 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.
[1073] 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.
[1074] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Qiagen),
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.
[1075] 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
filtered PS20 with 5% 0.2 .mu.m diafiltered fetal 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 3 L
production spinner is seeded at 1.2.times.10.sup.6 cells/mL. On day
0, the cell number pH ie 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.
[1076] 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.
[1077] 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.
Example 22
Expression of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 in Yeast
[1078] The following method describes recombinant expression of
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 in yeast.
[1079] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 from the ADH2/GAPDH
promoter. DNA encoding PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 and the promoter is inserted
into suitable restriction enzyme sites in the selected plasmid to
direct intracellular expression of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801. For secretion, DNA
encoding PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 can be cloned into the selected plasmid,
together with DNA encoding the ADH2/GAPDH promoter, a native
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801.
[1080] Yeast cells, such as yeast strain AB110, 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.
[1081] Recombinant PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
may further be purified using selected column chromatography
resins.
Example 23
Expression of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 in Baculovirus-Infected Insect
Cells
[1082] The following method describes recombinant expression of
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 in Baculovirus-infected insect cells.
[1083] The sequence coding for PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 or the
desired portion of the coding sequence of PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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.
[1084] 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).
[1085] Expressed poly-his tagged PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 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 .mu.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.280 baseline
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 PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
are pooled and dialyzed against loading buffer.
[1086] Alternatively, purification of the IgG tagged (or Fc tagged)
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
Example 24
Preparation of Antibodies that Bind PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801Polypeptides
[1087] This example illustrates preparation of monoclonal
antibodies which can specifically bind PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides.
[1088] 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptides, fusion proteins containing PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides, and
cells expressing recombinant PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides on the cell
surface. Selection of the immunogen can be made by the skilled
artisan without undue experimentation.
[1089] Mice, such as Balb/c, are immunized with the PRO224,
PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178,
PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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-PRO224, anti-PRO9783, anti-PRO1108, anti-PRO34000,
anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178,
anti-PRO1199, anti-PRO4333, anti-PRO1336, anti-PRO19598,
anti-PRO1083, anti-hu TRPM2 or anti-PRO1801 antibodies.
[1090] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801. 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.
[1091] The hybridoma cells will be screened in an ELISA for
reactivity against PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083; hu TRPM2 or PRO1801. Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 is within the skill in the art.
[1092] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO224, anti-PRO9783, anti-PRO1108,
anti-PRO34000, anti-PRO240, anti-PRO943, anti-hu A33, anti-PRO230,
anti-PRO178, anti-PRO1199, anti-PRO4333, anti-PRO1336,
anti-PRO19598, anti-PRO1083, anti-hu TRPM2 or anti-PRO1801
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 25
Purification of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 Polypeptides Using Specific
Antibodies
[1093] Native or recombinant PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides may be purified
by a variety of standard techniques in the art of protein
purification. For example, pro-PRO224, pro-PRO9783, pro-PRO1108,
pro-PRO34000, pro-PRO240, pro-PRO943, pro-hu A33, pro-PRO230,
pro-PRO178, pro-PRO1199, pro-PRO4333, pro-PRO1336, pro-PRO19598,
pro-PRO1083, pro-hu TRPM2 or pro-PRO1801 polypeptide, mature
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, or pre-PRO224, pre-PRO9783, pre-PRO1108,
pre-PRO34000, pre-PRO240, pre-PRO943, pre-hu A33, pre-PRO230,
pre-PRO178, pre-PRO1199, pre-PRO4333, pre-PRO1336, pre-PRO19598,
pre-PRO1083, pre-hu TRPM2 or pre-PRO1801 polypeptide is purified by
immunoaffinity chromatography using antibodies specific for the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide of interest. In general, an immunoaffinity
column is constructed by covalently coupling the anti-PRO224,
anti-PRO9783, anti-PRO1108, anti-PRO34000, anti-PRO240,
anti-PRO943, anti-hu A33, anti-PRO230, anti-PRO178, anti-PRO1199,
anti-PRO4333, anti-PRO1336, anti-PRO19598, anti-PRO1083, anti-hu
TRPM2 or anti-PRO1801 polypeptide antibody to an activated
chromatographic resin.
[1094] 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.
[1095] Such an immunoaffinity column is utilized in the
purification of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide by preparing a fraction
from cells containing PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide containing a signal sequence may be secreted in useful
quantity into the medium in which the cells are grown.
[1096] A soluble PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide-containing
preparation is passed over the immunoaffinity column, and the
column is washed under conditions that allow the preferential
absorbance of PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943,
hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598,
PRO1083, hu TRPM2 or PRO1801 polypeptide (e.g., high ionic strength
buffers in the presence of detergent). Then, the column is eluted
under conditions that disrupt antibody/PRO224, antibody/PRO9783,
antibody/PRO1108, antibody/PRO34000, antibody/PRO240,
antibody/PRO943, antibody/hu A33, antibody/PRO230, antibody/PRO178,
antibody/PRO1199, antibody/PRO4333, antibody/PRO1336,
antibody/PRO19598, antibody/PRO1083, antibody/hu TRPM2 or
antibody/PRO1801 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 PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide is
collected.
Example 26
Drug Screening
[1097] This invention is particularly useful for screening
compounds by using PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptides or binding
fragment thereof in any of a variety of drug screening techniques.
The PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide or a fragment and the agent being tested.
Alternatively, one can examine the diminution in complex formation
between the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide and its target cell or target
receptors caused by the agent being tested.
[1098] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide or
fragment thereof and assaying (i) for the presence of a complex
between the agent and the PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide or fragment, or
(ii) for the presence of a complex between the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide or fragment and the cell, by methods well known in the
art. In such competitive binding assays, the PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide or fragment is typically labeled. After suitable
incubation, free PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide or to interfere
with the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide/cell complex.
[1099] 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide, the peptide test compounds are
reacted with PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu
A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083,
hu TRPM2 or PRO1801 polypeptide and washed. Bound PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide is detected by methods well known in the art. Purified
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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.
[1100] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide specifically compete with a test
compound for binding to PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide.
Example 27
Rational Drug Design
[1101] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 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 PRO224, PRO9783, PRO1108, PRO34000, PRO240,
PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide or which enhance
or interfere with the function of the PRO224, PRO9783, PRO1108,
PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333,
PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801 polypeptide in vivo
(c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
[1102] In one approach, the three-dimensional structure of the
PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230,
PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or
PRO1801 polypeptide, or of a PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
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 PRO224, PRO9783,
PRO1108, PRO34000, PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199,
PRO4333, PRO1336, PRO19598, PRO1083, hu TRPM2 or PRO1801
polypeptide may be gained by modeling based on the structure of
homologous proteins. In both cases, relevant structural information
is used to design analogous PRO224, PRO9783, PRO1108, PRO34000,
PRO240, PRO943, hu A33, PRO230, PRO178, PRO1199, PRO4333, PRO1336,
PRO19598, PRO1083, hu TRPM2 or PRO1801 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).
[1103] 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.
[1104] By virtue of the present invention, sufficient amounts of
the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the PRO224, PRO9783, PRO1108, PRO34000, PRO240, PRO943, hu A33,
PRO230, PRO178, PRO1199, PRO4333, PRO1336, PRO19598, PRO1083, hu
TRPM2 or PRO1801 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.
Sequence CWU 1
1
7011210DNAHomo sapiens 1cagcgcgtgg ccggcgccgc tgtggggaca gcatgagcgg
cggttggatg 50gcgcaggttg gagcgtggcg aacaggggct ctgggcctgg cgctgctgct
100gctgctcggc ctcggactag gcctggaggc cgccgcgagc ccgctttcca
150ccccgacctc tgcccaggcc gcaggcccca gctcaggctc gtgcccaccc
200accaagttcc agtgccgcac cagtggctta tgcgtgcccc tcacctggcg
250ctgcgacagg gacttggact gcagcgatgg cagcgatgag gaggagtgca
300ggattgagcc atgtacccag aaagggcaat gcccaccgcc ccctggcctc
350ccctgcccct gcaccggcgt cagtgactgc tctgggggaa ctgacaagaa
400actgcgcaac tgcagccgcc tggcctgcct agcaggcgag ctccgttgca
450cgctgagcga tgactgcatt ccactcacgt ggcgctgcga cggccaccca
500gactgtcccg actccagcga cgagctcggc tgtggaacca atgagatcct
550cccggaaggg gatgccacaa ccatggggcc ccctgtgacc ctggagagtg
600tcacctctct caggaatgcc acaaccatgg ggccccctgt gaccctggag
650agtgtcccct ctgtcgggaa tgccacatcc tcctctgccg gagaccagtc
700tggaagccca actgcctatg gggttattgc agctgctgcg gtgctcagtg
750caagcctggt caccgccacc ctcctccttt tgtcctggct ccgagcccag
800gagcgcctcc gcccactggg gttactggtg gccatgaagg agtccctgct
850gctgtcagaa cagaagacct cgctgccctg aggacaagca cttgccacca
900ccgtcactca gccctgggcg tagccggaca ggaggagagc agtgatgcgg
950atgggtaccc gggcacacca gccctcagag acctgagttc ttctggccac
1000gtggaacctc gaacccgagc tcctgcagaa gtggccctgg agattgaggg
1050tccctggaca ctccctatgg agatccgggg agctaggatg gggaacctgc
1100cacagccaga actgaggggc tggccccagg cagctcccag ggggtagaac
1150ggccctgtgc ttaagacact ccctgctgcc ccgtctgagg gtggcgatta
1200aagttgcttc 12102282PRTHomo sapiens 2Met Ser Gly Gly Trp Met Ala
Gln Val Gly Ala Trp Arg Thr Gly1 5 10 15Ala Leu Gly Leu Ala Leu Leu
Leu Leu Leu Gly Leu Gly Leu Gly 20 25 30Leu Glu Ala Ala Ala Ser Pro
Leu Ser Thr Pro Thr Ser Ala Gln 35 40 45Ala Ala Gly Pro Ser Ser Gly
Ser Cys Pro Pro Thr Lys Phe Gln 50 55 60Cys Arg Thr Ser Gly Leu Cys
Val Pro Leu Thr Trp Arg Cys Asp 65 70 75Arg Asp Leu Asp Cys Ser Asp
Gly Ser Asp Glu Glu Glu Cys Arg 80 85 90Ile Glu Pro Cys Thr Gln Lys
Gly Gln Cys Pro Pro Pro Pro Gly 95 100 105Leu Pro Cys Pro Cys Thr
Gly Val Ser Asp Cys Ser Gly Gly Thr 110 115 120Asp Lys Lys Leu Arg
Asn Cys Ser Arg Leu Ala Cys Leu Ala Gly 125 130 135Glu Leu Arg Cys
Thr Leu Ser Asp Asp Cys Ile Pro Leu Thr Trp 140 145 150Arg Cys Asp
Gly His Pro Asp Cys Pro Asp Ser Ser Asp Glu Leu 155 160 165Gly Cys
Gly Thr Asn Glu Ile Leu Pro Glu Gly Asp Ala Thr Thr 170 175 180Met
Gly Pro Pro Val Thr Leu Glu Ser Val Thr Ser Leu Arg Asn 185 190
195Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val Pro Ser 200
205 210Val Gly Asn Ala Thr Ser Ser Ser Ala Gly Asp Gln Ser Gly Ser
215 220 225Pro Thr Ala Tyr Gly Val Ile Ala Ala Ala Ala Val Leu Ser
Ala 230 235 240Ser Leu Val Thr Ala Thr Leu Leu Leu Leu Ser Trp Leu
Arg Ala 245 250 255Gln Glu Arg Leu Arg Pro Leu Gly Leu Leu Val Ala
Met Lys Glu 260 265 270Ser Leu Leu Leu Ser Glu Gln Lys Thr Ser Leu
Pro 275 28031867DNAHomo sapiens 3acactggcca aacactcgca tcccagggcg
tctccggctg ctcccattga 50gctgtctgct cgctgtgccc gctgtgcctg ctgtgcccgc
gctgtcgccg 100ctgctaccgc gtctgctgga cgcgggagac gccagcgagc
tggtgattgg 150agccctgcgg agagctcaag cgcccagctc tgcccgagga
gcccaggctg 200ccccgtgagt cccatagttg ctgcaggagt ggagcctttc
cctttgcgat 250cgctaaacca ccatgagctg cgtcctgggt ggtgtcatcc
ccttggggct 300gctgttcctg gtctgcggat cccaaggcta cctcctgccc
aacgtcactc 350tcttagagga gctgctcagc aaataccagc acaacgagtc
tcactcccgg 400gtccgcagag ccatccccag ggaggacaag gaggagatcc
tcatgctgca 450caacaagctt cggggccagg tgcagcctca ggcctccaac
atggagtaca 500tgacctggga tgacgaactg gagaagtctg ctgcagcgtg
ggccagtcag 550tgcatctggg agcacgggcc caccagtctg ctggtgtcca
tcgggcagaa 600cctgggcgct cactggggca ggtatcgctc tccggggttc
catgtgcagt 650cctggtatga cgaggtgaag gactacacct acccctaccc
gagcgagtgc 700aacccctggt gtccagagag gtgctcgggg cctatgtgca
cgcactacac 750acagatagtt tgggccacca ccaacaagat cggttgtgct
gtgaacacct 800gccggaagat gactgtctgg ggagaagttt gggagaacgc
ggtctacttt 850gtctgcaatt attctccaaa ggggaactgg attggagaag
ccccctacaa 900gaatggccgg ccctgctctg agtgcccacc cagctatgga
ggcagctgca 950ggaacaactt gtgttaccga gaagaaacct acactccaaa
acctgaaacg 1000gacgagatga atgaggtgga aacggctccc attcctgaag
aaaaccatgt 1050ttggctccaa ccgagggtga tgagacccac caagcccaag
aaaacctctg 1100cggtcaacta catgacccaa gtcgtcagat gtgacaccaa
gatgaaggac 1150aggtgcaaag ggtccacgtg taacaggtac cagtgcccag
caggctgcct 1200gaaccacaag gcgaagatct ttggaagtct gttctatgaa
agctcgtcta 1250gcatatgccg cgccgccatc cactacggga tcctggatga
caagggaggc 1300ctggtggata tcaccaggaa cgggaaggtc cccttcttcg
tgaagtctga 1350gagacacggc gtgcagtccc tcagcaaata caaaccttcc
agctcattca 1400tggtgtcaaa agtgaaagtg caggatttgg actgctacac
gaccgttgct 1450cagctgtgcc cgtttgaaaa gccagcaact cactgcccaa
gaatccattg 1500tccggcacac tgcaaagacg aaccttccta ctgggctccg
gtgtttggaa 1550ccaacatcta tgcagatacc tcaagcatct gcaagacagc
tgtgcacgcg 1600ggagtcatca gcaacgagag tgggggtgac gtggacgtga
tgcccgtgga 1650taaaaagaag acctacgtgg gctcgctcag gaatggagtt
cagtctgaaa 1700gcctggggac tcctcgggat ggaaaggcct tccggatctt
tgctgtcagg 1750catttccctt tgcggccgcg tgaatttcca gcaccagggg
agaaggggcg 1800tcttcaggag ggcttcgggg ttttgctttt atttttattt
tgtcattgcg 1850gggtatatgg agagtca 18674497PRTHomo sapiens 4Met Ser
Cys Val Leu Gly Gly Val Ile Pro Leu Gly Leu Leu Phe1 5 10 15Leu Val
Cys Gly Ser Gln Gly Tyr Leu Leu Pro Asn Val Thr Leu 20 25 30Leu Glu
Glu Leu Leu Ser Lys Tyr Gln His Asn Glu Ser His Ser 35 40 45Arg Val
Arg Arg Ala Ile Pro Arg Glu Asp Lys Glu Glu Ile Leu 50 55 60Met Leu
His Asn Lys Leu Arg Gly Gln Val Gln Pro Gln Ala Ser 65 70 75Asn Met
Glu Tyr Met Thr Trp Asp Asp Glu Leu Glu Lys Ser Ala 80 85 90Ala Ala
Trp Ala Ser Gln Cys Ile Trp Glu His Gly Pro Thr Ser 95 100 105Leu
Leu Val Ser Ile Gly Gln Asn Leu Gly Ala His Trp Gly Arg 110 115
120Tyr Arg Ser Pro Gly Phe His Val Gln Ser Trp Tyr Asp Glu Val 125
130 135Lys Asp Tyr Thr Tyr Pro Tyr Pro Ser Glu Cys Asn Pro Trp Cys
140 145 150Pro Glu Arg Cys Ser Gly Pro Met Cys Thr His Tyr Thr Gln
Ile 155 160 165Val Trp Ala Thr Thr Asn Lys Ile Gly Cys Ala Val Asn
Thr Cys 170 175 180Arg Lys Met Thr Val Trp Gly Glu Val Trp Glu Asn
Ala Val Tyr 185 190 195Phe Val Cys Asn Tyr Ser Pro Lys Gly Asn Trp
Ile Gly Glu Ala 200 205 210Pro Tyr Lys Asn Gly Arg Pro Cys Ser Glu
Cys Pro Pro Ser Tyr 215 220 225Gly Gly Ser Cys Arg Asn Asn Leu Cys
Tyr Arg Glu Glu Thr Tyr 230 235 240Thr Pro Lys Pro Glu Thr Asp Glu
Met Asn Glu Val Glu Thr Ala 245 250 255Pro Ile Pro Glu Glu Asn His
Val Trp Leu Gln Pro Arg Val Met 260 265 270Arg Pro Thr Lys Pro Lys
Lys Thr Ser Ala Val Asn Tyr Met Thr 275 280 285Gln Val Val Arg Cys
Asp Thr Lys Met Lys Asp Arg Cys Lys Gly 290 295 300Ser Thr Cys Asn
Arg Tyr Gln Cys Pro Ala Gly Cys Leu Asn His 305 310 315Lys Ala Lys
Ile Phe Gly Ser Leu Phe Tyr Glu Ser Ser Ser Ser 320 325 330Ile Cys
Arg Ala Ala Ile His Tyr Gly Ile Leu Asp Asp Lys Gly 335 340 345Gly
Leu Val Asp Ile Thr Arg Asn Gly Lys Val Pro Phe Phe Val 350 355
360Lys Ser Glu Arg His Gly Val Gln Ser Leu Ser Lys Tyr Lys Pro 365
370 375Ser Ser Ser Phe Met Val Ser Lys Val Lys Val Gln Asp Leu Asp
380 385 390Cys Tyr Thr Thr Val Ala Gln Leu Cys Pro Phe Glu Lys Pro
Ala 395 400 405Thr His Cys Pro Arg Ile His Cys Pro Ala His Cys Lys
Asp Glu 410 415 420Pro Ser Tyr Trp Ala Pro Val Phe Gly Thr Asn Ile
Tyr Ala Asp 425 430 435Thr Ser Ser Ile Cys Lys Thr Ala Val His Ala
Gly Val Ile Ser 440 445 450Asn Glu Ser Gly Gly Asp Val Asp Val Met
Pro Val Asp Lys Lys 455 460 465Lys Thr Tyr Val Gly Ser Leu Arg Asn
Gly Val Gln Ser Glu Ser 470 475 480Leu Gly Thr Pro Arg Asp Gly Lys
Ala Phe Arg Ile Phe Ala Val 485 490 495Arg Gln 52359DNAHomo sapiens
5ctgtcaggaa ggaccatctg aaggctgcaa tttgttctta gggaggcagg
50tgctggcctg gcctggatct tccaccatgt tcctgttgct gccttttgat
100agcctgattg tcaaccttct gggcatctcc ctgactgtcc tcttcaccct
150ccttctcgtt ttcatcatag tgccagccat ttttggagtc tcctttggta
200tccgcaaact ctacatgaaa agtctgttaa aaatctttgc gtgggctacc
250ttgagaatgg agcgaggagc caaggagaag aaccaccagc tttacaagcc
300ctacaccaac ggaatcattg caaaggatcc cacttcacta gaagaagaga
350tcaaagagat tcgtcgaagt ggtagtagta aggctctgga caacactcca
400gagttcgagc tctctgacat tttctacttt tgccggaaag gaatggagac
450cattatggat gatgaggtga caaagagatt ctcagcagaa gaactggagt
500cctggaacct gctgagcaga accaattata acttccagta catcagcctt
550cggctcacgg tcctgtgggg gttaggagtg ctgattcggt actgctttct
600gctgccgctc aggatagcac tggctttcac agggattagc cttctggtgg
650tgggcacaac tgtggtggga tacttgccaa atgggaggtt taaggaattc
700atgagtaaac atgttcactt aatgtgttac cggatctgcg tgcgagcgct
750gacagccatc atcacctacc atgacaggga aaacagacca agaaatggtg
800gcatctgtgt ggccaatcat acctcaccga tcgatgtgat catcttggcc
850agcgatggct attatgccat ggtgggtcaa gtgcacgggg gactcatggg
900tgtgattcag agagccatgg tgaaggcctg cccacacgtc tggtttgagc
950gctcggaagt gaaggatcgc cacctggtgg ctaagagact gactgaacat
1000gtgcaagata aaagcaagct gcctatcctc atcttcccag aaggaacctg
1050catcaataat acatcggtga tgatgttcaa aaagggaagt tttgaaattg
1100gagccacagt ttaccctgtt gctatcaagt atgaccctca atttggcgat
1150gccttctgga acagcagcaa atacgggatg gtgacgtacc tgctgcgaat
1200gatgaccagc tgggccattg tctgcagcgt gtggtacctg cctcccatga
1250ctagagaggc agatgaagat gctgtccagt ttgcgaatag ggtgaaatct
1300gccattgcca ggcagggagg acttgtggac ctgctgtggg atgggggcct
1350gaagagggag aaggtgaagg acacgttcaa ggaggagcag cagaagctgt
1400acagcaagat gatcgtgggg aaccacaagg acaggagccg ctcctgagcc
1450tgcctccagc tggctggggc caccgtgcgg ggtgccaacg ggctcagagc
1500tggagttgcc gccgccgccc ccactgctgt gtcctttcca gactccaggg
1550ctccccgggc tgctctggat cccaggactc cggctttcgc cgagccgcag
1600cgggatccct gtgcacccgg cgcagcctac ccttggtggt ctaaacggat
1650gctgctgggt gttgcgaccc aggacgagat gccttgtttc ttttacaata
1700agtcgttgga ggaatgccat taaagtgaac tccccacctt tgcacgctgt
1750gcgggctgag tggttgggga gatgtggcca tggtcttgtg ctagagatgg
1800cggtacaaga gtctgttatg caagcccgtg tgccagggat gtgctggggg
1850cggccacccg ctctccagga aaggcacagc tgaggcactg tggctggctt
1900cggcctcaac atcgccccca gccttggagc tctgcagaca tgataggaag
1950gaaactgtca tctgcagggg ctttcagcaa aatgaagggt tagattttta
2000tgctgctgct gatggggtta ctaaagggag gggaagaggc caggtgggcc
2050gctgactggg ccatggggag aacgtgtgtt cgtactccag gctaaccctg
2100aactccccat gtgatgcgcg ctttgttgaa tgtgtgtctc ggtttcccca
2150tctgtaatat gagtcggggg gaatggtggt gattcctacc tcacagggct
2200gttgtgggga ttaaagtgct gcgggtgagt gaaggacaca tcacgttcag
2250tgtttcaagt acaggcccac aaaacggggc acggcaggcc tgagctcaga
2300gctgctgcac tgggctttgg atttgttctt gtgagtaaat aaaactggct
2350ggtgaatga 23596456PRTHomo sapiens 6Met Phe Leu Leu Leu Pro Phe
Asp Ser Leu Ile Val Asn Leu Leu1 5 10 15Gly Ile Ser Leu Thr Val Leu
Phe Thr Leu Leu Leu Val Phe Ile 20 25 30Ile Val Pro Ala Ile Phe Gly
Val Ser Phe Gly Ile Arg Lys Leu 35 40 45Tyr Met Lys Ser Leu Leu Lys
Ile Phe Ala Trp Ala Thr Leu Arg 50 55 60Met Glu Arg Gly Ala Lys Glu
Lys Asn His Gln Leu Tyr Lys Pro 65 70 75Tyr Thr Asn Gly Ile Ile Ala
Lys Asp Pro Thr Ser Leu Glu Glu 80 85 90Glu Ile Lys Glu Ile Arg Arg
Ser Gly Ser Ser Lys Ala Leu Asp 95 100 105Asn Thr Pro Glu Phe Glu
Leu Ser Asp Ile Phe Tyr Phe Cys Arg 110 115 120Lys Gly Met Glu Thr
Ile Met Asp Asp Glu Val Thr Lys Arg Phe 125 130 135Ser Ala Glu Glu
Leu Glu Ser Trp Asn Leu Leu Ser Arg Thr Asn 140 145 150Tyr Asn Phe
Gln Tyr Ile Ser Leu Arg Leu Thr Val Leu Trp Gly 155 160 165Leu Gly
Val Leu Ile Arg Tyr Cys Phe Leu Leu Pro Leu Arg Ile 170 175 180Ala
Leu Ala Phe Thr Gly Ile Ser Leu Leu Val Val Gly Thr Thr 185 190
195Val Val Gly Tyr Leu Pro Asn Gly Arg Phe Lys Glu Phe Met Ser 200
205 210Lys His Val His Leu Met Cys Tyr Arg Ile Cys Val Arg Ala Leu
215 220 225Thr Ala Ile Ile Thr Tyr His Asp Arg Glu Asn Arg Pro Arg
Asn 230 235 240Gly Gly Ile Cys Val Ala Asn His Thr Ser Pro Ile Asp
Val Ile 245 250 255Ile Leu Ala Ser Asp Gly Tyr Tyr Ala Met Val Gly
Gln Val His 260 265 270Gly Gly Leu Met Gly Val Ile Gln Arg Ala Met
Val Lys Ala Cys 275 280 285Pro His Val Trp Phe Glu Arg Ser Glu Val
Lys Asp Arg His Leu 290 295 300Val Ala Lys Arg Leu Thr Glu His Val
Gln Asp Lys Ser Lys Leu 305 310 315Pro Ile Leu Ile Phe Pro Glu Gly
Thr Cys Ile Asn Asn Thr Ser 320 325 330Val Met Met Phe Lys Lys Gly
Ser Phe Glu Ile Gly Ala Thr Val 335 340 345Tyr Pro Val Ala Ile Lys
Tyr Asp Pro Gln Phe Gly Asp Ala Phe 350 355 360Trp Asn Ser Ser Lys
Tyr Gly Met Val Thr Tyr Leu Leu Arg Met 365 370 375Met Thr Ser Trp
Ala Ile Val Cys Ser Val Trp Tyr Leu Pro Pro 380 385 390Met Thr Arg
Glu Ala Asp Glu Asp Ala Val Gln Phe Ala Asn Arg 395 400 405Val Lys
Ser Ala Ile Ala Arg Gln Gly Gly Leu Val Asp Leu Leu 410 415 420Trp
Asp Gly Gly Leu Lys Arg Glu Lys Val Lys Asp Thr Phe Lys 425 430
435Glu Glu Gln Gln Lys Leu Tyr Ser Lys Met Ile Val Gly Asn His 440
445 450Lys Asp Arg Ser Arg Ser 45573493DNAHomo sapiens 7ctccctttca
tctggtggcc ctagcgccac aagctgccgc ttaggaagtc 50cctgccggga gcagaagtgg
agacatcagc aggatggcat cggcaagtcg 100ctcccctccc gggcctcatc
tgccaaacga tcatctcctc ctccgaagtt 150gtatgcatga caggcgagtg
gaaacttcac taaaatgaag gcgattgaca 200caacagaagg aactccatcc
tttcgggggc ttacgaaaat
aataagttta 250aaaaaaatag gaagggaatt ccctcgctcc atgatcactg
agcgctctcc 300taaggaaaag gaaatctccc ggggggtgcc gactacgggc
ggcgggctta 350ggatgctccc acgctccccg acccccaatc cccaggaccc
gcaggacctc 400cggaggaacg cccgccagcc cgcccggagc cacgcggcac
aaggtgacac 450ggaccgcgcc gcgcgggccc ctcagccgcc tgggcgaggc
cgggagcagg 500gagaggggca tccgccggcc cgcggtacct tgtacttatc
aaagccagcc 550agctgctccg ggctcacgta ttcgtagcca gccatgacga
cccgaaaact 600gagcgcccac tcggcagcga ctcccggcta caaggctgtg
acacacaagc 650accacaccgg ctgggcaagg atggcaaaga ctgggctgcc
cgagaaggga 700cagagtcagg ctggagggga atctggatct gggcagctcc
tggaccaaga 750gaatggagca ggggaatcag cgctggtctc cgtctatgta
catctggact 800ttccagataa gacctggccc cctgaactct ccaggacact
gactctccct 850gctgcctcag cttcctcttc cccaaggcct cttctcactg
gcctcagact 900cacaacaggt gagtacatga gctgcttcga ggcccagggc
ttcaagtgga 950acctgtatga ggtggtgagg gtgcccttga aggcgacaga
tgtggctcga 1000cttccatacc agctgtccat ctcctgtgtc acctcccctg
gcttccagct 1050gagctgctgc atccccagca caaacctggc ctacaccgcg
gcctggagcc 1100ctggagaggg cagcaaagct tcctccttca acgagtcagg
ctctcagtgc 1150tttgtgctgg ctgttcagcg ctgcccgatg gctgacacca
cgtacacttg 1200tgacctgcag agcctgggcc tggctccact cagggtcccc
atctccatca 1250ccatcatcca ggatggagac atcacctgcc ctgaggacgc
ctcggtgctc 1300acctggaatg tcaccaaggc tggccacgtg gcacaggccc
catgtcctga 1350gagcaagagg ggcatagtga ggaggctctg tggggctgac
ggagtctggg 1400ggccggtcca cagcagctgc acagatgcga ggctcctggc
cttgttcact 1450agaaccaagc tgctgcaggc aggccagggc agtcctgctg
aggaggtgcc 1500acagatcctg gcacagctgc cagggcaggc ggcagaggca
agttcaccct 1550ccgacttact gaccctgctg agcaccatga aatacgtggc
caaggtggtg 1600gcagaggcca gaatacagct tgaccgcaga gccctgaaga
atctcctgat 1650tgccacagac aaggtcctag atatggacac caggtctctg
tggaccctgg 1700cccaagcccg gaagccctgg gcaggctcga ctctcctgct
ggctgtggag 1750accctggcat gcagcctgtg cccacaggac taccccttcg
ccttcagctt 1800acccaatgtg ctgctgcaga gccagctgtt tggacccacg
tttcctgctg 1850actacagcat ctccttccct actcgtcccc cactgcaggc
tcagattccc 1900aggcactcac tggccccatt ggtccgtaat ggaactgaaa
taagtattac 1950tagcctggtg ctgcgaaaac tggaccacct tctgccctca
aactatggac 2000aagggctggg ggattccctc tatgccactc ctggcctggt
ccttgtcatt 2050tccatcatgg caggtgaccg ggccttcagc cagggagagg
tcatcatgga 2100ctttgggaac acagatggtt cccctcactg tgtcttctgg
gatcacagtc 2150tcttccaggg cagggggggt tggtccaaag aagggtgcca
ggcacaggtg 2200gccagtgcca gccccactgc tcagtgcctc tgccagcacc
tcactgcctt 2250ctccgtcctc atgtccccac acactgttcc ggaagaaccc
gctctggcgc 2300tgctgactca agtgggcttg ggagcttcca tactggcgct
gcttgtgtgc 2350ctgggtgtgt actggctggt gtggagagtc gtggtgcgga
acaagatctc 2400ctatttccgc cacgccgccc tgctcaacat ggtgttctgc
ttgctggccg 2450cagacacttg cttcctgggc gccccattcc tctctccagg
gccccgaagc 2500ccgctctgcc ttgctgccgc cttcctctgt catttcctct
acctggccac 2550ctttttctgg atgctggcgc aggccctggt gttggcccac
cagctgctat 2600ttgtctttca ccagctggca aagcaccgag ttctccccct
catggtgctc 2650ctgggctacc tgtgcccact ggggttggca ggtgtcaccc
tggggctcta 2700cctacctcaa gggcaatacc tgagggaggg ggaatgctgg
ttggatggga 2750agggaggggc gttatacacc ttcgtggggc cagtgctggc
catcataggc 2800gtgaatgggc tggtactagc catggccatg ctgaagttgc
tgagaccttc 2850gctgtcagag ggacccccag cagagaagcg ccaagctctg
ctgggggtga 2900tcaaagccct gctcattctt acacccatct ttggcctcac
ctgggggctg 2950ggcctggcca ctctgttaga ggaagtctcc acggtccctc
attacatctt 3000caccattctc aacaccctcc agggcgtctt catcctattg
tttggttgcc 3050tcatggacag gaagatacaa gaagctttgc gcaaacgctt
ctgccgcgcc 3100caagccccca gctccaccat ctccctggcc acaaatgaag
gctgcatctt 3150ggaacacagc aaaggaggaa gcgacactgc caggaagaca
gatgcttcag 3200agtgaaccac acacggaccc atgttcctgc aagggagttg
aggctgtgtg 3250cttgaaccca ccagatgagc cctggcccaa tgctctgaac
tcttcccgcc 3300tcccggagct cagcccttga gaaagttatg aagaaaggat
gacttacttg 3350acaggaacct ctgatctttc aaacattgga gatgaagggc
agaatttggt 3400ttgtcttttc aagtttagga aaaggtgaag ttaattggtc
cctctttctt 3450taacctttaa aaaatcaata taaaatgtaa gtttcttaac cat
34938873PRTHomo sapiens 8Met Thr Thr Arg Lys Leu Ser Ala His Ser
Ala Ala Thr Pro Gly1 5 10 15Tyr Lys Ala Val Thr His Lys His His Thr
Gly Trp Ala Arg Met 20 25 30Ala Lys Thr Gly Leu Pro Glu Lys Gly Gln
Ser Gln Ala Gly Gly 35 40 45Glu Ser Gly Ser Gly Gln Leu Leu Asp Gln
Glu Asn Gly Ala Gly 50 55 60Glu Ser Ala Leu Val Ser Val Tyr Val His
Leu Asp Phe Pro Asp 65 70 75Lys Thr Trp Pro Pro Glu Leu Ser Arg Thr
Leu Thr Leu Pro Ala 80 85 90Ala Ser Ala Ser Ser Ser Pro Arg Pro Leu
Leu Thr Gly Leu Arg 95 100 105Leu Thr Thr Gly Glu Tyr Met Ser Cys
Phe Glu Ala Gln Gly Phe 110 115 120Lys Trp Asn Leu Tyr Glu Val Val
Arg Val Pro Leu Lys Ala Thr 125 130 135Asp Val Ala Arg Leu Pro Tyr
Gln Leu Ser Ile Ser Cys Val Thr 140 145 150Ser Pro Gly Phe Gln Leu
Ser Cys Cys Ile Pro Ser Thr Asn Leu 155 160 165Ala Tyr Thr Ala Ala
Trp Ser Pro Gly Glu Gly Ser Lys Ala Ser 170 175 180Ser Phe Asn Glu
Ser Gly Ser Gln Cys Phe Val Leu Ala Val Gln 185 190 195Arg Cys Pro
Met Ala Asp Thr Thr Tyr Thr Cys Asp Leu Gln Ser 200 205 210Leu Gly
Leu Ala Pro Leu Arg Val Pro Ile Ser Ile Thr Ile Ile 215 220 225Gln
Asp Gly Asp Ile Thr Cys Pro Glu Asp Ala Ser Val Leu Thr 230 235
240Trp Asn Val Thr Lys Ala Gly His Val Ala Gln Ala Pro Cys Pro 245
250 255Glu Ser Lys Arg Gly Ile Val Arg Arg Leu Cys Gly Ala Asp Gly
260 265 270Val Trp Gly Pro Val His Ser Ser Cys Thr Asp Ala Arg Leu
Leu 275 280 285Ala Leu Phe Thr Arg Thr Lys Leu Leu Gln Ala Gly Gln
Gly Ser 290 295 300Pro Ala Glu Glu Val Pro Gln Ile Leu Ala Gln Leu
Pro Gly Gln 305 310 315Ala Ala Glu Ala Ser Ser Pro Ser Asp Leu Leu
Thr Leu Leu Ser 320 325 330Thr Met Lys Tyr Val Ala Lys Val Val Ala
Glu Ala Arg Ile Gln 335 340 345Leu Asp Arg Arg Ala Leu Lys Asn Leu
Leu Ile Ala Thr Asp Lys 350 355 360Val Leu Asp Met Asp Thr Arg Ser
Leu Trp Thr Leu Ala Gln Ala 365 370 375Arg Lys Pro Trp Ala Gly Ser
Thr Leu Leu Leu Ala Val Glu Thr 380 385 390Leu Ala Cys Ser Leu Cys
Pro Gln Asp Tyr Pro Phe Ala Phe Ser 395 400 405Leu Pro Asn Val Leu
Leu Gln Ser Gln Leu Phe Gly Pro Thr Phe 410 415 420Pro Ala Asp Tyr
Ser Ile Ser Phe Pro Thr Arg Pro Pro Leu Gln 425 430 435Ala Gln Ile
Pro Arg His Ser Leu Ala Pro Leu Val Arg Asn Gly 440 445 450Thr Glu
Ile Ser Ile Thr Ser Leu Val Leu Arg Lys Leu Asp His 455 460 465Leu
Leu Pro Ser Asn Tyr Gly Gln Gly Leu Gly Asp Ser Leu Tyr 470 475
480Ala Thr Pro Gly Leu Val Leu Val Ile Ser Ile Met Ala Gly Asp 485
490 495Arg Ala Phe Ser Gln Gly Glu Val Ile Met Asp Phe Gly Asn Thr
500 505 510Asp Gly Ser Pro His Cys Val Phe Trp Asp His Ser Leu Phe
Gln 515 520 525Gly Arg Gly Gly Trp Ser Lys Glu Gly Cys Gln Ala Gln
Val Ala 530 535 540Ser Ala Ser Pro Thr Ala Gln Cys Leu Cys Gln His
Leu Thr Ala 545 550 555Phe Ser Val Leu Met Ser Pro His Thr Val Pro
Glu Glu Pro Ala 560 565 570Leu Ala Leu Leu Thr Gln Val Gly Leu Gly
Ala Ser Ile Leu Ala 575 580 585Leu Leu Val Cys Leu Gly Val Tyr Trp
Leu Val Trp Arg Val Val 590 595 600Val Arg Asn Lys Ile Ser Tyr Phe
Arg His Ala Ala Leu Leu Asn 605 610 615Met Val Phe Cys Leu Leu Ala
Ala Asp Thr Cys Phe Leu Gly Ala 620 625 630Pro Phe Leu Ser Pro Gly
Pro Arg Ser Pro Leu Cys Leu Ala Ala 635 640 645Ala Phe Leu Cys His
Phe Leu Tyr Leu Ala Thr Phe Phe Trp Met 650 655 660Leu Ala Gln Ala
Leu Val Leu Ala His Gln Leu Leu Phe Val Phe 665 670 675His Gln Leu
Ala Lys His Arg Val Leu Pro Leu Met Val Leu Leu 680 685 690Gly Tyr
Leu Cys Pro Leu Gly Leu Ala Gly Val Thr Leu Gly Leu 695 700 705Tyr
Leu Pro Gln Gly Gln Tyr Leu Arg Glu Gly Glu Cys Trp Leu 710 715
720Asp Gly Lys Gly Gly Ala Leu Tyr Thr Phe Val Gly Pro Val Leu 725
730 735Ala Ile Ile Gly Val Asn Gly Leu Val Leu Ala Met Ala Met Leu
740 745 750Lys Leu Leu Arg Pro Ser Leu Ser Glu Gly Pro Pro Ala Glu
Lys 755 760 765Arg Gln Ala Leu Leu Gly Val Ile Lys Ala Leu Leu Ile
Leu Thr 770 775 780Pro Ile Phe Gly Leu Thr Trp Gly Leu Gly Leu Ala
Thr Leu Leu 785 790 795Glu Glu Val Ser Thr Val Pro His Tyr Ile Phe
Thr Ile Leu Asn 800 805 810Thr Leu Gln Gly Val Phe Ile Leu Leu Phe
Gly Cys Leu Met Asp 815 820 825Arg Lys Ile Gln Glu Ala Leu Arg Lys
Arg Phe Cys Arg Ala Gln 830 835 840Ala Pro Ser Ser Thr Ile Ser Leu
Ala Thr Asn Glu Gly Cys Ile 845 850 855Leu Glu His Ser Lys Gly Gly
Ser Asp Thr Ala Arg Lys Thr Asp 860 865 870Ala Ser Glu9932DNAHomo
sapiensUnsure911Unknown base 9gggaacggaa aatggcgcct cacggcccgg
gtagtcttac gaccctggtg 50ccctgggctg ccgccctgct cctcgctctg ggcgtggaaa
gggctctggc 100gctacccgag atatgcaccc aatgtccagg gagcgtgcaa
aatttgtcaa 150aagtggcctt ttattgtaaa acgacacgag agctaatgct
gcatgcccgt 200tgctgcctga atcagaaggg caccatcttg gggctggatc
tccagaactg 250ttctctggag gaccctggtc caaactttca tcaggcacat
accactgtca 300tcatagacct gcaagcaaac cccctcaaag gtgacttggc
caacaccttc 350cgtggcttta ctcagctcca gactctgata ctgccacaac
atgtcaactg 400tcctggagga attaatgcct ggaatactat cacctcttat
atagacaacc 450aaatctgtca agggcaaaag aacctttgca ataacactgg
ggacccagaa 500atgtgtcctg agaatggatc ttgtgtacct gatggtccag
gtcttttgca 550gtgtgtttgt gctgatggtt tccatggata caagtgtatg
cgccagggct 600cgttctcact gcttatgttc ttcgggattc tgggagccac
cactctatcc 650gtctccattc tgctttgggc gacccagcgc cgaaaagcca
agacttcatg 700aactacatag gtcttaccat tgacctaaga tcaatctgaa
ctatcttagc 750ccagtcaggg agctctgctt cctagaaagg catctttcgc
cagtggattc 800gcctcaaggt tgaggccgcc attggaagat gaaaaattgc
actcccttgg 850tgtagacaaa taccagttcc cattggtgtt gttgcctata
ataaacactt 900tttctttttt naaaaaaaaa aaaaaaaaaa aa 93210229PRTHomo
sapiens 10Met Ala Pro His Gly Pro Gly Ser Leu Thr Thr Leu Val Pro
Trp1 5 10 15Ala Ala Ala Leu Leu Leu Ala Leu Gly Val Glu Arg Ala Leu
Ala 20 25 30Leu Pro Glu Ile Cys Thr Gln Cys Pro Gly Ser Val Gln Asn
Leu 35 40 45Ser Lys Val Ala Phe Tyr Cys Lys Thr Thr Arg Glu Leu Met
Leu 50 55 60His Ala Arg Cys Cys Leu Asn Gln Lys Gly Thr Ile Leu Gly
Leu 65 70 75Asp Leu Gln Asn Cys Ser Leu Glu Asp Pro Gly Pro Asn Phe
His 80 85 90Gln Ala His Thr Thr Val Ile Ile Asp Leu Gln Ala Asn Pro
Leu 95 100 105Lys Gly Asp Leu Ala Asn Thr Phe Arg Gly Phe Thr Gln
Leu Gln 110 115 120Thr Leu Ile Leu Pro Gln His Val Asn Cys Pro Gly
Gly Ile Asn 125 130 135Ala Trp Asn Thr Ile Thr Ser Tyr Ile Asp Asn
Gln Ile Cys Gln 140 145 150Gly Gln Lys Asn Leu Cys Asn Asn Thr Gly
Asp Pro Glu Met Cys 155 160 165Pro Glu Asn Gly Ser Cys Val Pro Asp
Gly Pro Gly Leu Leu Gln 170 175 180Cys Val Cys Ala Asp Gly Phe His
Gly Tyr Lys Cys Met Arg Gln 185 190 195Gly Ser Phe Ser Leu Leu Met
Phe Phe Gly Ile Leu Gly Ala Thr 200 205 210Thr Leu Ser Val Ser Ile
Leu Leu Trp Ala Thr Gln Arg Arg Lys 215 220 225Ala Lys Thr Ser
113402DNAHomo sapiens 11gccgccccgc cccgagaccg ggcccggggg cgcggggcgg
cgggatgcgg 50cgcccggggc ggcgatgacc gcggagcgca cgccgcgggc ccggccctga
100ccccgccgcc cgcccgctga gccccccgcc gaggtccgga caggccgaga
150tgacgccgag ccccctgttg ctgctcctgc tgccgccgct gctgctgggg
200gccttcccac cggccgccgc cgcccgaggc cccccaaaga tggcggacaa
250ggtggtccca cggcaggtgg cccggctggg ccgcactgtg cggctgcagt
300gcccagtgga gggggacccg ccgccgctga ccatgtggac caaggatggc
350cgcaccatcc acagcggctg gagccgcttc cgcgtgctgc cgcaggggct
400gaaggtgaag caggtggagc gggaggatgc cggcgtgtac gtgtgcaagg
450ccaccaacgg cttcggcagc ctgagcgtca actacaccct cgtcgtgctg
500gatgacatta gcccagggaa ggagagcctg gggcccgaca gctcctctgg
550gggtcaagag gaccccgcca gccagcagtg ggcacgaccg cgcttcacac
600agccctccaa gatgaggcgc cgggtgatcg cacggcccgt gggtagctcc
650gtgcggctca agtgcgtggc cagcgggcac cctcggcccg acatcacgtg
700gatgaaggac gaccaggcct tgacgcgccc agaggccgct gagcccagga
750agaagaagtg gacactgagc ctgaagaacc tgcggccgga ggacagcggc
800aaatacacct gccgcgtgtc gaaccgcgcg ggcgccatca acgccaccta
850caaggtggat gtgatccagc ggacccgttc caagcccgtg ctcacaggca
900cgcaccccgt gaacacgacg gtggacttcg gggggaccac gtccttccag
950tgcaaggtgc gcagcgacgt gaagccggtg atccagtggc tgaagcgcgt
1000ggagtacggc gccgagggcc gccacaactc caccatcgat gtgggcggcc
1050agaagtttgt ggtgctgccc acgggtgacg tgtggtcgcg gcccgacggc
1100tcctacctca ataagctgct catcacccgt gcccgccagg acgatgcggg
1150catgtacatc tgccttggcg ccaacaccat gggctacagc ttccgcagcg
1200ccttcctcac cgtgctgcca gacccaaaac cgccagggcc acctgtggcc
1250tcctcgtcct cggccactag cctgccgtgg cccgtggtca tcggcatccc
1300agccggcgct gtcttcatcc tgggcaccct gctcctgtgg ctttgccagg
1350cccagaagaa gccgtgcacc cccgcgcctg cccctcccct gcctgggcac
1400cgcccgccgg ggacggcccg cgaccgcagc ggagacaagg accttccctc
1450gttggccgcc ctcagcgctg gccctggtgt ggggctgtgt gaggagcatg
1500ggtctccggc agccccccag cacttactgg gcccaggccc agttgctggc
1550cctaagttgt accccaaact ctacacagac atccacacac acacacacac
1600acactctcac acacactcac acgtggaggg caaggtccac cagcacatcc
1650actatcagtg ctagacggca ccgtatctgc agtgggcacg ggggggccgg
1700ccagacaggc agactgggag gatggaggac ggagctgcag acgaaggcag
1750gggacccatg gcgaggagga atggccagca ccccaggcag tctgtgtgtg
1800aggcatagcc cctggacaca cacacacaga cacacacact acctggatgc
1850atgtatgcac acacatgcgc gcacacgtgc tccctgaagg cacacgtacg
1900cacacgcaca tgcacagata tgccgcctgg gcacacagat aagctgccca
1950aatgcacgca cacgcacaga gacatgccag aacatacaag gacatgctgc
2000ctgaacatac acacgcacac ccatgcgcag atgtgctgcc tggacacaca
2050cacacacacg gatatgctgt ctggacgcac acacgtgcag atatggtatc
2100cggacacaca cgtgcacaga tatgctgcct ggacacacag ataatgctgc
2150cttgacacac acatgcacgg atattgcctg gacacacaca cacacacacg
2200cgtgcacaga tatgctgtct ggacacgcac acacatgcag atatgctgcc
2250tggacacaca cttccagaca cacgtgcaca ggcgcagata tgctgcctgg
2300acacacgcag atatgctgtc tagtcacaca cacacgcaga
catgctgtcc 2350ggacacacac acgcatgcac agatatgctg tccggacaca
cacacgcacg 2400cagatatgct gcctggacac acacacagat aatgctgcct
caacactcac 2450acacgtgcag atattgcctg gacacacaca tgtgcacaga
tatgctgtct 2500ggacatgcac acacgtgcag atatgctgtc cggatacaca
cgcacgcaca 2550catgcagata tgctgcctgg gcacacactt ccggacacac
atgcacacac 2600aggtgcagat atgctgcctg gacacacaca cagataatgc
tgcctcaaca 2650ctcacacacg tgcagatatt gcctggacac acacatgtgc
acagatatgc 2700tgtctggaca tgcacacacg tgcagatatg ctgtccggat
acacacgcac 2750gcacacatgc agatatgctg cctgggcaca cacttccgga
cacacatgca 2800cacacaggtg cagatatgct gcctggacac acgcagactg
acgtgctttt 2850gggagggtgt gccgtgaagc ctgcagtacg tgtgccgtga
ggctcatagt 2900tgatgaggga ctttccctgc tccaccgtca ctcccccaac
tctgcccgcc 2950tctgtccccg cctcagtccc cgcctccatc cccgcctctg
tcccctggcc 3000ttggcggcta tttttgccac ctgccttggg tgcccaggag
tcccctactg 3050ctgtgggctg gggttggggg cacagcagcc ccaagcctga
gaggctggag 3100cccatggcta gtggctcatc cccagtgcat tctccccctg
acacagagaa 3150ggggccttgg tatttatatt taagaaatga agataatatt
aataatgatg 3200gaaggaagac tgggttgcag ggactgtggt ctctcctggg
gcccgggacc 3250cgcctggtct ttcagccatg ctgatgacca caccccgtcc
aggccagaca 3300ccacccccca ccccactgtc gtggtggccc cagatctctg
taattttatg 3350tagagtttga gctgaagccc cgtatattta atttattttg
ttaaacacaa 3400aa 340212504PRTHomo sapiens 12Met Thr Pro Ser Pro
Leu Leu Leu Leu Leu Leu Pro Pro Leu Leu1 5 10 15Leu Gly Ala Phe Pro
Pro Ala Ala Ala Ala Arg Gly Pro Pro Lys 20 25 30Met Ala Asp Lys Val
Val Pro Arg Gln Val Ala Arg Leu Gly Arg 35 40 45Thr Val Arg Leu Gln
Cys Pro Val Glu Gly Asp Pro Pro Pro Leu 50 55 60Thr Met Trp Thr Lys
Asp Gly Arg Thr Ile His Ser Gly Trp Ser 65 70 75Arg Phe Arg Val Leu
Pro Gln Gly Leu Lys Val Lys Gln Val Glu 80 85 90Arg Glu Asp Ala Gly
Val Tyr Val Cys Lys Ala Thr Asn Gly Phe 95 100 105Gly Ser Leu Ser
Val Asn Tyr Thr Leu Val Val Leu Asp Asp Ile 110 115 120Ser Pro Gly
Lys Glu Ser Leu Gly Pro Asp Ser Ser Ser Gly Gly 125 130 135Gln Glu
Asp Pro Ala Ser Gln Gln Trp Ala Arg Pro Arg Phe Thr 140 145 150Gln
Pro Ser Lys Met Arg Arg Arg Val Ile Ala Arg Pro Val Gly 155 160
165Ser Ser Val Arg Leu Lys Cys Val Ala Ser Gly His Pro Arg Pro 170
175 180Asp Ile Thr Trp Met Lys Asp Asp Gln Ala Leu Thr Arg Pro Glu
185 190 195Ala Ala Glu Pro Arg Lys Lys Lys Trp Thr Leu Ser Leu Lys
Asn 200 205 210Leu Arg Pro Glu Asp Ser Gly Lys Tyr Thr Cys Arg Val
Ser Asn 215 220 225Arg Ala Gly Ala Ile Asn Ala Thr Tyr Lys Val Asp
Val Ile Gln 230 235 240Arg Thr Arg Ser Lys Pro Val Leu Thr Gly Thr
His Pro Val Asn 245 250 255Thr Thr Val Asp Phe Gly Gly Thr Thr Ser
Phe Gln Cys Lys Val 260 265 270Arg Ser Asp Val Lys Pro Val Ile Gln
Trp Leu Lys Arg Val Glu 275 280 285Tyr Gly Ala Glu Gly Arg His Asn
Ser Thr Ile Asp Val Gly Gly 290 295 300Gln Lys Phe Val Val Leu Pro
Thr Gly Asp Val Trp Ser Arg Pro 305 310 315Asp Gly Ser Tyr Leu Asn
Lys Leu Leu Ile Thr Arg Ala Arg Gln 320 325 330Asp Asp Ala Gly Met
Tyr Ile Cys Leu Gly Ala Asn Thr Met Gly 335 340 345Tyr Ser Phe Arg
Ser Ala Phe Leu Thr Val Leu Pro Asp Pro Lys 350 355 360Pro Pro Gly
Pro Pro Val Ala Ser Ser Ser Ser Ala Thr Ser Leu 365 370 375Pro Trp
Pro Val Val Ile Gly Ile Pro Ala Gly Ala Val Phe Ile 380 385 390Leu
Gly Thr Leu Leu Leu Trp Leu Cys Gln Ala Gln Lys Lys Pro 395 400
405Cys Thr Pro Ala Pro Ala Pro Pro Leu Pro Gly His Arg Pro Pro 410
415 420Gly Thr Ala Arg Asp Arg Ser Gly Asp Lys Asp Leu Pro Ser Leu
425 430 435Ala Ala Leu Ser Ala Gly Pro Gly Val Gly Leu Cys Glu Glu
His 440 445 450Gly Ser Pro Ala Ala Pro Gln His Leu Leu Gly Pro Gly
Pro Val 455 460 465Ala Gly Pro Lys Leu Tyr Pro Lys Leu Tyr Thr Asp
Ile His Thr 470 475 480His Thr His Thr His Ser His Thr His Ser His
Val Glu Gly Lys 485 490 495Val His Gln His Ile His Tyr Gln Cys
500132197DNAHomo sapiens 13cggacgcgtg ggcgtccggc ggtcgcagag
ccaggaggcg gaggcgcgcg 50ggccagcctg ggccccagcc cacaccttca ccagggccca
ggagccacca 100tgtggcgatg tccactgggg ctactgctgt tgctgccgct
ggctggccac 150ttggctctgg gtgcccagca gggtcgtggg cgccgggagc
tagcaccggg 200tctgcacctg cggggcatcc gggacgcggg aggccggtac
tgccaggagc 250aggacctgtg ctgccgcggc cgtgccgacg actgtgccct
gccctacctg 300ggcgccatct gttactgtga cctcttctgc aaccgcacgg
tctccgactg 350ctgccctgac ttctgggact tctgcctcgg cgtgccaccc
ccttttcccc 400cgatccaagg atgtatgcat ggaggtcgta tctatccagt
cttgggaacg 450tactgggaca actgtaaccg ttgcacctgc caggagaaca
ggcagtggca 500tggtggatcc agacatgatc aaagccatca accagggcaa
ctatggctgg 550caggctggga accacagcgc cttctggggc atgaccctgg
atgagggcat 600tcgctaccgc ctgggcacca tccgcccatc ttcctcggtc
atgaacatgc 650atgaaattta tacagtgctg aacccagggg aggtgcttcc
cacagccttc 700gaggcctctg agaagtggcc caacctgatt catgagcctc
ttgaccaagg 750caactgtgca ggctcctggg ccttctccac agcagctgtg
gcatccgatc 800gtgtctcaat ccattctctg ggacacatga cgcctgtcct
gtcgccccag 850aacctgctgt cttgtgacac ccaccagcag cagggctgcc
gcggtgggcg 900tctcgatggt gcctggtggt tcctgcgtcg ccgaggggtg
gtgtctgacc 950actgctaccc cttctcgggc cgtgaacgag acgaggctgg
ccctgcgccc 1000ccctgtatga tgcacagccg agccatgggt cggggcaagc
gccaggccac 1050tgcccactgc cccaacagct atgttaataa caatgacatc
taccaggtca 1100ctcctgtcta ccgcctcggc tccaacgaca aggagatcat
gaaggagctg 1150atggagaatg gccctgtcca agccctcatg gaggtgcatg
aggacttctt 1200cctatacaag ggaggcatct acagccacac gccagtgagc
cttgggaggc 1250cagagagata ccgccggcat gggacccact cagtcaagat
cacaggatgg 1300ggagaggaga cgctgccaga tggaaggacg ctcaaatact
ggactgcggc 1350caactcctgg ggcccagcct ggggcgagag gggccacttc
cgcatcgtgc 1400gcggcgtcaa tgagtgcgac atcgagagct tcgtgctggg
cgtctggggc 1450cgcgtgggca tggaggacat gggtcatcac tgaggctgcg
ggcaccacgc 1500ggggtccggc ctgggatcca ggctaagggc cggcggaaga
ggccccaatg 1550gggcggtgac cccagcctcg cccgacagag cccggggcgc
aggcgggcgc 1600cagggcgcta atcccggcgc gggttccgct gacgcagcgc
cccgcctggg 1650agccgcgggc aggcgagact ggcggagccc ccagacctcc
cagtggggac 1700ggggcagggc ctggcctggg aagagcacag ctgcagatcc
caggcctctg 1750gcgcccccac tcaagactac caaagccagg acacctcaag
tctccagccc 1800caatacccca ccccaatccc gtattctttt tttttttttt
ttagacaggg 1850tcttgctccg ttgcccaggt tggagtgcag tggcccatca
gggctcactg 1900taacctccga ctcctgggtt caagtgaccc tcccacctca
gcctctcaag 1950tagctgggac tacaggtgca ccaccacacc tggctaattt
ttgtattttt 2000tgtaaagagg ggggtctcac tgtgttgccc aggctggttt
cgaactcctg 2050ggctcaagcg gtccacctgc ctccgcctcc caaagtgctg
ggattgcagg 2100catgagccac tgcacccagc cctgtattct tattcttcag
atatttattt 2150ttcttttcac tgttttaaaa taaaaccaaa gtattgataa aaaaaaa
219714164PRTHomo sapiens 14Met Trp Arg Cys Pro Leu Gly Leu Leu Leu
Leu Leu Pro Leu Ala1 5 10 15Gly His Leu Ala Leu Gly Ala Gln Gln Gly
Arg Gly Arg Arg Glu 20 25 30Leu Ala Pro Gly Leu His Leu Arg Gly Ile
Arg Asp Ala Gly Gly 35 40 45Arg Tyr Cys Gln Glu Gln Asp Leu Cys Cys
Arg Gly Arg Ala Asp 50 55 60Asp Cys Ala Leu Pro Tyr Leu Gly Ala Ile
Cys Tyr Cys Asp Leu 65 70 75Phe Cys Asn Arg Thr Val Ser Asp Cys Cys
Pro Asp Phe Trp Asp 80 85 90Phe Cys Leu Gly Val Pro Pro Pro Phe Pro
Pro Ile Gln Gly Cys 95 100 105Met His Gly Gly Arg Ile Tyr Pro Val
Leu Gly Thr Tyr Trp Asp 110 115 120Asn Cys Asn Arg Cys Thr Cys Gln
Glu Asn Arg Gln Trp His Gly 125 130 135Gly Ser Arg His Asp Gln Ser
His Gln Pro Gly Gln Leu Trp Leu 140 145 150Ala Gly Trp Glu Pro Gln
Arg Leu Leu Gly His Asp Pro Gly 155 160151780DNAHomo sapiens
15ggctcagagg ccccactgga ccctcggctc ttccttggac ttcttgtgtg
50ttctgtgagc ttcgctggat tcagggtctt gggcatcaga ggtgagaggg
100tgggaaggtc cgccgcgatg gggaagccct ggctgcgtgc gctacagctg
150ctgctcctgc tgggcgcgtc gtgggcgcgg gcgggcgccc cgcgctgcac
200ctacaccttc gtgctgcccc cgcagaagtt cacgggcgct gtgtgctgga
250gcggccccgc atccacgcgg gcgacgcccg aggccgccaa cgccagcgag
300ctggcggcgc tgcgcatgcg cgtcggccgc cacgaggagc tgttacgcga
350gctgcagagg ctggcggcgg ccgacggcgc cgtggccggc gaggtgcgcg
400cgctgcgcaa ggagagccgc ggcctgagcg cgcgcctggg ccagttgcgc
450gcgcagctgc agcacgaggc ggggcccggg gcgggcccgg gggcggatct
500gggggcggag cctgccgcgg cgctggcgct gctcggggag cgcgtgctca
550acgcgtccgc cgaggctcag cgcgcagccg cccggttcca ccagctggac
600gtcaagttcc gcgagctggc gcagctcgtc acccagcaga gcagtctcat
650cgcccgcctg gagcgcctgt gcccgggagg cgcgggcggg cagcagcagg
700tcctgccgcc acccccactg gtgcctgtgg ttccggtccg tcttgtgggt
750agcaccagtg acaccagtag gatgctggac ccagccccag agccccagag
800agaccagacc cagagacagc aggagcccat ggcttctccc atgcctgcag
850gtcaccctgc ggtccccacc aagcctgtgg gcccgtggca ggattgtgca
900gaggcccgcc aggcaggcca tgaacagagt ggagtgtatg aactgcgagt
950gggccgtcac gtagtgtcag tatggtgtga gcagcaactg gagggtggag
1000gctggactgt gatccagcgg aggcaagatg gttcagtcaa cttcttcact
1050acctggcagc actataaggc gggctttggg cggccagacg gagaatactg
1100gctgggcctt gaacccgtgt atcagctgac cagccgtggg gaccatgagc
1150tgctggttct cctggaggac tgggggggcc gtggagcacg tgcccactat
1200gatggcttct ccctggaacc cgagagcgac cactaccgcc tgcggcttgg
1250ccagtaccat ggtgatgctg gagactctct ttcctggcac aatgacaagc
1300ccttcagcac cgtggatagg gaccgagact cctattctgg taactgtgcc
1350ctgtaccagc ggggaggctg gtggtaccat gcctgtgccc actccaacct
1400caacggtgtg tggcaccacg gcggccacta ccgaagccgc taccaggatg
1450gtgtctactg ggctgagttt cgtggtgggg catattctct caggaaggcc
1500gccatgctca ttcggcccct gaagctgtga ctctgtgttc ctctgtcccc
1550taggccctag aggacattgg tcagcaggag cccaagttgt tctggccaca
1600ccttctttgt ggctcagtgc caatgtgtcc cacagaactt cccactgtgg
1650atctgtgacc ctgggcgctg aaaatgggac ccaggaatcc cccccgtcaa
1700tatcttggcc tcagatggct ccccaaggtc attcatatct cggtttgagc
1750tcatatctta taataacaca aagtagccac 178016470PRTHomo sapiens 16Met
Gly Lys Pro Trp Leu Arg Ala Leu Gln Leu Leu Leu Leu Leu1 5 10 15Gly
Ala Ser Trp Ala Arg Ala Gly Ala Pro Arg Cys Thr Tyr Thr 20 25 30Phe
Val Leu Pro Pro Gln Lys Phe Thr Gly Ala Val Cys Trp Ser 35 40 45Gly
Pro Ala Ser Thr Arg Ala Thr Pro Glu Ala Ala Asn Ala Ser 50 55 60Glu
Leu Ala Ala Leu Arg Met Arg Val Gly Arg His Glu Glu Leu 65 70 75Leu
Arg Glu Leu Gln Arg Leu Ala Ala Ala Asp Gly Ala Val Ala 80 85 90Gly
Glu Val Arg Ala Leu Arg Lys Glu Ser Arg Gly Leu Ser Ala 95 100
105Arg Leu Gly Gln Leu Arg Ala Gln Leu Gln His Glu Ala Gly Pro 110
115 120Gly Ala Gly Pro Gly Ala Asp Leu Gly Ala Glu Pro Ala Ala Ala
125 130 135Leu Ala Leu Leu Gly Glu Arg Val Leu Asn Ala Ser Ala Glu
Ala 140 145 150Gln Arg Ala Ala Ala Arg Phe His Gln Leu Asp Val Lys
Phe Arg 155 160 165Glu Leu Ala Gln Leu Val Thr Gln Gln Ser Ser Leu
Ile Ala Arg 170 175 180Leu Glu Arg Leu Cys Pro Gly Gly Ala Gly Gly
Gln Gln Gln Val 185 190 195Leu Pro Pro Pro Pro Leu Val Pro Val Val
Pro Val Arg Leu Val 200 205 210Gly Ser Thr Ser Asp Thr Ser Arg Met
Leu Asp Pro Ala Pro Glu 215 220 225Pro Gln Arg Asp Gln Thr Gln Arg
Gln Gln Glu Pro Met Ala Ser 230 235 240Pro Met Pro Ala Gly His Pro
Ala Val Pro Thr Lys Pro Val Gly 245 250 255Pro Trp Gln Asp Cys Ala
Glu Ala Arg Gln Ala Gly His Glu Gln 260 265 270Ser Gly Val Tyr Glu
Leu Arg Val Gly Arg His Val Val Ser Val 275 280 285Trp Cys Glu Gln
Gln Leu Glu Gly Gly Gly Trp Thr Val Ile Gln 290 295 300Arg Arg Gln
Asp Gly Ser Val Asn Phe Phe Thr Thr Trp Gln His 305 310 315Tyr Lys
Ala Gly Phe Gly Arg Pro Asp Gly Glu Tyr Trp Leu Gly 320 325 330Leu
Glu Pro Val Tyr Gln Leu Thr Ser Arg Gly Asp His Glu Leu 335 340
345Leu Val Leu Leu Glu Asp Trp Gly Gly Arg Gly Ala Arg Ala His 350
355 360Tyr Asp Gly Phe Ser Leu Glu Pro Glu Ser Asp His Tyr Arg Leu
365 370 375Arg Leu Gly Gln Tyr His Gly Asp Ala Gly Asp Ser Leu Ser
Trp 380 385 390His Asn Asp Lys Pro Phe Ser Thr Val Asp Arg Asp Arg
Asp Ser 395 400 405Tyr Ser Gly Asn Cys Ala Leu Tyr Gln Arg Gly Gly
Trp Trp Tyr 410 415 420His Ala Cys Ala His Ser Asn Leu Asn Gly Val
Trp His His Gly 425 430 435Gly His Tyr Arg Ser Arg Tyr Gln Asp Gly
Val Tyr Trp Ala Glu 440 445 450Phe Arg Gly Gly Ala Tyr Ser Leu Arg
Lys Ala Ala Met Leu Ile 455 460 465Arg Pro Leu Lys Leu
47017462DNAHomo sapiens 17agcccaccga gaggcgcctg caggatgaaa
gctctctgtc tcctcctcct 50ccctgtcctg gggctgttgg tgtctagcaa gaccctgtgc
tccatggaag 100aagccatcaa tgagaggatc caggaggtcg ccggctccct
aatatttagg 150gcaataagca gcattggcct ggagtgccag agcgtcacct
ccagggggga 200cctggctact tgcccccgag gcttcgccgt caccggctgc
acttgtggct 250ccgcctgtgg ctcgtgggat gtgcgcgccg agaccacatg
tcactgccag 300tgcgcgggca tggactggac cggagcgcgc tgctgtcgtg
tgcagccctg 350aggtcgcgcg cagcgcgtgc acagcgcggg cggaggcggc
tccaggtccg 400gaggggttgc gggggagctg gaaataaacc tggagatgat
gatgatgatg 450atgatggaaa aa 46218108PRTHomo sapiens 18Met Lys Ala
Leu Cys Leu Leu Leu Leu Pro Val Leu Gly Leu Leu1 5 10 15Val Ser Ser
Lys Thr Leu Cys Ser Met Glu Glu Ala Ile Asn Glu 20 25 30Arg Ile Gln
Glu Val Ala Gly Ser Leu Ile Phe Arg Ala Ile Ser 35 40 45Ser Ile Gly
Leu Glu Cys Gln Ser Val Thr Ser Arg Gly Asp Leu 50 55 60Ala Thr Cys
Pro Arg Gly Phe Ala Val Thr Gly Cys Thr Cys Gly 65 70 75Ser Ala Cys
Gly Ser Trp Asp Val Arg Ala Glu Thr Thr Cys His 80 85 90Cys Gln Cys
Ala Gly Met Asp Trp Thr Gly Ala Arg Cys Cys Arg 95 100 105Val Gln
Pro193228DNAHomo sapiens 19tcagggtcag gtgattctcc cacctcagcc
tcctgagtag ctgggagtac 50aggcacatgc caccacaccc agataatttt taaatttttt
gtagagatgg 100ggtctcactg tgttgcccag gctggtctcg agctcctggg
ctgaagtgat 150ccatccacct ccgtccacca aagtgctggg attacaggtg
tgagccaccg 200tgccctgcct gcatttcttt taatagacat gtctctggat
ggtcaactgg 250acagttgtgc tcaccctcca catttcctcc cctctactca
caccccaagg 300tgataatgga ttggcaaccc tgggtgactt aagaattcca
tgagcttcat 350aaatgtcaaa taagctgtct taccctacta acccctctac
catcacaatg 400atcaaagcaa agccaaatag cccacacctc tcatcccaca
cataccatag 450tcatcctttc atccatttat ccatccattt accaacttaa
aatattcatt 500gattacctac tacatatgac actttacttg accaaaattg
gcaaaacaca 550tagcttaaag ttgtctttct tcctagttag gtggaatgat
gcctggtata 600tcgtatcatt agaaatggct gaatgaatga gctcactgtt
tattaggaga 650atgagacaca gaccaaataa ctcaagcata agagagaatg
tagttagtgt 700cctaaaagag atccagagtg ctgtgtgagt tcaaaggtga
gaaagagccc 750ttctgactga agaaatcagg aaagacttca tagaagcagt
ggtgatatat 800gagctgactt ctaaaggaca agtaagatta taatagcaga
tatagtaggg 850gaagggaatt accgaggggg tggcatcaat aaagttttgg
gggtaaggaa 900gtatgagttc tgggtatgaa aatatgtcta acttggtcag
agcataaatt 950acgtattctg gaaggtagac tgtgtttgag aacaaatgct
agaatacctt 1000aaaagctaat ttgttaggtt ctgcagagtc agtgatagga
cccaagcaga 1050caagtaatta ggaagactaa tttggcaaag atattataaa
atgttggggc 1100tgaacaatta ttacatataa taagagaatt aacaaggtgc
ctgagtgaaa 1150tgtaataaac agaaaacaac aaattttgta tgtcaaccaa
acctagcagt 1200caaaaggatt aataacaata agtcatgtag gatactatga
attcataaca 1250caaagaaatg ctaggggaaa tatttgcaat gcttatcaca
tccaaaagtt 1300cctttcccta atatacaaag atctgctaga agtcaacaag
ctaaagatca 1350acagctcaat agaaatatgg ccaaacggct ggacgtggtg
gctcatgcct 1400gtaatcccag cattttggga gactgaggca ggattgcttg
agcccaggaa 1450ttcaagacca gcctggtcaa cgtagcgaga ttctgtgtct
atatttttaa 1500aaatttatta aaaaaagaaa tacgggcaaa tgagctacct
agtctcagaa 1550aagaaaatat atatgatgtg caactatatt aaaagatttt
caatttcact 1600aataattttt ttttttgaga cagagtcttg ctctgtcgcc
aggctggagt 1650gcagtggcac catcttggct cactgcaagc tctgcctccc
gagttcacca 1700ttctcctgcc tcagcctccc aagtagctgg gattacaggc
gcacaccacc 1750acacctggct aatgttttgt atttttagta gaggcggggt
ttcaccgtgt 1800tagccaggat ggcctcgatc tcttgacctc gtgatcagcc
caccttggcc 1850tctcaaagtg ctgtgattac aggcgtgagc caccgcgcct
ggccaaattt 1900cactaataat tttaaaaagt aaattatata tacatgggat
atcatgttca 1950cttagattgg cgatgagcag aaagtttgat aactgtgtca
taaacacttg 2000gtaactgtgt tagtgagtgt gtggggagat aggtatcctt
atatgctgct 2050aataggagtg taggctgtaa aattctcatg gtagctagtt
tagcaatatc 2100tataaaaatt acaaatatgc ataactttca gtgagtcaga
aattttactt 2150ttaagaattt atcttacatg tataatcaca acacgtgtga
aatatcgtac 2200acataataga tattggttgc aatcttttca tagttgtgaa
agatgaggaa 2250aaacaatctt aaaagtagtt tggttaaata aatcatgtca
ctcatataca 2300gtgaaatatc atccccattt taaaaagatg atggtggtgg
tgctatacat 2350accgatacag aaagctttct aaaacctttc attaaatgaa
aaatgaataa 2400atcattgcag aacagtgtat atatatctaa aatatctatg
gaagaaacca 2450gcaacagcca ctgctcctgg agaattatgg tcccacacca
ctgatcattc 2500tttcagtagg gtgaccatca tccaaatttg cttgggactg
agggggttcc 2550ttttggtttg aaaaccagga cagtcctagg aaaagtgaga
caagttggtc 2600acatgtcccc aagatgatct tctttcactt atgaacttgc
tactttccca 2650gtcagaatat aaactctgag gggggagact tcctgttttc
ttcatgacta 2700tatctcttgc gcactgtggg gtggaggctg tagaagagga
gagaagtaga 2750gaaacagatc acattgtgtc ttgaagtgtt tcagcaaata
tgggcaacac 2800ccttctttta ctagcttgga accctacctc tgagtgcatt
tcccttttta 2850ttatttattt cctgtcagtt ataagagagg cctacccctt
tgtgagcagt 2900ctaggacttt gtacacctgc taagtaggga gaaggcaggg
gaggtggctg 2950gtttaagggg aacttgaggg aagtagggaa gactcctctc
gggacctttg 3000gagtaggtga cacatgagcc cagccccagc tcacctgcca
atccagctga 3050ggagctcacc tgccaatcca gctgaggctg ggcagaggtg
ggtgagaaga 3100gggaaaattg cagggacctc cagttgggcc aggccagaag
ctgctgtagc 3150tttaaccaga cagctcagac ctgtatggag gctgccagtg
acaggttagg 3200tttagggcag agaagaagca agaccatg 322820319PRTHomo
sapiens 20Met Val Gly Lys Met Trp Pro Val Leu Trp Thr Leu Cys Ala
Val1 5 10 15Arg Val Thr Val Asp Ala Ile Ser Val Glu Thr Pro Gln Asp
Val 20 25 30Leu Arg Ala Ser Gln Gly Lys Ser Val Thr Leu Pro Cys Thr
Tyr 35 40 45His Thr Ser Thr Ser Ser Arg Glu Gly Leu Ile Gln Trp Asp
Lys 50 55 60Leu Leu Leu Thr His Thr Glu Arg Val Val Ile Trp Pro Phe
Ser 65 70 75Asn Lys Asn Tyr Ile His Gly Glu Leu Tyr Lys Asn Arg Val
Ser 80 85 90Ile Ser Asn Asn Ala Glu Gln Ser Asp Ala Ser Ile Thr Ile
Asp 95 100 105Gln Leu Thr Met Ala Asp Asn Gly Thr Tyr Glu Cys Ser
Val Ser 110 115 120Leu Met Ser Asp Leu Glu Gly Asn Thr Lys Ser Arg
Val Arg Leu 125 130 135Leu Val Leu Val Pro Pro Ser Lys Pro Glu Cys
Gly Ile Glu Gly 140 145 150Glu Thr Ile Ile Gly Asn Asn Ile Gln Leu
Thr Cys Gln Ser Lys 155 160 165Glu Gly Ser Pro Thr Pro Gln Tyr Ser
Trp Lys Arg Tyr Asn Ile 170 175 180Leu Asn Gln Glu Gln Pro Leu Ala
Gln Pro Ala Ser Gly Gln Pro 185 190 195Val Ser Leu Lys Asn Ile Ser
Thr Asp Thr Ser Gly Tyr Tyr Ile 200 205 210Cys Thr Ser Ser Asn Glu
Glu Gly Thr Gln Phe Cys Asn Ile Thr 215 220 225Val Ala Val Arg Ser
Pro Ser Met Asn Val Ala Leu Tyr Val Gly 230 235 240Ile Ala Val Gly
Val Val Ala Ala Leu Ile Ile Ile Gly Ile Ile 245 250 255Ile Tyr Cys
Cys Cys Cys Arg Gly Lys Asp Asp Asn Thr Glu Asp 260 265 270Lys Glu
Asp Ala Arg Pro Asn Arg Glu Ala Tyr Glu Glu Pro Pro 275 280 285Glu
Gln Leu Arg Glu Leu Ser Arg Glu Arg Glu Glu Glu Asp Asp 290 295
300Tyr Arg Gln Glu Glu Gln Arg Ser Thr Gly Arg Glu Ser Pro Asp 305
310 315His Leu Asp Gln 212870DNAHomo sapiens 21cgcggagccc
tgcgctggga ggtgcacggt gtgcacgctg gactggaccc 50ccatgcaacc ccgcgccctg
cgccttaacc aggactgctc cgcgcgcccc 100tgagcctcgg gctccggccc
ggacctgcag cctcccaggt ggctgggaag 150aactctccaa caataaatac
atttgataag aaagatggct ttaaaagtgc 200tactagaaca agagaaaacg
tttttcactc ttttagtatt actaggctat 250ttgtcatgta aagtgacttg
tgaatcagga gactgtagac agcaagaatt 300cagggatcgg tctggaaact
gtgttccctg caaccagtgt gggccaggca 350tggagttgtc taaggaatgt
ggcttcggct atggggagga tgcacagtgt 400gtgacgtgcc ggctgcacag
gttcaaggag gactggggct tccagaaatg 450caagccctgt ctggactgcg
cagtggtgaa ccgctttcag aaggcaaatt 500gttcagccac cagtgatgcc
atctgcgggg actgcttgcc aggattttat 550aggaagacga aacttgtcgg
ctttcaagac atggagtgtg tgccttgtgg 600agaccctcct cctccttacg
aaccgcactg tgccagcaag gtcaacctcg 650tgaagatcgc gtccacggcc
tccagcccac gggacacggc gctggctgcc 700gttatctgca gcgctctggc
caccgtcctg ctggccctgc tcatcctctg 750tgtcatctat tgtaagagac
agtttatgga gaagaaaccc agctggtctc 800tgcggtcgca ggacattcag
tacaacggct ctgagctgtc gtgttttgac 850agacctcagc tccacgaata
tgcccacaga gcctgctgcc agtgccgccg 900tgactcagtg cagacctgcg
ggccggtgcg cttgctccca tccatgtgct 950gtgaggaggc ctgcagcccc
aacccggcga ctcttggttg tggggtgcat 1000tctgcagcca gtcttcaggc
aagaaacgca ggcccagccg gggagatggt 1050gccgactttc ttcggatccc
tcacgcagtc catctgtggc gagttttcag 1100atgcctggcc tctgatgcag
aatcccatgg gtggtgacaa catctctttt 1150tgtgactctt atcctgaact
cactggagaa gacattcatt ctctcaatcc 1200agaacttgaa agctcaacgt
ctttggattc aaatagcagt caagatttgg 1250ttggtggggc tgttccagtc
cagtctcatt ctgaaaactt tacagcagct 1300actgatttat ctagatataa
caacacactg gtagaatcag catcaactca 1350ggatgcacta actatgagaa
gccagctaga tcaggagagt ggcgctgtca 1400tccacccagc cactcagacg
tccctccagg aagcttaaag aacctgcttc 1450tttctgcagt agaagcgtgt
gctggaaccc aaagagtact cctttgttag 1500gcttatggac tgagcagtct
ggaccttgca tggcttctgg ggcaaaaata 1550aatctgaacc aaactgacgg
catttgaagc ctttcagcca gttgcttctg 1600agccagacca gctgtaagct
gaaacctcaa tgaataacaa gaaaagactc 1650caggccgact catgatactc
tgcatctttc ctacatgaga agcttctctg 1700ccacaaaagt gacttcaaag
actgatgggt tgagctggca gcctatgaga 1750ttgtggacat ataacaagaa
acagaaatgc cctcatgctt attttcatgg 1800tgattgtggt tttacaagac
tgaagaccca gagtatactt tttctttcca 1850gaaataattt cataccgcct
atgaaatatc agataaatta ccttagcttt 1900tatgtagaat gggttcaaaa
gtgagtgttt ctatttgaga aggacacttt 1950ttcatcatct aaactgattc
gcataggtgg ttagaatggc cctcatattg 2000cctgcctaaa tcttgggttt
attagatgaa gtttactgaa tcagaggaat 2050cagacagagg aggatagctc
tttccagaat ccacacttct gacctcagcc 2100tcggtctcat gaacacccgc
tgatctcagg agaacacctg ggctagggaa 2150tgtggtcgag aaagggcagc
ccattgccca gaattaacac atattgtaga 2200gacttgtatg caaaggttgg
catatttata tgaaaattag ttgctataga 2250aacatttgtt gcatctgtcc
ctctgcctga gcttagaagg ttatagaaaa 2300agggtattta taaacataaa
tgacctttta cttgcattgt atcttatact 2350aaaggcttta gaaattacaa
catatcaggt tcccctacta ctgaagtagc 2400cttccgtgag aacacaccac
atgttaggac tagaagaaaa tgcacaattt 2450gtaggggttt ggatgaagca
gctgtaactg ccctagtgta gtttgaccag 2500gacattgtcg tgctccttcc
aattgtgtaa gattagttag cacatcatct 2550cctactttag ccatccggtg
ttggatttaa gaggacggtg cttctttcta 2600ttaaagtgct ccatccccta
ccatctacac attagcattg tctctagagc 2650taagacagaa attaaccccg
ttcagtcaca aagcagggaa tggttcattt 2700actcttaatc tttatgccct
ggagaagacc tacttgaaca gggcatattt 2750tttagacttc tgaacatcag
tatgttcgag ggtactatga tattttggtt 2800tggaattgcc ctgcccaagt
cactgtcttt taacttttaa actgaatatt 2850aaaatgtatc tgtctttcct
287022417PRTHomo sapiens 22Met Ala Leu Lys Val Leu Leu Glu Gln Glu
Lys Thr Phe Phe Thr1 5 10 15Leu Leu Val Leu Leu Gly Tyr Leu Ser Cys
Lys Val Thr Cys Glu 20 25 30Ser Gly Asp Cys Arg Gln Gln Glu Phe Arg
Asp Arg Ser Gly Asn 35 40 45Cys Val Pro Cys Asn Gln Cys Gly Pro Gly
Met Glu Leu Ser Lys 50 55 60Glu Cys Gly Phe Gly Tyr Gly Glu Asp Ala
Gln Cys Val Thr Cys 65 70 75Arg Leu His Arg Phe Lys Glu Asp Trp Gly
Phe Gln Lys Cys Lys 80 85 90Pro Cys Leu Asp Cys Ala Val Val Asn Arg
Phe Gln Lys Ala Asn 95 100 105Cys Ser Ala Thr Ser Asp Ala Ile Cys
Gly Asp Cys Leu Pro Gly 110 115 120Phe Tyr Arg Lys Thr Lys Leu Val
Gly Phe Gln Asp Met Glu Cys 125 130 135Val Pro Cys Gly Asp Pro Pro
Pro Pro Tyr Glu Pro His Cys Ala 140 145 150Ser Lys Val Asn Leu Val
Lys Ile Ala Ser Thr Ala Ser Ser Pro 155 160 165Arg Asp Thr Ala Leu
Ala Ala Val Ile Cys Ser Ala Leu Ala Thr 170 175 180Val Leu Leu Ala
Leu Leu Ile Leu Cys Val Ile Tyr Cys Lys Arg 185 190 195Gln Phe Met
Glu Lys Lys Pro Ser Trp Ser Leu Arg Ser Gln Asp 200 205 210Ile Gln
Tyr Asn Gly Ser Glu Leu Ser Cys Phe Asp Arg Pro Gln 215 220 225Leu
His Glu Tyr Ala His Arg Ala Cys Cys Gln Cys Arg Arg Asp 230 235
240Ser Val Gln Thr Cys Gly Pro Val Arg Leu Leu Pro Ser Met Cys 245
250 255Cys Glu Glu Ala Cys Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly
260 265 270Val His Ser Ala Ala Ser Leu Gln Ala Arg Asn Ala Gly Pro
Ala 275 280 285Gly Glu Met Val Pro Thr Phe Phe Gly Ser Leu Thr Gln
Ser Ile 290 295 300Cys Gly Glu Phe Ser Asp Ala Trp Pro Leu Met Gln
Asn Pro Met 305 310 315Gly Gly Asp Asn Ile Ser Phe Cys Asp Ser Tyr
Pro Glu Leu Thr 320 325 330Gly Glu Asp Ile His Ser Leu Asn Pro Glu
Leu Glu Ser Ser Thr 335 340 345Ser Leu Asp Ser Asn Ser Ser Gln Asp
Leu Val Gly Gly Ala Val 350 355 360Pro Val Gln Ser His Ser Glu Asn
Phe Thr Ala Ala Thr Asp Leu 365 370 375Ser Arg Tyr Asn Asn Thr Leu
Val Glu Ser Ala Ser Thr Gln Asp 380 385 390Ala Leu Thr Met Arg Ser
Gln Leu Asp Gln Glu Ser Gly Ala Val 395 400 405Ile His Pro Ala Thr
Gln Thr Ser Leu Gln Glu Ala 410 415234842DNAHomo sapiens
23cgcgctcccc gcgcgcctcc tcgggctcca cgcgtcttgc cccgcagagg
50cagcctcctc caggagcggg gccctgcaca ccatggcccc cgggtgggca
100ggggtcggcg ccgccgtgcg cgcccgcctg gcgctggcct tggcgctggc
150gagcgtcctg agtgggcctc cagccgtcgc ctgccccacc aagtgtacct
200gctccgctgc cagcgtggac tgccacgggc tgggcctccg cgcggttcct
250cggggcatcc cccgcaacgc tgagcgcctt gacctggaca gaaataatat
300caccaggatc accaagatgg acttcgctgg gctcaagaac ctccgagtct
350tgcatctgga agacaaccag gtcagcgtca tcgagagagg cgccttccag
400gacctgaagc agctagagcg actgcgcctg aacaagaata agctgcaagt
450ccttccagaa ttgcttttcc agagcacgcc gaagctcacc agactagatt
500tgagtgaaaa ccagatccag gggatcccga ggaaggcgtt ccgcggcatc
550accgatgtga agaacctgca actggacaac aaccacatca gctgcattga
600agatggagcc ttccgagcgc tgcgcgattt ggagatcctt accctcaaca
650acaacaacat cagtcgcatc ctggtcacca gcttcaacca catgccgaag
700atccgaactc tgcgcctcca ctccaaccac ctctactgcg actgccacct
750ggcctggctc tcggattggc tgcgacagcg acggacagtt ggccagttca
800cactctgcat ggctcctgtg catttgaggg gcttcaacgt ggcggatgtg
850cagaagaagg agtacgtgtg cccagccccc cactcggagc ccccatcctg
900caatgccaac tccatctcct gcccttcgcc ctgcacgtgc agcaataaca
950tcgtggactg tcgaggaaag ggcttgatgg agattcctgc caacttgccg
1000gagggcatcg tcgaaatacg cctagaacag aactccatca aagccatccc
1050tgcaggagcc ttcacccagt acaagaaact gaagcgaata gacatcagca
1100agaatcagat atcggatatt gctccagatg ccttccaggg cctgaaatca
1150ctcacatcgc tggtcctgta tgggaacaag atcaccgaga ttgccaaggg
1200actgtttgat gggctggtgt ccctacagct gctcctcctc aatgccaaca
1250agatcaactg cctgcgggtg aacacgtttc aggacctgca gaacctcaac
1300ttgctctccc tgtatgacaa caagctgcag accatcagca aggggctctt
1350cgcccctctg cagtccatcc agacactcca cttagcccaa aacccatttg
1400tgtgcgactg ccacttgaag tggctggccg actacctcca ggacaacccc
1450atcgagacaa gcggggcccg ctgcagcagc ccgcgccgac tcgccaacaa
1500gcgcatcagc cagatcaaga gcaagaagtt ccgctgctca ggctccgagg
1550attaccgcag caggttcagc agcgagtgct tcatggacct cgtgtgcccc
1600gagaagtgtc gctgtgaggg cacgattgtg gactgctcca accagaagct
1650ggtccgcatc ccaagccacc tccctgaata tgtcaccgac ctgcgactga
1700atgacaatga ggtatctgtt ctggaggcca ctggcatctt caagaagttg
1750cccaacctgc ggaaaataaa tctgagtaac aataagatca aggaggtgcg
1800agagggagct ttcgatggag cagccagcgt gcaggagctg atgctgacag
1850ggaaccagct ggagaccgtg cacgggcgcg tgttccgtgg cctcagtggc
1900ctcaaaacct tgatgctgag gagtaacttg atcagctgtg tgagtaatga
1950cacctttgcc ggcctgagtt cggtgagact gctgtccctc tatgacaatc
2000ggatcaccac catcacccct ggggccttca ccacgcttgt ctccctgtcc
2050accataaacc tcctgtccaa ccccttcaac tgcaactgcc acctggcctg
2100gctcggcaag tggttgagga agaggcggat cgtcagtggg aaccctaggt
2150gccagaagcc atttttcctc aaggagattc ccatccagga tgtggccatc
2200caggacttca cctgtgatgg caacgaggag agtagctgcc agctgagccc
2250gcgctgcccg gagcagtgca cctgtatgga gacagtggtg cgatgcagca
2300acaaggggct ccgcgccctc cccagaggca tgcccaagga tgtgaccgag
2350ctgtacctgg aaggaaacca cctaacagcc gtgcccagag agctgtccgc
2400cctccgacac ctgacgctta ttgacctgag caacaacagc atcagcatgc
2450tgaccaatta caccttcagt aacatgtctc acctctccac tctgatcctg
2500agctacaacc ggctgaggtg catccccgtc
cacgccttca acgggctgcg 2550gtccctgcga gtgctaaccc tccatggcaa
tgacatttcc agcgttcctg 2600aaggctcctt caacgacctc acatctcttt
cccatctggc gctgggaacc 2650aacccactcc actgtgactg cagtcttcgg
tggctgtcgg agtgggtgaa 2700ggcggggtac aaggagcctg gcatcgcccg
ctgcagtagc cctgagccca 2750tggctgacag gctcctgctc accaccccaa
cccaccgctt ccagtgcaaa 2800gggccagtgg acatcaacat tgtggccaaa
tgcaatgcct gcctctccag 2850cccgtgcaag aataacggga catgcaccca
ggaccctgtg gagctgtacc 2900gctgtgcctg cccctacagc tacaagggca
aggactgcac tgtgcccatc 2950aacacctgca tccagaaccc ctgtcagcat
ggaggcacct gccacctgag 3000tgacagccac aaggatgggt tcagctgctc
ctgccctctg ggctttgagg 3050ggcagcggtg tgagatcaac ccagatgact
gtgaggacaa cgactgcgaa 3100aacaatgcca cctgcgtgga cgggatcaac
aactacgtgt gtatctgtcc 3150gcctaactac acaggtgagc tatgcgacga
ggtgattgac cactgtgtgc 3200ctgagctgaa cctctgtcag catgaggcca
agtgcatccc cctggacaaa 3250ggattcagct gcgagtgtgt ccctggctac
agcgggaagc tctgtgagac 3300agacaatgat gactgtgtgg cccacaagtg
ccgccacggg gcccagtgcg 3350tggacacaat caatggctac acatgcacct
gcccccaggg cttcagtgga 3400cccttctgtg aacacccccc acccatggtc
ctactgcaga ccagcccatg 3450cgaccagtac gagtgccaga acggggccca
gtgcatcgtg gtgcagcagg 3500agcccacctg ccgctgccca ccaggcttcg
ccggccccag atgcgagaag 3550ctcatcactg tcaacttcgt gggcaaagac
tcctacgtgg aactggcctc 3600cgccaaggtc cgaccccagg ccaacatctc
cctgcaggtg gccactgaca 3650aggacaacgg catccttctc tacaaaggag
acaatgaccc cctggcactg 3700gagctgtacc agggccacgt gcggctggtc
tatgacagcc tgagttcccc 3750tccaaccaca gtgtacagtg tggagacagt
gaatgatggg cagtttcaca 3800gtgtggagct ggtgacgcta aaccagaccc
tgaacctagt agtggacaaa 3850ggaactccaa agagcctggg gaagctccag
aagcagccag cagtgggcat 3900caacagcccc ctctaccttg gaggcatccc
cacctccacc ggcctctccg 3950ccttgcgcca gggcacggac cggcctctag
gcggcttcca cggatgcatc 4000catgaggtgc gcatcaacaa cgagctgcag
gacttcaagg ccctcccacc 4050acagtccctg ggggtgtcac caggctgcaa
gtcctgcacc gtgtgcaagc 4100acggcctgtg ccgctccgtg gagaaggaca
gcgtggtgtg cgagtgccgc 4150ccaggctgga ccggcccact ctgcgaccag
gaggcccggg acccctgcct 4200cggccacaga tgccaccatg gaaaatgtgt
ggcaactggg acctcataca 4250tgtgcaagtg tgccgagggc tatggagggg
acttgtgtga caacaagaat 4300gactctgcca atgcctgctc agccttcaag
tgtcaccatg ggcagtgcca 4350catctcagac caaggggagc cctactgcct
gtgccagccc ggctttagcg 4400gcgagcactg ccaacaagag aatccgtgcc
tgggacaagt agtccgagag 4450gtgatccgcc gccagaaagg ttatgcatca
tgtgccacag cctccaaggt 4500gcccatcatg gaatgtcgtg ggggctgtgg
gccccagtgc tgccagccca 4550cccgcagcaa gcggcggaaa tacgtcttcc
agtgcacgga cggctcctcg 4600tttgtagaag aggtggagag acacttagag
tgcggctgcc tcgcgtgttc 4650ctaagcccct gcccgcctgc ctgccacctc
tcggactcca gcttgatgga 4700gttgggacag ccatgtggga ccccctggtg
attcagcatg aaggaaatga 4750agctggagag gaaggtaaag aagaagagaa
tattaagtat attgtaaaat 4800aaacaaaaaa tagaacttaa aaaaaaaaaa
aaaaaaaaaa aa 4842241523PRTHomo sapiens 24Met Ala Pro Gly Trp Ala
Gly Val Gly Ala Ala Val Arg Ala Arg1 5 10 15Leu Ala Leu Ala Leu Ala
Leu Ala Ser Val Leu Ser Gly Pro Pro 20 25 30Ala Val Ala Cys Pro Thr
Lys Cys Thr Cys Ser Ala Ala Ser Val 35 40 45Asp Cys His Gly Leu Gly
Leu Arg Ala Val Pro Arg Gly Ile Pro 50 55 60Arg Asn Ala Glu Arg Leu
Asp Leu Asp Arg Asn Asn Ile Thr Arg 65 70 75Ile Thr Lys Met Asp Phe
Ala Gly Leu Lys Asn Leu Arg Val Leu 80 85 90His Leu Glu Asp Asn Gln
Val Ser Val Ile Glu Arg Gly Ala Phe 95 100 105Gln Asp Leu Lys Gln
Leu Glu Arg Leu Arg Leu Asn Lys Asn Lys 110 115 120Leu Gln Val Leu
Pro Glu Leu Leu Phe Gln Ser Thr Pro Lys Leu 125 130 135Thr Arg Leu
Asp Leu Ser Glu Asn Gln Ile Gln Gly Ile Pro Arg 140 145 150Lys Ala
Phe Arg Gly Ile Thr Asp Val Lys Asn Leu Gln Leu Asp 155 160 165Asn
Asn His Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu 170 175
180Arg Asp Leu Glu Ile Leu Thr Leu Asn Asn Asn Asn Ile Ser Arg 185
190 195Ile Leu Val Thr Ser Phe Asn His Met Pro Lys Ile Arg Thr Leu
200 205 210Arg Leu His Ser Asn His Leu Tyr Cys Asp Cys His Leu Ala
Trp 215 220 225Leu Ser Asp Trp Leu Arg Gln Arg Arg Thr Val Gly Gln
Phe Thr 230 235 240Leu Cys Met Ala Pro Val His Leu Arg Gly Phe Asn
Val Ala Asp 245 250 255Val Gln Lys Lys Glu Tyr Val Cys Pro Ala Pro
His Ser Glu Pro 260 265 270Pro Ser Cys Asn Ala Asn Ser Ile Ser Cys
Pro Ser Pro Cys Thr 275 280 285Cys Ser Asn Asn Ile Val Asp Cys Arg
Gly Lys Gly Leu Met Glu 290 295 300Ile Pro Ala Asn Leu Pro Glu Gly
Ile Val Glu Ile Arg Leu Glu 305 310 315Gln Asn Ser Ile Lys Ala Ile
Pro Ala Gly Ala Phe Thr Gln Tyr 320 325 330Lys Lys Leu Lys Arg Ile
Asp Ile Ser Lys Asn Gln Ile Ser Asp 335 340 345Ile Ala Pro Asp Ala
Phe Gln Gly Leu Lys Ser Leu Thr Ser Leu 350 355 360Val Leu Tyr Gly
Asn Lys Ile Thr Glu Ile Ala Lys Gly Leu Phe 365 370 375Asp Gly Leu
Val Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys 380 385 390Ile Asn
Cys Leu Arg Val Asn Thr Phe Gln Asp Leu Gln Asn Leu 395 400 405Asn
Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ser Lys 410 415
420Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln Thr Leu His Leu Ala 425
430 435Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys Trp Leu Ala Asp
440 445 450Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys
Ser 455 460 465Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile
Lys Ser 470 475 480Lys Lys Phe Arg Cys Ser Gly Ser Glu Asp Tyr Arg
Ser Arg Phe 485 490 495Ser Ser Glu Cys Phe Met Asp Leu Val Cys Pro
Glu Lys Cys Arg 500 505 510Cys Glu Gly Thr Ile Val Asp Cys Ser Asn
Gln Lys Leu Val Arg 515 520 525Ile Pro Ser His Leu Pro Glu Tyr Val
Thr Asp Leu Arg Leu Asn 530 535 540Asp Asn Glu Val Ser Val Leu Glu
Ala Thr Gly Ile Phe Lys Lys 545 550 555Leu Pro Asn Leu Arg Lys Ile
Asn Leu Ser Asn Asn Lys Ile Lys 560 565 570Glu Val Arg Glu Gly Ala
Phe Asp Gly Ala Ala Ser Val Gln Glu 575 580 585Leu Met Leu Thr Gly
Asn Gln Leu Glu Thr Val His Gly Arg Val 590 595 600Phe Arg Gly Leu
Ser Gly Leu Lys Thr Leu Met Leu Arg Ser Asn 605 610 615Leu Ile Ser
Cys Val Ser Asn Asp Thr Phe Ala Gly Leu Ser Ser 620 625 630Val Arg
Leu Leu Ser Leu Tyr Asp Asn Arg Ile Thr Thr Ile Thr 635 640 645Pro
Gly Ala Phe Thr Thr Leu Val Ser Leu Ser Thr Ile Asn Leu 650 655
660Leu Ser Asn Pro Phe Asn Cys Asn Cys His Leu Ala Trp Leu Gly 665
670 675Lys Trp Leu Arg Lys Arg Arg Ile Val Ser Gly Asn Pro Arg Cys
680 685 690Gln Lys Pro Phe Phe Leu Lys Glu Ile Pro Ile Gln Asp Val
Ala 695 700 705Ile Gln Asp Phe Thr Cys Asp Gly Asn Glu Glu Ser Ser
Cys Gln 710 715 720Leu Ser Pro Arg Cys Pro Glu Gln Cys Thr Cys Met
Glu Thr Val 725 730 735Val Arg Cys Ser Asn Lys Gly Leu Arg Ala Leu
Pro Arg Gly Met 740 745 750Pro Lys Asp Val Thr Glu Leu Tyr Leu Glu
Gly Asn His Leu Thr 755 760 765Ala Val Pro Arg Glu Leu Ser Ala Leu
Arg His Leu Thr Leu Ile 770 775 780Asp Leu Ser Asn Asn Ser Ile Ser
Met Leu Thr Asn Tyr Thr Phe 785 790 795Ser Asn Met Ser His Leu Ser
Thr Leu Ile Leu Ser Tyr Asn Arg 800 805 810Leu Arg Cys Ile Pro Val
His Ala Phe Asn Gly Leu Arg Ser Leu 815 820 825Arg Val Leu Thr Leu
His Gly Asn Asp Ile Ser Ser Val Pro Glu 830 835 840Gly Ser Phe Asn
Asp Leu Thr Ser Leu Ser His Leu Ala Leu Gly 845 850 855Thr Asn Pro
Leu His Cys Asp Cys Ser Leu Arg Trp Leu Ser Glu 860 865 870Trp Val
Lys Ala Gly Tyr Lys Glu Pro Gly Ile Ala Arg Cys Ser 875 880 885Ser
Pro Glu Pro Met Ala Asp Arg Leu Leu Leu Thr Thr Pro Thr 890 895
900His Arg Phe Gln Cys Lys Gly Pro Val Asp Ile Asn Ile Val Ala 905
910 915Lys Cys Asn Ala Cys Leu Ser Ser Pro Cys Lys Asn Asn Gly Thr
920 925 930Cys Thr Gln Asp Pro Val Glu Leu Tyr Arg Cys Ala Cys Pro
Tyr 935 940 945Ser Tyr Lys Gly Lys Asp Cys Thr Val Pro Ile Asn Thr
Cys Ile 950 955 960Gln Asn Pro Cys Gln His Gly Gly Thr Cys His Leu
Ser Asp Ser 965 970 975His Lys Asp Gly Phe Ser Cys Ser Cys Pro Leu
Gly Phe Glu Gly 980 985 990Gln Arg Cys Glu Ile Asn Pro Asp Asp Cys
Glu Asp Asn Asp Cys 995 1000 1005Glu Asn Asn Ala Thr Cys Val Asp
Gly Ile Asn Asn Tyr Val Cys 1010 1015 1020Ile Cys Pro Pro Asn Tyr
Thr Gly Glu Leu Cys Asp Glu Val Ile 1025 1030 1035Asp His Cys Val
Pro Glu Leu Asn Leu Cys Gln His Glu Ala Lys 1040 1045 1050Cys Ile
Pro Leu Asp Lys Gly Phe Ser Cys Glu Cys Val Pro Gly 1055 1060
1065Tyr Ser Gly Lys Leu Cys Glu Thr Asp Asn Asp Asp Cys Val Ala
1070 1075 1080His Lys Cys Arg His Gly Ala Gln Cys Val Asp Thr Ile
Asn Gly 1085 1090 1095Tyr Thr Cys Thr Cys Pro Gln Gly Phe Ser Gly
Pro Phe Cys Glu 1100 1105 1110His Pro Pro Pro Met Val Leu Leu Gln
Thr Ser Pro Cys Asp Gln 1115 1120 1125Tyr Glu Cys Gln Asn Gly Ala
Gln Cys Ile Val Val Gln Gln Glu 1130 1135 1140Pro Thr Cys Arg Cys
Pro Pro Gly Phe Ala Gly Pro Arg Cys Glu 1145 1150 1155Lys Leu Ile
Thr Val Asn Phe Val Gly Lys Asp Ser Tyr Val Glu 1160 1165 1170Leu
Ala Ser Ala Lys Val Arg Pro Gln Ala Asn Ile Ser Leu Gln 1175 1180
1185Val Ala Thr Asp Lys Asp Asn Gly Ile Leu Leu Tyr Lys Gly Asp
1190 1195 1200Asn Asp Pro Leu Ala Leu Glu Leu Tyr Gln Gly His Val
Arg Leu 1205 1210 1215Val Tyr Asp Ser Leu Ser Ser Pro Pro Thr Thr
Val Tyr Ser Val 1220 1225 1230Glu Thr Val Asn Asp Gly Gln Phe His
Ser Val Glu Leu Val Thr 1235 1240 1245Leu Asn Gln Thr Leu Asn Leu
Val Val Asp Lys Gly Thr Pro Lys 1250 1255 1260Ser Leu Gly Lys Leu
Gln Lys Gln Pro Ala Val Gly Ile Asn Ser 1265 1270 1275Pro Leu Tyr
Leu Gly Gly Ile Pro Thr Ser Thr Gly Leu Ser Ala 1280 1285 1290Leu
Arg Gln Gly Thr Asp Arg Pro Leu Gly Gly Phe His Gly Cys 1295 1300
1305Ile His Glu Val Arg Ile Asn Asn Glu Leu Gln Asp Phe Lys Ala
1310 1315 1320Leu Pro Pro Gln Ser Leu Gly Val Ser Pro Gly Cys Lys
Ser Cys 1325 1330 1335Thr Val Cys Lys His Gly Leu Cys Arg Ser Val
Glu Lys Asp Ser 1340 1345 1350Val Val Cys Glu Cys Arg Pro Gly Trp
Thr Gly Pro Leu Cys Asp 1355 1360 1365Gln Glu Ala Arg Asp Pro Cys
Leu Gly His Arg Cys His His Gly 1370 1375 1380Lys Cys Val Ala Thr
Gly Thr Ser Tyr Met Cys Lys Cys Ala Glu 1385 1390 1395Gly Tyr Gly
Gly Asp Leu Cys Asp Asn Lys Asn Asp Ser Ala Asn 1400 1405 1410Ala
Cys Ser Ala Phe Lys Cys His His Gly Gln Cys His Ile Ser 1415 1420
1425Asp Gln Gly Glu Pro Tyr Cys Leu Cys Gln Pro Gly Phe Ser Gly
1430 1435 1440Glu His Cys Gln Gln Glu Asn Pro Cys Leu Gly Gln Val
Val Arg 1445 1450 1455Glu Val Ile Arg Arg Gln Lys Gly Tyr Ala Ser
Cys Ala Thr Ala 1460 1465 1470Ser Lys Val Pro Ile Met Glu Cys Arg
Gly Gly Cys Gly Pro Gln 1475 1480 1485Cys Cys Gln Pro Thr Arg Ser
Lys Arg Arg Lys Tyr Val Phe Gln 1490 1495 1500Cys Thr Asp Gly Ser
Ser Phe Val Glu Glu Val Glu Arg His Leu 1505 1510 1515Glu Cys Gly
Cys Leu Ala Cys Ser 1520251318DNAHomo sapiens 25cagtttcttc
atctgtaaca tcaaatgaat aataatacca atctcctaga 50cttcataaga ggattaacaa
agacaaaata tgggaaaaac ataacatggc 100gtcccataat tattagatct
tattattgac actaaaatgg cattaaaatt 150accaaaagga agacagcatc
tgtttcctct ttggtcctga gctggttaaa 200aggaacactg gttgcctgaa
cagtcacact tgcaaccatg atgcctaaac 250attgctttct aggcttcctc
atcagtttct tccttactgg tgtagcagga 300actcagtcaa cgcatgagtc
tctgaagcct cagagggtac aatttcagtc 350ccgaaatttt cacaacattt
tgcaatggca gcctgggagg gcacttactg 400gcaacagcag tgtctatttt
gtgcagtaca aaatcatgtt ctcatgcagc 450atgaaaagct ctcaccagaa
gccaagtgga tgctggcagc acatttcttg 500taacttccca ggctgcagaa
cattggctaa atatggacag agacaatgga 550aaaataaaga agactgttgg
ggtactcaag aactctcttg tgaccttacc 600agtgaaacct cagacataca
ggaaccttat tacgggaggg tgagggcggc 650ctcggctggg agctactcag
aatggagcat gacgccgcgg ttcactccct 700ggtgggaaac aaaaatagat
cctccagtca tgaatataac ccaagtcaat 750ggctctttgt tggtaattct
ccatgctcca aatttaccat atagatacca 800aaaggaaaaa aatgtatcta
tagaagatta ctatgaacta ctataccgag 850tttttataat taacaattca
ctagaaaagg agcaaaaggt ttatgaaggg 900gctcacagag cggttgaaat
tgaagctcta acaccacact ccagctactg 950tgtagtggct gaaatatatc
agcccatgtt agacagaaga agtcagagaa 1000gtgaagagag atgtgtggaa
attccatgac ttgtggaatt tggcattcag 1050caatgtggaa attctaaagc
tccctgagaa caggatgact cgtgtttgaa 1100ggatcttatt taaaattgtt
tttgtatttt cttaaagcaa tattcactgt 1150tacaccttgg ggacttcttt
gtttatccat tcttttatcc tttatatttc 1200atttgtaaac tatatttgaa
cgacattccc cccgaaaaat tgaaatgtaa 1250agatgaggca gagaataaag
tgttctatga aaaaaaaaaa aaaaaaaaaa 1300aaaaaaaaaa aaaaaaaa
131826262PRTHomo sapiens 26Met Pro Lys His Cys Phe Leu Gly Phe Leu
Ile Ser Phe Phe Leu1 5 10 15Thr Gly Val Ala Gly Thr Gln Ser Thr His
Glu Ser Leu Lys Pro 20 25 30Gln Arg Val Gln Phe Gln Ser Arg Asn Phe
His Asn Ile Leu Gln 35 40 45Trp Gln Pro Gly Arg Ala Leu Thr Gly Asn
Ser Ser Val Tyr Phe 50 55 60Val Gln Tyr Lys Ile Met Phe Ser Cys Ser
Met Lys Ser Ser His 65 70 75Gln Lys Pro Ser Gly Cys Trp Gln His Ile
Ser Cys Asn Phe Pro 80 85 90Gly Cys Arg Thr Leu Ala Lys Tyr Gly Gln
Arg Gln Trp Lys Asn
95 100 105Lys Glu Asp Cys Trp Gly Thr Gln Glu Leu Ser Cys Asp Leu
Thr 110 115 120Ser Glu Thr Ser Asp Ile Gln Glu Pro Tyr Tyr Gly Arg
Val Arg 125 130 135Ala Ala Ser Ala Gly Ser Tyr Ser Glu Trp Ser Met
Thr Pro Arg 140 145 150Phe Thr Pro Trp Trp Glu Thr Lys Ile Asp Pro
Pro Val Met Asn 155 160 165Ile Thr Gln Val Asn Gly Ser Leu Leu Val
Ile Leu His Ala Pro 170 175 180Asn Leu Pro Tyr Arg Tyr Gln Lys Glu
Lys Asn Val Ser Ile Glu 185 190 195Asp Tyr Tyr Glu Leu Leu Tyr Arg
Val Phe Ile Ile Asn Asn Ser 200 205 210Leu Glu Lys Glu Gln Lys Val
Tyr Glu Gly Ala His Arg Ala Val 215 220 225Glu Ile Glu Ala Leu Thr
Pro His Ser Ser Tyr Cys Val Val Ala 230 235 240Glu Ile Tyr Gln Pro
Met Leu Asp Arg Arg Ser Gln Arg Ser Glu 245 250 255Glu Arg Cys Val
Glu Ile Pro 260273819DNAHomo sapiens 27ggaaggggag gagcaggcca
cacaggcaca ggccggtgag ggacctgccc 50agacctggag ggtctcgctc tgtcacacag
gctggagtgc agtggtgtga 100tcttggctca tcgtaacctc cacctcccgg
gttcaagtga ttctcatgcc 150tcagcctccc gagtagctgg gattacaggt
ggtgacttcc aagagtgact 200ccgtcggagg aaaatgactc cccagtcgct
gctgcagacg acactgttcc 250tgctgagtct gctcttcctg gtccaaggtg
cccacggcag gggccacagg 300gaagactttc gcttctgcag ccagcggaac
cagacacaca ggagcagcct 350ccactacaaa cccacaccag acctgcgcat
ctccatcgag aactccgaag 400aggccctcac agtccatgcc cctttccctg
cagcccaccc tgcttcccga 450tccttccctg accccagggg cctctaccac
ttctgcctct actggaaccg 500acatgctggg agattacatc ttctctatgg
caagcgtgac ttcttgctga 550gtgacaaagc ctctagcctc ctctgcttcc
agcaccagga ggagagcctg 600gctcagggcc ccccgctgtt agccacttct
gtcacctcct ggtggagccc 650tcagaacatc agcctgccca gtgccgccag
cttcaccttc tccttccaca 700gtcctcccca cacggccgct cacaatgcct
cggtggacat gtgcgagctc 750aaaagggacc tccagctgct cagccagttc
ctgaagcatc cccagaaggc 800ctcaaggagg ccctcggctg cccccgccag
ccagcagttg cagagcctgg 850agtcgaaact gacctctgtg agattcatgg
gggacatggt gtccttcgag 900gaggaccgga tcaacgccac ggtgtggaag
ctccagccca cagccggcct 950ccaggacctg cacatccact cccggcagga
ggaggagcag agcgagatca 1000tggagtactc ggtgctgctg cctcgaacac
tcttccagag gacgaaaggc 1050cggagcgggg aggctgagaa gagactcctc
ctggtggact tcagcagcca 1100agccctgttc caggacaaga attccagcca
agtcctgggt gagaaggtct 1150tggggattgt ggtacagaac accaaagtag
ccaacctcac ggagcccgtg 1200gtgctcactt tccagcacca gctacagccg
aagaatgtga ctctgcaatg 1250tgtgttctgg gttgaagacc ccacattgag
cagcccgggg cattggagca 1300gtgctgggtg tgagaccgtc aggagagaaa
cccaaacatc ctgcttctgc 1350aaccacttga cctactttgc agtgctgatg
gtctcctcgg tggaggtgga 1400cgccgtgcac aagcactacc tgagcctcct
ctcctacgtg ggctgtgtcg 1450tctctgccct ggcctgcctt gtcaccattg
ccgcctacct ctgctccagg 1500gtgcccctgc cgtgcaggag gaaacctcgg
gactacacca tcaaggtgca 1550catgaacctg ctgctggccg tcttcctgct
ggacacgagc ttcctgctca 1600gcgagccggt ggccctgaca ggctctgagg
ctggctgccg agccagtgcc 1650atcttcctgc acttctccct gctcacctgc
ctttcctgga tgggcctcga 1700ggggtacaac ctctaccgac tcgtggtgga
ggtctttggc acctatgtcc 1750ctggctacct actcaagctg agcgccatgg
gctggggctt ccccatcttt 1800ctggtgacgc tggtggccct ggtggatgtg
gacaactatg gccccatcat 1850cttggctgtg cataggactc cagagggcgt
catctaccct tccatgtgct 1900ggatccggga ctccctggtc agctacatca
ccaacctggg cctcttcagc 1950ctggtgtttc tgttcaacat ggccatgcta
gccaccatgg tggtgcagat 2000cctgcggctg cgcccccaca cccaaaagtg
gtcacatgtg ctgacactgc 2050tgggcctcag cctggtcctt ggcctgccct
gggccttgat cttcttctcc 2100tttgcttctg gcaccttcca gcttgtcgtc
ctctaccttt tcagcatcat 2150cacctccttc caaggcttcc tcatcttcat
ctggtactgg tccatgcggc 2200tgcaggcccg gggtggcccc tcccctctga
agagcaactc agacagcgcc 2250aggctcccca tcagctcggg cagcacctcg
tccagccgca tctaggcctc 2300cagcccacct gcccatgtga tgaagcagag
atgcggcctc gtcgcacact 2350gcctgtggcc cccgagccag gcccagcccc
aggccagtca gccgcagact 2400ttggaaagcc caacgaccat ggagagatgg
gccgttgcca tggtggacgg 2450actcccgggc tgggcttttg aattggcctt
ggggactact cggctctcac 2500tcagctccca cgggactcag aagtgcgccg
ccatgctgcc tagggtactg 2550tccccacatc tgtcccaacc cagctggagg
cctggtctct ccttacaacc 2600cctgggccca gccctcattg ctgggggcca
ggccttggat cttgagggtc 2650tggcacatcc ttaatcctgt gcccctgcct
gggacagaaa tgtggctcca 2700gttgctctgt ctctcgtggt caccctgagg
gcactctgca tcctctgtca 2750ttttaacctc aggtggcacc cagggcgaat
ggggcccagg gcagaccttc 2800agggccagag ccctggcgga ggagaggccc
tttgccagga gcacagcagc 2850agctcgccta cctctgagcc caggccccct
ccctccctca gccccccagt 2900cctccctcca tcttccctgg ggttctcctc
ctctcccagg gcctccttgc 2950tccttcgttc acagctgggg gtccccgatt
ccaatgctgt tttttgggga 3000gtggtttcca ggagctgcct ggtgtctgct
gtaaatgttt gtctactgca 3050caagcctcgg cctgcccctg agccaggctc
ggtaccgatg cgtgggctgg 3100gctaggtccc tctgtccatc tgggcctttg
tatgagctgc attgcccttg 3150ctcaccctga ccaagcacac gcctcagagg
ggccctcagc ctctcctgaa 3200gccctcttgt ggcaagaact gtggaccatg
ccagtcccgt ctggtttcca 3250tcccaccact ccaaggactg agactgacct
cctctggtga cactggccta 3300gagcctgaca ctctcctaag aggttctctc
caagccccca aatagctcca 3350ggcgccctcg gccgcccatc atggttaatt
ctgtccaaca aacacacacg 3400ggtagattgc tggcctgttg taggtggtag
ggacacagat gaccgacctg 3450gtcactcctc ctgccaacat tcagtctggt
atgtgaggcg tgcgtgaagc 3500aagaactcct ggagctacag ggacagggag
ccatcattcc tgcctgggaa 3550tcctggaaga cttcctgcag gagtcagcgt
tcaatcttga ccttgaagat 3600gggaaggatg ttctttttac gtaccaattc
ttttgtcttt tgatattaaa 3650aagaagtaca tgttcattgt agagaatttg
gaaactgtag aagagaatca 3700agaagaaaaa taaaaatcag ctgttgtaat
cgcctagcaa aaaaaaaaaa 3750aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 3800aaaaaaaaaa aaaaaaaaa 381928693PRTHomo
sapiens 28Met Thr Pro Gln Ser Leu Leu Gln Thr Thr Leu Phe Leu Leu
Ser1 5 10 15Leu Leu Phe Leu Val Gln Gly Ala His Gly Arg Gly His Arg
Glu 20 25 30Asp Phe Arg Phe Cys Ser Gln Arg Asn Gln Thr His Arg Ser
Ser 35 40 45Leu His Tyr Lys Pro Thr Pro Asp Leu Arg Ile Ser Ile Glu
Asn 50 55 60Ser Glu Glu Ala Leu Thr Val His Ala Pro Phe Pro Ala Ala
His 65 70 75Pro Ala Ser Arg Ser Phe Pro Asp Pro Arg Gly Leu Tyr His
Phe 80 85 90Cys Leu Tyr Trp Asn Arg His Ala Gly Arg Leu His Leu Leu
Tyr 95 100 105Gly Lys Arg Asp Phe Leu Leu Ser Asp Lys Ala Ser Ser
Leu Leu 110 115 120Cys Phe Gln His Gln Glu Glu Ser Leu Ala Gln Gly
Pro Pro Leu 125 130 135Leu Ala Thr Ser Val Thr Ser Trp Trp Ser Pro
Gln Asn Ile Ser 140 145 150Leu Pro Ser Ala Ala Ser Phe Thr Phe Ser
Phe His Ser Pro Pro 155 160 165His Thr Ala Ala His Asn Ala Ser Val
Asp Met Cys Glu Leu Lys 170 175 180Arg Asp Leu Gln Leu Leu Ser Gln
Phe Leu Lys His Pro Gln Lys 185 190 195Ala Ser Arg Arg Pro Ser Ala
Ala Pro Ala Ser Gln Gln Leu Gln 200 205 210Ser Leu Glu Ser Lys Leu
Thr Ser Val Arg Phe Met Gly Asp Met 215 220 225Val Ser Phe Glu Glu
Asp Arg Ile Asn Ala Thr Val Trp Lys Leu 230 235 240Gln Pro Thr Ala
Gly Leu Gln Asp Leu His Ile His Ser Arg Gln 245 250 255Glu Glu Glu
Gln Ser Glu Ile Met Glu Tyr Ser Val Leu Leu Pro 260 265 270Arg Thr
Leu Phe Gln Arg Thr Lys Gly Arg Ser Gly Glu Ala Glu 275 280 285Lys
Arg Leu Leu Leu Val Asp Phe Ser Ser Gln Ala Leu Phe Gln 290 295
300Asp Lys Asn Ser Ser Gln Val Leu Gly Glu Lys Val Leu Gly Ile 305
310 315Val Val Gln Asn Thr Lys Val Ala Asn Leu Thr Glu Pro Val Val
320 325 330Leu Thr Phe Gln His Gln Leu Gln Pro Lys Asn Val Thr Leu
Gln 335 340 345Cys Val Phe Trp Val Glu Asp Pro Thr Leu Ser Ser Pro
Gly His 350 355 360Trp Ser Ser Ala Gly Cys Glu Thr Val Arg Arg Glu
Thr Gln Thr 365 370 375Ser Cys Phe Cys Asn His Leu Thr Tyr Phe Ala
Val Leu Met Val 380 385 390Ser Ser Val Glu Val Asp Ala Val His Lys
His Tyr Leu Ser Leu 395 400 405Leu Ser Tyr Val Gly Cys Val Val Ser
Ala Leu Ala Cys Leu Val 410 415 420Thr Ile Ala Ala Tyr Leu Cys Ser
Arg Val Pro Leu Pro Cys Arg 425 430 435Arg Lys Pro Arg Asp Tyr Thr
Ile Lys Val His Met Asn Leu Leu 440 445 450Leu Ala Val Phe Leu Leu
Asp Thr Ser Phe Leu Leu Ser Glu Pro 455 460 465Val Ala Leu Thr Gly
Ser Glu Ala Gly Cys Arg Ala Ser Ala Ile 470 475 480Phe Leu His Phe
Ser Leu Leu Thr Cys Leu Ser Trp Met Gly Leu 485 490 495Glu Gly Tyr
Asn Leu Tyr Arg Leu Val Val Glu Val Phe Gly Thr 500 505 510Tyr Val
Pro Gly Tyr Leu Leu Lys Leu Ser Ala Met Gly Trp Gly 515 520 525Phe
Pro Ile Phe Leu Val Thr Leu Val Ala Leu Val Asp Val Asp 530 535
540Asn Tyr Gly Pro Ile Ile Leu Ala Val His Arg Thr Pro Glu Gly 545
550 555Val Ile Tyr Pro Ser Met Cys Trp Ile Arg Asp Ser Leu Val Ser
560 565 570Tyr Ile Thr Asn Leu Gly Leu Phe Ser Leu Val Phe Leu Phe
Asn 575 580 585Met Ala Met Leu Ala Thr Met Val Val Gln Ile Leu Arg
Leu Arg 590 595 600Pro His Thr Gln Lys Trp Ser His Val Leu Thr Leu
Leu Gly Leu 605 610 615Ser Leu Val Leu Gly Leu Pro Trp Ala Leu Ile
Phe Phe Ser Phe 620 625 630Ala Ser Gly Thr Phe Gln Leu Val Val Leu
Tyr Leu Phe Ser Ile 635 640 645Ile Thr Ser Phe Gln Gly Phe Leu Ile
Phe Ile Trp Tyr Trp Ser 650 655 660Met Arg Leu Gln Ala Arg Gly Gly
Pro Ser Pro Leu Lys Ser Asn 665 670 675Ser Asp Ser Ala Arg Leu Pro
Ile Ser Ser Gly Ser Thr Ser Ser 680 685 690Ser Arg Ile296220DNAHomo
sapiens 29tgtgcagaat tgtacagttg cgaaaccatg tcgctggcag ctggtgctgg
50cggtggagac ttccctgtgc ggtgctcagt gcatctgcac ccgtggggga
100gggagctctt tctctggccc tgcagtcacc tgaggttgtt accattatga
150acggccgctg ggacccccgc atgtgcatgt actcccccag agtgtccggg
200ggccccagcc aagggacaca tctcacgcag ctgggaacat gtgcaggctg
250atgaagagaa ccggatgagg gcttcacatg aggaagcatg tggccaggtc
300ctctcagaac atcagcctca tcttcctgtc tctgatctat ttcaccaacc
350accccatgtg tctctagaac cccagtgtag cgagctggag agaggactgt
400cctgagggca gcaggcctgg ttgcagctgg cgtgggggtc tcagaatgga
450gccctcagcc ctgaggaaag ctggctcgga gcaggaggag ggctttgagg
500ggctgcccag aagggtcact gacctgggga tggtctccaa tctccggcgc
550agcaacagca gcctcttcaa gagctggagg ctacagtgcc ccttcggcaa
600caatgacaag caagaaagcc tcagttcgtg gattcctgaa aacatcaaga
650agaaagaatg cgtgtatttt gtggaaagtt ccaaactgtc tgatgctggg
700aaggtggtgt gtcagtgtgg ctacacgcat gagcagcact tggaggaggc
750taccaagccc cacaccttcc agggcacaca gtgggaccca aagaaacatg
800tccaggagat gccaaccgat gcctttggcg acatcgtctt cacgggcctg
850agccagaagg tgaaaaagta cgtccgagtc tcccaggaca cgccctccag
900cgtgatctac cacctcatga cccagcactg ggggctggac gtccccaatc
950tcttgatctc ggtgaccggg ggggccaaga acttcaacat gaagccgcgg
1000ctgaagagca ttttccgcag aggcctggtc aaggtggctc agaccacagg
1050ggcctggatc atcacagggg ggtcccacac cggcgtcatg aagcaggtag
1100gcgaggcggt gcgggacttc agcctgagca gcagctacaa ggaaggcgag
1150ctcatcacca tcggagtcgc cacctggggc actgtccacc gccgcgaggg
1200cctgatccat cccacgggca gcttccccgc cgagtacata ctggatgagg
1250atggccaagg gaacctgacc tgcctagaca gcaaccactc tcacttcatc
1300ctcgtggacg acgggaccca cggccagtac ggggtggaga ttcctctgag
1350gaccaggctg gagaagttca tatcggagca gaccaaggaa agaggaggtg
1400tggccatcaa gatccccatc gtgtgcgtgg tgctggaggg cggcccgggc
1450acgttgcaca ccatcgacaa cgccaccacc aacggcaccc cctgtgtggt
1500tgtggagggc tcgggccgcg tggccgacgt cattgcccag gtggccaacc
1550tgcctgtctc ggacatcact atctccctga tccagcagaa actgagcgtg
1600ttcttccagg agatgtttga gaccttcacg gaaagcagga ttgtcgagtg
1650gaccaaaaag atccaagata ttgtccggag gcggcagctg ctgactgtct
1700tccgggaagg caaggatggt cagcaggacg tggatgtggc catcttgcag
1750gccttgctga aagcctcacg gagccaagac cactttggcc acgagaactg
1800ggaccaccag ctgaaactgg cagtggcatg gaatcgcgtg gacattgccc
1850gcagtgagat cttcatggat gagtggcagt ggaagccttc agatctgcac
1900cccacgatga cagctgcact catctccaac aagcctgagt ttgtgaagct
1950cttcctggaa aacggggtgc agctgaagga gtttgtcacc tgggacacct
2000tgctctacct gtacgagaac ctggacccct cctgcctgtt ccacagcaag
2050ctgcaaaagg tgctggtgga ggatcccgag cgcccggctt gcgcgcccgc
2100ggcgccccgc ctgcagatgc accacgtggc ccaggtgctg cgggagctgc
2150tgggggactt cacgcagccg ctttatcccc ggccccggca caacgaccgg
2200ctgcggctcc tgctgcccgt tccccacgtc aagctcaacg tgcagggagt
2250gagcctccgg tccctctaca agcgttcctc aggccatgtg accttcacca
2300tggaccccat ccgtgacctt ctcatttggg ccattgtcca gaaccgtcgg
2350gagctggcag gaatcatctg ggctcagagc caggactgca tcgcagcggc
2400cttggcctgc agcaagatcc tgaaggaact gtccaaggag gaggaggaca
2450cggacagctc ggaggagatg ctggcgctgg cggaggagta tgagcacaga
2500gccatcgggg tcttcaccga gtgctaccgg aaggacgaag agagagccca
2550gaaactgctc acccgcgtgt ccgaggcctg ggggaagacc acctgcctgc
2600agctcgccct ggaggccaag gacatgaagt ttgtgtctca cgggggcatc
2650caggccttcc tgaccaaggt gtggtggggc cagctctccg tggacaatgg
2700gctgtggcgt gtgaccctgt gcatgctggc cttcccgctg ctcctcaccg
2750gcctcatctc cttcagggag aagaggctgc aggatgtggg cacccccgcg
2800gcccgcgccc gtgccttctt caccgcaccc gtggtggtct tccacctgaa
2850catcctctcc tacttcgcct tcctctgcct gttcgcctac gtgctcatgg
2900tggacttcca gcctgtgccc tcctggtgcg agtgtgccat ctacctctgg
2950ctcttctcct tggtgtgcga ggagatgcgg cagctcttct atgaccctga
3000cgagtgcggg ctgatgaaga aggcagcctt gtacttcagt gacttctgga
3050ataagctgga cgtcggcgca atcttgctct tcgtggcagg gctgacctgc
3100aggctcatcc cggcgacgct gtaccccggg cgcgtcatcc tctctctgga
3150cttcatcctg ttctgcctcc ggctcatgca catttttacc atcagtaaga
3200cgctggggcc caagatcatc attgtgaagc ggatgatgaa ggacgtcttc
3250ttcttcctct tcctgctggc tgtgtgggtg gtgtccttcg gggtggccaa
3300gcaggccatc ctcatccaca acgagcgccg ggtggactgg ctgttccgag
3350gggccgtcta ccactcctac ctcaccatct tcgggcagat cccgggctac
3400atcgacggtg tgaacttcaa cccggagcac tgcagcccca atggcaccga
3450cccctacaag cctaagtgcc ccgagagcga cgcgacgcag cagaggccgg
3500ccttccctga gtggctgacg gtcctcctac tctgcctcta cctgctcttc
3550accaacatcc tgctgctcaa cctcctcatc gccatgttca actacacctt
3600ccagcaggtg caggagcaca cggaccagat ttggaagttc cagcgccatg
3650acctgatcga ggagtaccac ggccgccccg ccgcgccgcc ccccttcatc
3700ctcctcagcc acctgcagct cttcatcaag agggtggtcc tgaagactcc
3750ggccaagagg cacaagcagc tcaagaacaa gctggagaag aacgaggagg
3800cggccctgct atcctgggag atctacctga aggagaacta cctccagaac
3850cgacagttcc agcaaaagca gcggcccgag cagaagatcg aggacatcag
3900caataaggtt gacgccatgg tggacctgct ggacctggac ccactgaaga
3950ggtcgggctc catggagcag aggttggcct ccctggagga gcaggtggcc
4000cagacagccc gagccctgca ctggatcgtg aggacgctgc gggccagcgg
4050cttcagctcg gaggcggacg tccccactct ggcctcccag aaggccgcgg
4100aggagccgga tgctgagccg ggaggcagga agaagacgga ggagccgggc
4150gacagctacc acgtgaatgc ccggcacctc ctctacccca actgccctgt
4200cacgcgcttc cccgtgccca acgagaaggt gccctgggag acggagttcc
4250tgatctatga cccacccttt tacacggcag agaggaagga cgcggccgcc
4300atggacccca tgggagacac cctggagcca ctgtccacga tccagtacaa
4350cgtggtggat ggcctgaggg accgccggag cttccacggg ccgtacacag
4400tgcaggccgg gttgcccctg aaccccatgg gccgcacagg actgcgtggg
4450cgcgggagcc tcagctgctt cggacccaac cacacgctgt accccatggt
4500cacgcggtgg aggcggaacg aggatggagc catctgcagg aagagcataa
4550agaagatgct ggaagtgctg gtggtgaagc tccctctctc cgagcactgg
4600gccctgcctg ggggctcccg ggagccaggg gagatgctac ctcggaagct
4650gaagcggatc ctccggcagg agcactggcc gtcttttgaa aacttgctga
4700agtgcggcat ggaggtgtac aaaggctaca tggatgaccc gaggaacacg
4750gacaatgcct ggatcgagac ggtggccgtc agcgtccact tccaggacca
4800gaatgacgtg gagctgaaca ggctgaactc taacctgcac gcctgcgact
4850cgggggcctc catccgatgg caggtggtgg acaggcgcat cccactctat
4900gcgaaccaca agaccctcct ccagaaggca gccgctgagt tcggggctca
4950ctactgactg tgccctcagg ctgggcggct ccagtccata gacgttcccc
5000ccagaaacca gggcttctct ctcctgagcc tggccaggac tcaggctgtt
5050cctgggccct gcacatgatg gggtttggtg gacccagtgc ccctcacggc
5100tgccgcaagt ctgctgcaga tgacctcatg aactggaagg ggtcaaggtg
5150acccgggagg agagctcaag acagggcaca ggctactcag agctgagggg
5200cccctgggac ccttggccat caggcgaggg gctgggcctg tgcagctggg
5250cccttggcca gagtccactc ccttcctggc tgtgtcaccc cgagcagctc
5300atccaccatg gaggtcattg gcctgaggca agttccccgg agagtcggga
5350tcccctgtgg ccccctcagg cctatgtctg tgaggaaggg gccctgccac
5400tctccccaag agggcctcca tgtttcgagg tgcctcaaca tggagccttg
5450cctggcctgg gctaggggca ctgtctgaac tcctgactgt caggataaac
5500tccgtggggg tacaggagcc cagacaaagc ccaggcctgt caagagacgc
5550agagggcccc tgccagggtt ggccccaggg accctgggac gaggctgcag
5600aagctctccc tccctactcc ctgggagcca cgtgctggcc atgtggccag
5650ggacggcatg agcaggaggc ggggacgtgg gggccttctg gtttggtgtc
5700aacagctcac aggagcgtga accatgaggg ccctcaggag gggaacgtgg
5750taaaacccaa gacattaaat ctgccatctc aggcctggct ggctcttctg
5800tgctttccac aaataaagtt cctgacacgt ccagggccag gggctgtgtg
5850acggctgcct gaagttctcc tcgatccccc ggtgagcttc ctgcagcctg
5900tggatgtcct gcagcccctc agccctaccc ccaagtttct cctctgaccc
5950atcagctccc tgtcttcatt ttcctaaacc tgggctccag catcgtcccc
6000aagcccacca ggccaggatg caggcatcca catgccctcc tccttggctt
6050cccctgcgtg gtggtgccaa tgtgccctgg cacccctgca gaggctccgg
6100atggagcctg gggctgcctg gccactgagc actggccgag gtgatgccca
6150cccttccctg gacaggcctc tgtcttccac ctgacccaaa gctctctagc
6200cacccccttg tccccagtat 6220301503PRTHomo sapiens 30Met Glu Pro
Ser Ala Leu Arg Lys Ala Gly Ser Glu Gln Glu Glu1 5 10 15Gly Phe Glu
Gly Leu Pro Arg Arg Val Thr Asp Leu Gly Met Val 20 25 30Ser Asn Leu
Arg Arg Ser Asn Ser Ser Leu Phe Lys Ser Trp Arg 35 40 45Leu Gln Cys
Pro Phe Gly Asn Asn Asp Lys Gln Glu Ser Leu Ser 50 55 60Ser Trp Ile
Pro Glu Asn Ile Lys Lys Lys Glu Cys Val Tyr Phe 65 70 75Val Glu Ser
Ser Lys Leu Ser Asp Ala Gly Lys Val Val Cys Gln 80 85 90Cys Gly Tyr
Thr His Glu Gln His Leu Glu Glu Ala Thr Lys Pro 95 100 105His Thr
Phe Gln Gly Thr Gln Trp Asp Pro Lys Lys His Val Gln 110 115 120Glu
Met Pro Thr Asp Ala Phe Gly Asp Ile Val Phe Thr Gly Leu 125 130
135Ser Gln Lys Val Lys Lys Tyr Val Arg Val Ser Gln Asp Thr Pro 140
145 150Ser Ser Val Ile Tyr His Leu Met Thr Gln His Trp Gly Leu Asp
155 160 165Val Pro Asn Leu Leu Ile Ser Val Thr Gly Gly Ala Lys Asn
Phe 170 175 180Asn Met Lys Pro Arg Leu Lys Ser Ile Phe Arg Arg Gly
Leu Val 185 190 195Lys Val Ala Gln Thr Thr Gly Ala Trp Ile Ile Thr
Gly Gly Ser 200 205 210His Thr Gly Val Met Lys Gln Val Gly Glu Ala
Val Arg Asp Phe 215 220 225Ser Leu Ser Ser Ser Tyr Lys Glu Gly Glu
Leu Ile Thr Ile Gly 230 235 240Val Ala Thr Trp Gly Thr Val His Arg
Arg Glu Gly Leu Ile His 245 250 255Pro Thr Gly Ser Phe Pro Ala Glu
Tyr Ile Leu Asp Glu Asp Gly 260 265 270Gln Gly Asn Leu Thr Cys Leu
Asp Ser Asn His Ser His Phe Ile 275 280 285Leu Val Asp Asp Gly Thr
His Gly Gln Tyr Gly Val Glu Ile Pro 290 295 300Leu Arg Thr Arg Leu
Glu Lys Phe Ile Ser Glu Gln Thr Lys Glu 305 310 315Arg Gly Gly Val
Ala Ile Lys Ile Pro Ile Val Cys Val Val Leu 320 325 330Glu Gly Gly
Pro Gly Thr Leu His Thr Ile Asp Asn Ala Thr Thr 335 340 345Asn Gly
Thr Pro Cys Val Val Val Glu Gly Ser Gly Arg Val Ala 350 355 360Asp
Val Ile Ala Gln Val Ala Asn Leu Pro Val Ser Asp Ile Thr 365 370
375Ile Ser Leu Ile Gln Gln Lys Leu Ser Val Phe Phe Gln Glu Met 380
385 390Phe Glu Thr Phe Thr Glu Ser Arg Ile Val Glu Trp Thr Lys Lys
395 400 405Ile Gln Asp Ile Val Arg Arg Arg Gln Leu Leu Thr Val Phe
Arg 410 415 420Glu Gly Lys Asp Gly Gln Gln Asp Val Asp Val Ala Ile
Leu Gln 425 430 435Ala Leu Leu Lys Ala Ser Arg Ser Gln Asp His Phe
Gly His Glu 440 445 450Asn Trp Asp His Gln Leu Lys Leu Ala Val Ala
Trp Asn Arg Val 455 460 465Asp Ile Ala Arg Ser Glu Ile Phe Met Asp
Glu Trp Gln Trp Lys 470 475 480Pro Ser Asp Leu His Pro Thr Met Thr
Ala Ala Leu Ile Ser Asn 485 490 495Lys Pro Glu Phe Val Lys Leu Phe
Leu Glu Asn Gly Val Gln Leu 500 505 510Lys Glu Phe Val Thr Trp Asp
Thr Leu Leu Tyr Leu Tyr Glu Asn 515 520 525Leu Asp Pro Ser Cys Leu
Phe His Ser Lys Leu Gln Lys Val Leu 530 535 540Val Glu Asp Pro Glu
Arg Pro Ala Cys Ala Pro Ala Ala Pro Arg 545 550 555Leu Gln Met His
His Val Ala Gln Val Leu Arg Glu Leu Leu Gly 560 565 570Asp Phe Thr
Gln Pro Leu Tyr Pro Arg Pro Arg His Asn Asp Arg 575 580 585Leu Arg
Leu Leu Leu Pro Val Pro His Val Lys Leu Asn Val Gln 590 595 600Gly
Val Ser Leu Arg Ser Leu Tyr Lys Arg Ser Ser Gly His Val 605 610
615Thr Phe Thr Met Asp Pro Ile Arg Asp Leu Leu Ile Trp Ala Ile 620
625 630Val Gln Asn Arg Arg Glu Leu Ala Gly Ile Ile Trp Ala Gln Ser
635 640 645Gln Asp Cys Ile Ala Ala Ala Leu Ala Cys Ser Lys Ile Leu
Lys 650 655 660Glu Leu Ser Lys Glu Glu Glu Asp Thr Asp Ser Ser Glu
Glu Met 665 670 675Leu Ala Leu Ala Glu Glu Tyr Glu His Arg Ala Ile
Gly Val Phe 680 685 690Thr Glu Cys Tyr Arg Lys Asp Glu Glu Arg Ala
Gln Lys Leu Leu 695 700 705Thr Arg Val Ser Glu Ala Trp Gly Lys Thr
Thr Cys Leu Gln Leu 710 715 720Ala Leu Glu Ala Lys Asp Met Lys Phe
Val Ser His Gly Gly Ile 725 730 735Gln Ala Phe Leu Thr Lys Val Trp
Trp Gly Gln Leu Ser Val Asp 740 745 750Asn Gly Leu Trp Arg Val Thr
Leu Cys Met Leu Ala Phe Pro Leu 755 760 765Leu Leu Thr Gly Leu Ile
Ser Phe Arg Glu Lys Arg Leu Gln Asp 770 775 780Val Gly Thr Pro Ala
Ala Arg Ala Arg Ala Phe Phe Thr Ala Pro 785 790 795Val Val Val Phe
His Leu Asn Ile Leu Ser Tyr Phe Ala Phe Leu 800 805 810Cys Leu Phe
Ala Tyr Val Leu Met Val Asp Phe Gln Pro Val Pro 815 820 825Ser Trp
Cys Glu Cys Ala Ile Tyr Leu Trp Leu Phe Ser Leu Val 830 835 840Cys
Glu Glu Met Arg Gln Leu Phe Tyr Asp Pro Asp Glu Cys Gly 845 850
855Leu Met Lys Lys Ala Ala Leu Tyr Phe Ser Asp Phe Trp Asn Lys 860
865 870Leu Asp Val Gly Ala Ile Leu Leu Phe Val Ala Gly Leu Thr Cys
875 880 885Arg Leu Ile Pro Ala Thr Leu Tyr Pro Gly Arg Val Ile Leu
Ser 890 895 900Leu Asp Phe Ile Leu Phe Cys Leu Arg Leu Met His Ile
Phe Thr 905 910 915Ile Ser Lys Thr Leu Gly Pro Lys Ile Ile Ile Val
Lys Arg Met 920 925 930Met Lys Asp Val Phe Phe Phe Leu Phe Leu Leu
Ala Val Trp Val 935 940 945Val Ser Phe Gly Val Ala Lys Gln Ala Ile
Leu Ile His Asn Glu 950 955 960Arg Arg Val Asp Trp Leu Phe Arg Gly
Ala Val Tyr His Ser Tyr 965 970 975Leu Thr Ile Phe Gly Gln Ile Pro
Gly Tyr Ile Asp Gly Val Asn 980 985 990Phe Asn Pro Glu His Cys Ser
Pro Asn Gly Thr Asp Pro Tyr Lys 995 1000 1005Pro Lys Cys Pro Glu
Ser Asp Ala Thr Gln Gln Arg Pro Ala Phe 1010 1015 1020Pro Glu Trp
Leu Thr Val Leu Leu Leu Cys Leu Tyr Leu Leu Phe 1025 1030 1035Thr
Asn Ile Leu Leu Leu Asn Leu Leu Ile Ala Met Phe Asn Tyr 1040 1045
1050Thr Phe Gln Gln Val Gln Glu His Thr Asp Gln Ile Trp Lys Phe
1055 1060 1065Gln Arg His Asp Leu Ile Glu Glu Tyr His Gly Arg Pro
Ala Ala 1070 1075 1080Pro Pro Pro Phe Ile Leu Leu Ser His Leu Gln
Leu Phe Ile Lys 1085 1090 1095Arg Val Val Leu Lys Thr Pro Ala Lys
Arg His Lys Gln Leu Lys 1100 1105 1110Asn Lys Leu Glu Lys Asn Glu
Glu Ala Ala Leu Leu Ser Trp Glu 1115 1120 1125Ile Tyr Leu Lys Glu
Asn Tyr Leu Gln Asn Arg Gln Phe Gln Gln 1130 1135 1140Lys Gln Arg
Pro Glu Gln Lys Ile Glu Asp Ile Ser Asn Lys Val 1145 1150 1155Asp
Ala Met Val Asp Leu Leu Asp Leu Asp Pro Leu Lys Arg Ser 1160 1165
1170Gly Ser Met Glu Gln Arg Leu Ala Ser Leu Glu Glu Gln Val Ala
1175 1180 1185Gln Thr Ala Arg Ala Leu His Trp Ile Val Arg Thr Leu
Arg Ala 1190 1195 1200Ser Gly Phe Ser Ser Glu Ala Asp Val Pro Thr
Leu Ala Ser Gln 1205 1210 1215Lys Ala Ala Glu Glu Pro Asp Ala Glu
Pro Gly Gly Arg Lys Lys 1220 1225 1230Thr Glu Glu Pro Gly Asp Ser
Tyr His Val Asn Ala Arg His Leu 1235 1240 1245Leu Tyr Pro Asn Cys
Pro Val Thr Arg Phe Pro Val Pro Asn Glu 1250 1255 1260Lys Val Pro
Trp Glu Thr Glu Phe Leu Ile Tyr Asp Pro Pro Phe 1265 1270 1275Tyr
Thr Ala Glu Arg Lys Asp Ala Ala Ala Met Asp Pro Met Gly 1280 1285
1290Asp Thr Leu Glu Pro Leu Ser Thr Ile Gln Tyr Asn Val Val Asp
1295 1300 1305Gly Leu Arg Asp Arg Arg Ser Phe His Gly Pro Tyr Thr
Val Gln 1310 1315 1320Ala Gly Leu Pro Leu Asn Pro Met Gly Arg Thr
Gly Leu Arg Gly 1325 1330 1335Arg Gly Ser Leu Ser Cys Phe Gly Pro
Asn His Thr Leu Tyr Pro 1340 1345 1350Met Val Thr Arg Trp Arg Arg
Asn Glu Asp Gly Ala Ile Cys Arg 1355 1360 1365Lys Ser Ile Lys Lys
Met Leu Glu Val Leu Val Val Lys Leu Pro 1370 1375 1380Leu Ser Glu
His Trp Ala Leu Pro Gly Gly Ser Arg Glu Pro Gly 1385 1390 1395Glu
Met Leu Pro Arg Lys Leu Lys Arg Ile Leu Arg Gln Glu His 1400 1405
1410Trp Pro Ser Phe Glu Asn Leu Leu Lys Cys Gly Met Glu Val Tyr
1415 1420 1425Lys Gly Tyr Met Asp Asp Pro Arg Asn Thr Asp Asn Ala
Trp Ile 1430 1435 1440Glu Thr Val Ala Val Ser Val His Phe Gln Asp
Gln Asn Asp Val 1445 1450 1455Glu Leu Asn Arg Leu Asn Ser Asn Leu
His Ala Cys Asp Ser Gly 1460 1465 1470Ala Ser Ile Arg Trp Gln Val
Val Asp Arg Arg Ile Pro Leu Tyr 1475 1480 1485Ala Asn His Lys Thr
Leu Leu Gln Lys Ala Ala Ala Glu Phe Gly 1490 1495 1500Ala His
Tyr3143DNAArtificial sequenceoligonucleotide probe 31tgtaaaacga
cggccagtta aatagacctg caattattaa tct 433241DNAArtificial
sequenceoligonucleotide probe 32caggaaacag ctatgaccac ctgcacacct
gcaaatccat t 413324DNAArtificial sequenceoligonucleotide probe
33aagttccagt gccgcaccag tggc 243424DNAArtificial
sequenceoligonucleotide probe 34ttggttccac agccgagctc gtcg
243550DNAArtificial sequenceoligonucleotide probe 35gaggaggagt
gcaggattga gccatgtacc cagaaagggc aatgcccacc 503625DNAArtificial
sequenceoligonucleotide probe 36cgaaccttcc tactgggctc cggtg
253745DNAArtificial sequenceoligonucleotide probe 37ccaacatcta
tgcagatacc tcaagcatct gcaagacagc cgtgc 453824DNAArtificial
sequenceoligonucleotide probe 38gcctgacagc aaagatccgg aagg
243924DNAArtificial sequenceoligonucleotide probe 39tcagctccag
actctgatac tgcc 244024DNAArtificial sequenceoligonucleotide probe
40tgcctttcta ggaggcagag ctcc 244150DNAArtificial
sequenceoligonucleotide probe 41ggacccagaa atgtgtcctg agaatggatc
ttgtgtacct gatggtccag 504220DNAArtificial sequenceoligonucleotide
probe 42cgagatgacg ccgagccccc 204321DNAArtificial
sequenceoligonucleotide probe 43cggttcgaca cgcggcaggt g
214445DNAArtificial sequenceoligonucleotide probe 44tgctgctcct
gctgccgccg ctgctgctgg gggccttccc gccgg 454524DNAArtificial
sequenceoligonucleotide probe 45ttcgaggcct ctgagaagtg gccc
244622DNAArtificial sequenceoligonucleotide probe 46ggcggtatct
ctctggcctc cc 224750DNAArtificial sequenceoligonucleotide probe
47ttctccacag cagctgtggc atccgatcgt gtctcaatcc attctctggg
504834DNAArtificial sequenceoligonucleotide probe 48acgtagttcc
agtatggtgt gagcagcaac tgga 344926DNAArtificial
sequenceoligonucleotide probe 49agtccagcct ccaccctcca gttgct
265025DNAArtificial sequenceoligonucleotide probe 50ccccagtcct
ccaggagaac cagca 255127DNAArtificial sequenceoligonucleotide probe
51ggatttggtt agctgagccc accgaga 275226DNAArtificial
sequenceoligonucleotide probe 52gcactgcgcg cgacctcagg gctgca
265350DNAArtificial sequenceoligonucleotide probe 53cttattgccc
taaatattag ggagccggcg acctcctgga tcctctcatt 505424DNAArtificial
sequenceoligonucleotide probe 54gcaagaattc agggatcggt ctgg
245547DNAArtificial sequenceoligonucleotide
probe 55ctgtgttccc tgcaaccagt gtgggccagg catggagttg tctaagg
475624DNAArtificial sequenceoligonucleotide probe 56agatggcatc
actggtggct gaac 245726DNAArtificial sequenceoligonucleotide probe
57cagaaggcaa attgttcagc caccag 265827DNAArtificial
sequenceoligonucleotide probe 58acagtttcca gaccgatccc tgaattc
275924DNAArtificial sequenceoligonucleotide probe 59atggagattc
ctgccaactt gccg 246024DNAArtificial sequenceoligonucleotide probe
60ttgttggcat tgaggaggag cagc 246150DNAArtificial
sequenceoligonucleotide probe 61gagggcatcg tcgaaatacg cctagaacag
aactccatca aagccatccc 506227DNAArtificial sequenceoligonucleotide
probe 62ctggcaacag cagtgtctat tttgtgc 276321DNAArtificial
sequenceoligonucleotide probe 63taagtgccct cccaggctgc c
216452DNAArtificial sequenceoligonucleotide probe 64tcctccagtc
atgaatataa cccaagtcaa tggctctttg ttggtaattc 50tc
526522DNAArtificial sequenceoligonucleotide probe 65ggcattggag
cagtgctggg tg 226624DNAArtificial sequenceoligonucleotide probe
66tggaggccta gatgcggctg gacg 24671571DNAHomo sapiens 67gatggcgcag
ccacagcttc tgtgagattc gatttctccc cagttcccct 50gtgggtctga ggggaccaga
agggtgagct acgttggctt tctggaaggg 100gaggctatat gcgtcaattc
cccaaaacaa gttttgacat ttcccctgaa 150atgtcattct ctatctattc
actgcaagtg cctgctgttc caggccttac 200ctgctgggca ctaacggcgg
agccaggatg gggacagaat aaaggagcca 250cgacctgtgc caccaactcg
cactcagact ctgaactcag acctgaaatc 300ttctcttcac gggaggcttg
gcagtttttc ttactcctgt ggtctccaga 350tttcaggcct aagatgaaag
cctctagtct tgccttcagc cttctctctg 400ctgcgtttta tctcctatgg
actccttcca ctggactgaa gacactcaat 450ttgggaagct gtgtgatcgc
cacaaacctt caggaaatac gaaatggatt 500ttctgagata cggggcagtg
tgcaagccaa agatggaaac attgacatca 550gaatcttaag gaggactgag
tctttgcaag acacaaagcc tgcgaatcga 600tgctgcctcc tgcgccattt
gctaagactc tatctggaca gggtatttaa 650aaactaccag acccctgacc
attatactct ccggaagatc agcagcctcg 700ccaattcctt tcttaccatc
aagaaggacc tccggctctc tcatgcccac 750atgacatgcc attgtgggga
ggaagcaatg aagaaataca gccagattct 800gagtcacttt gaaaagctgg
aacctcaggc agcagttgtg aaggctttgg 850gggaactaga cattcttctg
caatggatgg aggagacaga ataggaggaa 900agtgatgctg ctgctaagaa
tattcgaggt caagagctcc agtcttcaat 950acctgcagag gaggcatgac
cccaaaccac catctcttta ctgtactagt 1000cttgtgctgg tcacagtgta
tcttatttat gcattacttg cttccttgca 1050tgattgtctt tatgcatccc
caatcttaat tgagaccata cttgtataag 1100atttttgtaa tatctttctg
ctattggata tatttattag ttaatatatt 1150tatttatttt ttgctattta
atgtatttat ttttttactt ggacatgaaa 1200ctttaaaaaa attcacagat
tatatttata acctgactag agcaggtgat 1250gtatttttat acagtaaaaa
aaaaaaacct tgtaaattct agaagagtgg 1300ctaggggggt tattcatttg
tattcaacta aggacatatt tactcatgct 1350gatgctctgt gagatatttg
aaattgaacc aatgactact taggatgggt 1400tgtggaataa gttttgatgt
ggaattgcac atctacctta caattactga 1450ccatccccag tagactcccc
agtcccataa ttgtgtatct tccagccagg 1500aatcctacac ggccagcatg
tatttctaca aataaagttt tctttgcata 1550ccaaaaaaaa aaaaaaaaaa a
157168176PRTHomo sapiens 68Met Lys Ala Ser Ser Leu Ala Phe Ser Leu
Leu Ser Ala Ala Phe1 5 10 15Tyr Leu Leu Trp Thr Pro Ser Thr Gly Leu
Lys Thr Leu Asn Leu 20 25 30Gly Ser Cys Val Ile Ala Thr Asn Leu Gln
Glu Ile Arg Asn Gly 35 40 45Phe Ser Glu Ile Arg Gly Ser Val Gln Ala
Lys Asp Gly Asn Ile 50 55 60Asp Ile Arg Ile Leu Arg Arg Thr Glu Ser
Leu Gln Asp Thr Lys 65 70 75Pro Ala Asn Arg Cys Cys Leu Leu Arg His
Leu Leu Arg Leu Tyr 80 85 90Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr
Pro Asp His Tyr Thr 95 100 105Leu Arg Lys Ile Ser Ser Leu Ala Asn
Ser Phe Leu Thr Ile Lys 110 115 120Lys Asp Leu Arg Leu Ser His Ala
His Met Thr Cys His Cys Gly 125 130 135Glu Glu Ala Met Lys Lys Tyr
Ser Gln Ile Leu Ser His Phe Glu 140 145 150Lys Leu Glu Pro Gln Ala
Ala Val Val Lys Ala Leu Gly Glu Leu 155 160 165Asp Ile Leu Leu Gln
Trp Met Glu Glu Thr Glu 170 1756928DNAArtificial
sequenceoligonucleotide probe 69ctcctgtggt ctccagattt caggccta
287026DNAArtificial sequenceoligonucleotide probe 70agtcctcctt
aagattctga tgtcaa 26
* * * * *