U.S. patent application number 12/182090 was filed with the patent office on 2009-01-15 for compositions and methods for the treatment of immune related diseases.
Invention is credited to Iqbal Grewal, Austin L. Gurney, Patricia A. Valdez.
Application Number | 20090017014 12/182090 |
Document ID | / |
Family ID | 40481851 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017014 |
Kind Code |
A1 |
Valdez; Patricia A. ; et
al. |
January 15, 2009 |
COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE RELATED
DISEASES
Abstract
The present invention relates to compositions containing a novel
protein and methods of using those compositions for the diagnosis
and treatment of immune related diseases.
Inventors: |
Valdez; Patricia A.; (San
Francisco, CA) ; Gurney; Austin L.; (Belmont, CA)
; Grewal; Iqbal; (Fremont, CA) |
Correspondence
Address: |
Goodwin Procter LLP;Attn: Patent Administrator
135 Commonwealth Drive
Menlo Park
CA
94025-1105
US
|
Family ID: |
40481851 |
Appl. No.: |
12/182090 |
Filed: |
July 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10136574 |
Apr 29, 2002 |
7419799 |
|
|
12182090 |
|
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Current U.S.
Class: |
424/130.1 ;
206/459.5; 514/1.1; 530/350; 530/387.3; 530/402 |
Current CPC
Class: |
A61P 11/02 20180101;
A61P 17/04 20180101; A61P 25/00 20180101; A61P 17/06 20180101; A61P
1/04 20180101; C07K 14/47 20130101; A61P 29/00 20180101; A61P 37/04
20180101; A61P 3/10 20180101; A61P 37/06 20180101; A61P 5/14
20180101; A61P 17/00 20180101; A61P 7/06 20180101; A61P 37/00
20180101; A61P 37/08 20180101; A61P 11/00 20180101; A61P 1/16
20180101; A61K 39/00 20130101; A61P 7/00 20180101; A61P 11/06
20180101; A61P 13/12 20180101; A61P 19/02 20180101; A61P 9/00
20180101 |
Class at
Publication: |
424/130.1 ;
530/350; 530/402; 530/387.3; 514/12; 206/459.5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/00 20060101 C07K014/00; C07K 19/00 20060101
C07K019/00; A61P 37/04 20060101 A61P037/04; B65D 85/00 20060101
B65D085/00; A61K 38/16 20060101 A61K038/16; C07K 16/46 20060101
C07K016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 2001 |
US |
PCT/US01/27099 |
Claims
1. An isolated polypeptide having at least 80% amino acid sequence
identity to: (a) an amino acid sequence of the polypeptide shown in
FIG. 2 (SEQ ID NO:2); (b) an amino acid sequence of the polypeptide
shown in FIG. 2 (SEQ ID NO:2); lacking its associated signal
peptide; (c) an amino acid sequence of an extracellular domain of
the polypeptide shown in FIG. 2 (SEQ ID NO:2); with its associated
signal peptide; or (d) an amino acid sequence of an extracellular
domain of the polypeptide shown in FIG. 2 (SEQ ID NO:2); lacking
its associated signal peptide.
2. An isolated polypeptide having at least 80% amino acid sequence
identity to an amino acid sequence encoded by the full-length
coding sequence of the DNA deposited under the ATCC accession
number PTA-1731.
3. A chimeric molecule comprising a polypeptide according to claim
1 fused to a heterologous amino acid sequence.
4. The chimeric molecule of claim 3, wherein said heterologous
amino acid sequence is an epitope tag sequence or an Fc region of
an immunoglobulin.
5. A composition of matter comprising (a) a polypeptide of claim 1,
(b) an agonist of said polypeptide, (c) an antagonist of said
polypeptide, or (d) an antibody that binds to said polypeptide, in
combination with a carrier.
6. The composition of matter of claim 5, wherein said carrier is a
pharmaceutically acceptable carrier.
7. The composition of matter of claim 5 comprising a
therapeutically effective amount of (a), (b), (c) or (d).
8. An article of manufacture, comprising: a container; a label on
said container; and a composition of matter comprising (a) a
polypeptide of claim 1, (b) an agonist of said polypeptide, (c) an
antagonist of said polypeptide, or (d) an antibody that binds to
said polypeptide, contained within said container, wherein label on
said container indicates that said composition of matter can be
used for treating an immune related disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of application Ser.
No. 10/136,574 filed Apr. 29, 2002, which claims priority under 35
U.S.C. .sctn. 120 to PCT international application no.
PCT/US01/27099, filed Aug. 29, 2001, the contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
useful for the diagnosis and treatment of immune related
diseases.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases, non-immune-mediated inflammatory diseases,
infectious diseases, immunodeficiency diseases, neoplasia, etc.
[0006] 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 id the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0007] A central event in both humoral and cell mediated immune
responses is the activation and clonal expansion of helper T cells.
Helper T cell activation is initiated by the interaction of the T
cell receptor (TCR)-CD28 complex with an antigen-MHC on the surface
of an antigen presenting cell. This interaction mediates a cascade
of biochemical events that induce the resting helper T cell to
enter a cell cycle (the G0 to G1 transition) and results in the
expression of a high affinity receptor for IL-2 and sometimes IL-4.
The activated T cell progresses through the cycle proliferating and
differentiating into memory cells or effector cells.
[0008] In addition to the signals mediated through CD28 [Carreno et
al., (2002) Annu Rev Immunol 20: 29-53], the molecule SLAM is also
able to provide the second signal for co-stimulation of T cells
necessary for proliferation [Aversa et al., (1997) J Immunol
158(9): 4036-4044, Aversa et al., (1997) Immunol Cell Biol 75(2):
202-205]. SLAM is a member of the family of molecules that contain
extracellular Ig-like repeats and also contain a SAP intracellular
binding site characterized by the motif TxYxxV/I. Tyrosine
phosphorylation of this motif greatly increases the binding of SAP
with SLAM. As SAP is associated with X-linked lymphoproliferative
disease, a greater understanding of the SAP/SLAM interaction may
lead to further insights into treatment of this disease [Howie et
al, (2002) Blood 99(3): 957-965, Latour et al., 2001, Nat Immuno
2(8): 681-690, Morra et al. 2001 J Biol Chem 276(39): 36809-36816,
Nichols et al., 2001 Nat Immunol 2(8): 665-666].
[0009] SLAM has been implicated in Th1 cell response, suggesting
that this molecule may play a role in inflammation and autoimmune
diseases [Cocks et al., (1995) Nature 376 (6537): 260-263, Aversa
et al., (1997) Immunol Cell Bio 75(2): 202-205]. SLAM and CD3
crosslinking on the surface of T cells induces proliferation of T
cells and an increase in Interferon gamma (IFN-( ) production. The
reasons above lead to the search and discovery of a new member of
the SLAM family, PRO20080. It is shown herein, that PRO20080 has
homology to SLAM, and like SLAM, has expression in B cells, T cells
and memory T cells. PRO20080 is upregulated on activated T cells,
exhibits homotypic binding, contains SAP binding motifs and
interacts with SAP in T cells. PRO20080 plus CD3 co-stimulation
induces the proliferation of T cells and increases the production
and secretion of IFN-(. Evidence is also shown for PRO20080 in the
Th1 mediated delayed-type hypersensitivity response, thus making
PRO20080 a potential target for treatment of inflammation and Th1
mediated autoimmune diseases.
SUMMARY OF THE INVENTION
A. Embodiments
[0010] The present invention concerns compositions and methods
useful for the diagnosis and treatment of immune related disease in
mammals, including humans. The present invention is based oil the
identification of PRO20080 (including agonist and antagonist
antibodies) which either stimulate or inhibit the immune response
in mammals. Immune related diseases can be treated by suppressing
or enhancing the immune response. Molecules that enhance the immune
response stimulate or potentiate the immune response to an antigen.
Molecules which stimulate the immune response can be used
therapeutically where enhancement of the immune response would be
beneficial. Alternatively, molecules that suppress the immune
response attenuate or reduce the immune response to an antigen
(e.g., neutralizing antibodies) can be used therapeutically where
attenuation of the immune response would be beneficial (e.g.,
inflammation). Accordingly, the PRO20080 polypeptides, agonists and
antagonists thereof are also useful to prepare medicines and
medicaments for the treatment of immune-related and inflammatory
diseases. In a specific aspect, such medicines and medicaments
comprise a therapeutically effective amount of a PRO20080
polypeptide, agonist or antagonist thereof with a pharmaceutically
acceptable carrier. Preferably, the admixture is sterile.
[0011] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists to a PRO20080 polypeptide which
comprises contacting the PRO20080 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
PRO20080 polypeptide. Preferably, the PRO20080 polypeptide is a
native sequence PRO20080 polypeptide. In a specific aspect, the
PRO20080 agonist or antagonist is an anti-PRO20080 antibody.
[0012] In another embodiment, the invention concerns a composition
of matter comprising a PRO20080 polypeptide or an agonist or
antagonist antibody which binds the polypeptide in admixture with a
carrier or excipient. In one aspect, the composition comprises a
therapeutically effective amount of the polypeptide or antibody. In
another aspect, when the composition comprises an immune
stimulating molecule, the composition is useful for: (a)
stimulating or enhancing an immune response in a mammal in need
thereof, (b) increasing the proliferation of T-lymphocytes in a
mammal in need thereof in response to an antigen, (c) stimulating
the activity of T-lymphocytes (d) production of INF-(. In another
aspect, the composition comprises a further active ingredient,
which may, for example, be a further antibody or a cytotoxic or
chemotherapeutic agent. Preferably, the composition is sterile.
[0013] In another embodiment, the invention concerns a method of
treating an immune related disorder in a mammal in need thereof,
comprising administering to the mammal an effective amount of a
PRO20080 polypeptide, an agonist thereof, or an antagonist thereto.
In a preferred aspect, the immune related disorder is selected from
the group consisting of: systemic lupus erythematosis, rheumatoid
arthritis, osteoarthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis, idiopathic inflammatory
myopathies, Sjogren's syndrome, systemic vasculitis, sarcoidosis,
autoimmune hemolytic anemia, autoimmune thrombocytopenia,
thyroiditis, diabetes mellitus, immune-mediated renal disease,
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, autoimmune chronic active hepatitis, primary biliary
cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,
inflammatory bowel 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,
transplantation associated diseases including graft rejection and
graft-versus-host-disease.
[0014] In another embodiment, the invention provides an antibody
which specifically binds to any of the above or below described
polypeptides. Optionally, the antibody is a monoclonal antibody,
humanized antibody, antibody fragment or single-chain antibody. In
one aspect, the present invention concerns an isolated antibody
which binds a PRO20080 polypeptide. In another aspect, the antibody
mimics the activity of a PRO20080 polypeptide (an agonist antibody)
or conversely the antibody inhibits or neutralizes the activity of
a PRO20080 polypeptide (an antagonist antibody). In another aspect,
the antibody is a monoclonal antibody, which preferably has
nonhuman complementarity determining region (CDR) residues and
human framework region (FR) residues. The antibody may be labeled
and may be immobilized on a solid support. In a further aspect, the
antibody is an antibody fragment, a monoclonal antibody, a
single-chain antibody, or an anti-idiotypic antibody.
[0015] In yet another embodiment, the present invention provides a
composition comprising an anti-PRO20080 antibody in admixture with
a pharmaceutically acceptable carrier. In one aspect, the
composition comprises a therapeutically effective amount of the
antibody. Preferably, the composition is sterile. The composition
may be administered in the form of a liquid pharmaceutical
formulation, which may be preserved to achieve extended storage
stability. Alternatively, the antibody is a monoclonal antibody, an
antibody fragment, a humanized antibody, or a single-chain
antibody.
[0016] In a further embodiment, the invention concerns an article
of manufacture, comprising:
[0017] (a) a composition of matter comprising a PRO20080
polypeptide or agonist or antagonist thereof;
[0018] (b) a container containing said composition; and
[0019] (c) a label affixed to said container, or a package insert
included in said container referring to the use of said PRO20080
polypeptide or agonist or antagonist thereof in the treatment of an
immune related disease. The composition may comprise a
therapeutically effective amount of the PRO20080 polypeptide or the
agonist or antagonist thereof.
[0020] In yet another embodiment, the present invention concerns a
method of diagnosing an immune related disease in a mammal,
comprising detecting the level of expression of a gene encoding a
PRO20080 polypeptide (a) in a test sample of tissue cells obtained
from the mammal, and (b) in a control sample of known normal tissue
cells of the same cell type, wherein a higher or lower expression
level in the test sample as compared to the control sample
indicates the presence of immune related disease in the mammal from
which the test tissue cells were obtained.
[0021] In another embodiment, the present invention concerns a
method of diagnosing an immune disease in a mammal, comprising (a)
contacting an anti-PRO20080 antibody with a test sample of tissue
cells obtained from the mammal, and (b) detecting the formation of
a complex between the antibody and a PRO20080 polypeptide, in the
test sample; wherein the formation of said complex is indicative of
the presence or absence of said disease. The detection may be
qualitative or quantitative, and may be performed in comparison
with monitoring the complex formation in a control sample of known
normal tissue cells of the same cell type. A larger quantity of
complexes formed in the test sample indicates the presence or
absence of an immune disease in the mammal from which the test
tissue cells were obtained. The antibody preferably carries a
detectable label. Complex formation can be monitored, for example,
by light microscopy, flow cytometry, fluorimetry, or other
techniques known in the art. The test sample is usually obtained
from an individual suspected of having a deficiency or abnormality
of the immune system.
[0022] In another embodiment, the invention provides a method for
determining the presence of a PRO20080 polypeptide in a sample
comprising exposing a test sample of cells suspected of containing
the PRO20080 polypeptide to an anti-PRO20080 antibody and
determining the binding of said antibody to said cell sample. In a
specific aspect, the sample comprises a cell suspected of
containing the PRO20080 polypeptide and the antibody binds to the
cell. The antibody is preferably detectably labeled and/or bound to
a solid support.
[0023] In another embodiment, the present invention concerns an
immune-related disease diagnostic kit, comprising an anti-PRO20080
antibody and a carrier in suitable packaging. The kit preferably
contains instructions for using the antibody to detect the presence
of the PRO20080 polypeptide. Preferably the carrier is
pharmaceutically acceptable.
[0024] In another embodiment, the present invention concerns a
diagnostic kit, containing an anti-PRO020080 antibody in suitable
packaging. The kit preferably contains instructions for using the
antibody to detect the PRO20080 polypeptide.
[0025] In another embodiment, the invention provides a method of
diagnosing an immune-related disease in a mammal which comprises
detecting the presence or absence or a PRO20080 polypeptide in a
test sample of tissue cells obtained from said mammal, wherein the
presence or absence of the PRO20080 polypeptide in said test sample
is indicative of the presence of an immune-related disease in said
mammal.
[0026] In another embodiment, the present invention concerns a
method for identifying an agonist of a PRO20080 polypeptide
comprising:
[0027] (a) contacting cells and a test compound to be screened
under conditions suitable for the induction of a cellular response
normally induced by a PRO20080 polypeptide; and
[0028] (b) determining the induction of said cellular response to
determine if the test compound is an effective agonist, wherein the
induction of said cellular response is indicative of said test
compound being an effective agonist.
[0029] In another embodiment, the invention concerns a method for
identifying a compound capable of inhibiting the activity of a
PRO20080 polypeptide comprising contacting a candidate compound
with a PRO20080 polypeptide under conditions and for a time
sufficient to allow these two components to interact and
determining whether the activity of the PRO20080 polypeptide is
inhibited. In a specific aspect, either the candidate compound or
the PRO20080 polypeptide is immobilized on a solid support. In
another aspect, the non-immobilized component carries a detectable
label. In a preferred aspect, this method comprises the steps
of:
[0030] (a) contacting cells and a test compound to be screened in
the presence of a PRO20080 polypeptide under conditions suitable
for the induction of a cellular response normally induced by a
PRO20080 polypeptide; and
[0031] (b) determining the induction of said cellular response to
determine if the test compound is an effective antagonist.
[0032] In another embodiment, the invention provides a method for
identifying a compound that inhibits the expression of a PRO20080
polypeptide in cells that normally express the polypeptide, wherein
the method comprises contacting the cells with a test compound and
determining whether the expression of the PRO20080 polypeptide is
inhibited. In a preferred aspect, this method comprises the steps
of:
[0033] (a) contacting cells and a test compound to be screened
under conditions suitable for allowing expression of the PRO20080
polypeptide; and
[0034] (b) determining the inhibition of expression of said
polypeptide.
[0035] In yet another embodiment, the present invention concerns a
method for treating an immune-related disorder in a mammal that
suffers therefrom comprising administering to the mammal a nucleic
acid molecule that codes for either (a) a PRO20080 polypeptide, (b)
an agonist of a PRO20080 polypeptide or (c) an antagonist of a
PRO20080 polypeptide, wherein said agonist or antagonist may be an
anti-PRO20080 antibody. In a preferred embodiment, the mammal is
human. In another preferred embodiment, the nucleic acid is
administered via ex vivo gene therapy. In a further preferred
embodiment, the nucleic acid is comprised within a vector, more
preferably an adenoviral, adeno-associated viral, lentiviral or
retroviral vector.
[0036] In yet another aspect, the invention provides a recombinant
viral particle comprising a viral vector consisting essentially of
a promoter, nucleic acid encoding (a) a PRO20080 polypeptide, (b)
an agonist polypeptide of a PRO20080 polypeptide, or (c) an
antagonist polypeptide of a PRO20080 polypeptide, and a signal
sequence for cellular secretion of the polypeptide, wherein the
viral vector is in association with viral structural proteins.
Preferably, the signal sequence is from a mammal, such as from a
native PRO20080 polypeptide.
[0037] In a still further embodiment, the invention concerns an ex
vivo producer cell comprising a nucleic acid construct that
expresses retroviral structural proteins and also comprises a
retroviral vector consisting essentially of a promoter, nucleic
acid encoding (a) a PRO20080 polypeptide, (b) an agonist
polypeptide of a PRO20080 polypeptide or (c) an antagonist
polypeptide of a PRO20080 polypeptide, and a signal sequence for
cellular secretion of the polypeptide, wherein said producer cell
packages the retroviral vector in association with the structural
proteins to produce recombinant retroviral particles.
[0038] In a still further embodiment, the invention provides a
method of increasing the activity of T-lymphocytes in a mammal
comprising administering to said mammal (a) a PRO20080 polypeptide,
(b) an agonist of a PRO20080 polypeptide, wherein the activity of
T-lymphocytes in the mammal is increased.
[0039] In a still further embodiment, the invention provides a
method of decreasing the activity of T-lymphocytes in a mammal
comprising administering to said mammal an antagonist of a PRO20080
polypeptide, wherein the activity of T-lymphocytes in the mammal is
decreased.
[0040] In a still further embodiment, the invention provides a
method of increasing the proliferation of T-lymphocytes in a mammal
comprising administering to said mammal (a) a PRO20080 polypeptide,
(b) an agonist of a PRO20080 polypeptide, wherein the proliferation
of T-lymphocytes in the mammal is increased.
[0041] In a still further embodiment, the invention provides a
method of decreasing the proliferation of 1-lymphocytes in a mammal
comprising administering to said mammal an antagonist of a PRO20080
polypeptide, wherein the proliferation of T-lymphocytes in the
mammal is decreased.
[0042] In a still further embodiment, the invention provides a
method of decreasing the Th1 mediated delayed type hypersensitive
response in a mammal comprising administering to said mammal an
antagonist of a PRO20080 polypeptide, wherein the Th1 mediated
delayed type hypersensitive response is decreased.
[0043] In a still further embodiment, the invention provides a
method of inhibiting the inflammatory immune response, said method
comprising contacting a T cell line containing a reporter construct
with a PRO20080 antagonist thereof, wherein said inflammatory
immune response is inhibited and measured.
B. Additional Embodiments
[0044] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described polypeptides. Host cell comprising any such vector are
also provided. By way of example, the host cells may be CHO cells,
E. coli, or yeast. A process for producing any of the herein
described polypeptides is further provided and comprises culturing
host cells under conditions suitable for expression of the desired
polypeptide and recovering the desired polypeptide from the cell
culture.
[0045] In other embodiments, the invention provides chimeric
molecules comprising any of the herein described polypeptides
fussed 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.
[0046] In another embodiment, the invention provides an antibody
which specifically binds to any of the above or below described
polypeptides. Optionally, the antibody is a monoclonal antibody,
humanized antibody, antibody fragment or single-chain antibody.
[0047] In yet other embodiments, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences or as antisense probes, wherein those probes
may be derived from any of the above or below described nucleotide
sequences.
[0048] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a PRO20080 polypeptide. 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
PRO20080 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).
[0049] 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 PRO20080 polypeptide cDNA as disclosed
herein, the coding sequence of a PRO20080 polypeptide lacking the
signal peptide as disclosed herein, the coding sequence of an
extracellular domain of a transmembrane PRO20080 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).
[0050] 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).
[0051] Another aspect the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO20080
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 PRO20080 polypeptides
are contemplated.
[0052] Another embodiment is directed to fragments of a PRO20080
polypeptide coding sequence, or the complement thereof, that may
find use as, for example, hybridization probes, for encoding
fragments of a PRO20080 polypeptide that may optionally encode a
polypeptide comprising a binding site for an anti-PRO20080 antibody
or as antisense oligonucleotide probes. Such nucleic acid fragments
are usually at least about 20 nucleotides in length, alternatively
at least about 30 nucleotides in length, alternatively at least
about 40 nucleotides in length, alternatively at least about 50
nucleotides in length, alternatively at least about 60 nucleotides
in length, alternatively at least about 70 nucleotides in length,
alternatively at least about 80 nucleotides in length,
alternatively at least about 90 nucleotides in length,
alternatively at least about 100 nucleotides in length,
alternatively at least about 110 nucleotides in length,
alternatively at least about 120 nucleotides in length,
alternatively at least about 130 nucleotides in length,
alternatively at least about 140 nucleotides in length,
alternatively at least about 150 nucleotides in length,
alternatively at least about 160 nucleotides in length,
alternatively at least about 170 nucleotides in length,
alternatively at least about 180 nucleotides in length,
alternatively at least about 190 nucleotides in length,
alternatively at least about 200 nucleotides in length,
alternatively at least about 250 nucleotides in length,
alternatively at least about 300 nucleotides in length,
alternatively at least about 350 nucleotides in length,
alternatively at least about 400 nucleotides in length,
alternatively at least about 450 nucleotides in length,
alternatively at least about 500 nucleotides in length,
alternatively at least about 600 nucleotides in length,
alternatively at least about 700 nucleotides in length,
alternatively at least about 800 nucleotides in length,
alternatively at least about 900 nucleotides in length and
alternatively 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 PRO20080 polypeptide-encoding
nucleotide sequence may be determined in a routine manner by
aligning the PRO20080 polypeptide-encoding nucleotide sequence with
other known nucleotide sequences using any of a number of well
known sequence alignment programs and determining which PRO20080
polypeptide-encoding nucleotide sequence fragment(s) are novel. All
of such PRO20080 polypeptide-encoding nucleotide sequences are
contemplated herein. Also contemplated are the PRO20080 polypeptide
fragments encoded by these nucleotide molecule fragments,
preferably those PRO20080 polypeptide fragments that comprise a
binding site for an anti-PRO20080 antibody.
[0053] In another embodiment, the invention provides isolated
PRO20080 polypeptide encoded by any of the isolated nucleic acid
sequences herein above identified.
[0054] In a certain aspect, the invention concerns an isolated
PRO20080 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 PRO20080
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.
[0055] In a further aspect, the invention concerns an isolated
PRO20080 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.
[0056] In a specific aspect, the invention provides an isolated
PRO20080 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 herein before
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 PRO20080 polypeptide and recovering the PRO20080 polypeptide
from the cell culture.
[0057] Another aspect the invention provides an isolated PRO20080
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 PRO20080 polypeptide and recovering
the PRO20080 polypeptide from the cell culture.
[0058] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO20080 polypeptide as defined herein.
In a particular embodiment, the agonist or antagonist is an
anti-PRO20080 antibody or a small molecule.
[0059] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists to a PRO20080 polypeptide which
comprise contacting the PRO20080 polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
PRO20080 polypeptide. Preferably, the PRO20080 polypeptide is a
native PRO20080 polypeptide.
[0060] In a still further embodiment, the invention concerns a
composition of matter comprising a PRO20080 polypeptide, or an
agonist or antagonist of a PRO20080 polypeptide as herein
described, or an anti-PRO20080 antibody, in combination with a
carrier. Optionally, the carrier is a pharmaceutically acceptable
carrier.
[0061] Another embodiment of the present invention is directed to
the use of a PRO20080 polypeptide, or an agonist or antagonist
thereof as herein before described, or an anti-PRO20080 antibody,
for the preparation of a medicament useful in the treatment of a
condition which is responsive to the PRO20080 polypeptide, an
agonist or antagonist thereof or an anti-PRO20080 antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native
sequence PRO20080 cDNA, wherein SEQ ID NO:1 is a clone designated
herein as "DNA161000-2896".
[0063] 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.
[0064] FIG. 3 shows a Northern Blot analysis of the expression of
PRO20080.
[0065] FIG. 4 shows the expression of PRO20080 in a panel of cell
lines.
[0066] FIG. 5a shows the expression of PRO20080 on human peripheral
r cells.
[0067] FIG. 5b shows high expression of PRO20080 on CD4+ CD45RO+
memory T-cells.
[0068] FIG. 6 shows an ELISA assay showing that labeled PRO20080
can bind to bound PRO20080.
[0069] FIG. 7 shows homotypic binding of PRO20080 at the cell
surface by immunofluorescence.
[0070] FIG. 8a shows the proliferation of T-cells and the
production of INF-(when T cells are stimulated with anti-PRO20080
antibody and anti-CD3 antibody.
[0071] FIG. 8b shows the proliferation of T-cells and the
production of INF-(when T cells are stimulated with PRO20080-Fc
fusion molecule.
[0072] FIG. 9a shows the phosphorylated state of PRO20080 and its
association with SAP.
[0073] FIG. 9b shows a time course of phosphorylation of PRO20080
upon co-stimulation treatment and addition of a cross linking
antibody.
[0074] FIG. 9c shows the phosphorylated state of PRO20080 and its
association with SAP upon co-stimulation.
[0075] FIG. 10a shows the effect of the murine homologue of
PRO20080 on T cell proliferation in vivo.
[0076] FIG. 10b shows the effect of the murine homologue of
PRO20080 on INF-(production in vivo.
[0077] FIG. 10c shows the effect of murine homologue of PRO20080 in
a delayed-type hypersensitivity response as measured by ear
swelling.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Definitions
[0078] The terms "PRO20080 polypeptide" and "PRO20080" as used
herein encompass native sequence polypeptides and polypeptide
variants (which are further defined herein). The PRO20080
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 "PRO20080
polypeptide" refers to each individual PRO20080/number polypeptide
disclosed herein. All disclosures in this specification which refer
to the "PRO20080 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 "PRO20080
polypeptide" also includes variants of the PRO20080 polypeptides
disclosed herein.
[0079] A "native sequence PRO20080 polypeptide" comprises a
polypeptide having the same amino acid sequence as the
corresponding PRO20080 polypeptide derived from nature. Such native
sequence PRO20080 polypeptides can be isolated from nature or can
be produced by recombinant or synthetic means. The term "native
sequence PRO20080 polypeptide" specifically encompasses
naturally-occurring truncated or secreted forms of the specific
PRO20080 polypeptide (e.g., an extracellular domain sequence),
naturally-occurring variant forms (e.g., alternatively spliced
forms) and naturally-occurring allelic variants of the polypeptide.
In various embodiments of the invention, the native sequence
PRO20080 polypeptides disclosed herein are mature or full-length
native sequence polypeptides comprising the full-length amino acids
sequences shown in the accompanying figures. Start and stop codons
are shown in bold font and underlined in the figures. However,
while the PRO20080 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 PRO20080
polypeptides.
[0080] The PRO20080 polypeptide "extracellular domain" or "ECD"
refers to a form of the PRO20080 polypeptide which is essentially
free of the transmembrane and cytoplasmic domains. Ordinarily, a
PRO20080 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 PRO20080 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 PRO20080 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.
[0081] The approximate location of the "signal peptides" of the
various PRO20080 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.
[0082] "PRO20080 polypeptide variant" means an active PRO20080
polypeptide as defined above or below having at least about 80%
amino acid sequence identity with a full-length native sequence
PRO20080 polypeptide sequence as disclosed herein, a PRO20080
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO20080 polypeptide, with or
without the signal peptide, as disclosed herein or any other
fragment of a full-length PRO20080 polypeptide sequence as
disclosed herein. Such PRO20080 polypeptide variants include, for
instance, PRO20080 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 PRO20080
polypeptide variant will have 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 full-length native sequence PRO20080
polypeptide sequence as disclosed herein, a PRO20080 polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO20080 polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length PRO20080 polypeptide sequence as
disclosed herein. Ordinarily, PRO20080 variant polypeptides are at
least about 10 amino acids in length, alternatively at least about
20 amino acids in length, alternatively at least about 30 amino
acids in length, alternatively at least about 40 amino acids in
length, alternatively at least about 50 amino acids in length,
alternatively at least about 60 amino acids in length,
alternatively at least about 70 amino acids in length,
alternatively at least about 80 amino acids in length,
alternatively at least about 90 amino acids in length,
alternatively at least about 100 amino acids in length,
alternatively at least about 150 amino acids in length,
alternatively at least about 200 amino acids in length,
alternatively at least about 300 amino acids in length, or
more.
[0083] "Percent (%) amino acid sequence identity" with respect to
the PRO20080 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 PRO20080
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.
[0084] 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 CA, 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 "PRO20080", wherein
"PRO20080" represents the amino acid sequence of a hypothetical
PRO20080 polypeptide of interest, "Comparison Protein" represents
the amino acid sequence of a polypeptide against which the
"PRO20080" polypeptide of interest is being compared, and "X, "Y"
and "Z" each represent different hypothetical amino acid
residues.
[0085] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program. However, % amino acid sequence identity values may also be
obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid
sequence identity value is determined by dividing (a) the number of
matching identical amino acid residues between the amino acid
sequence of the PRO20080 polypeptide of interest having a sequence
derived from the native PRO20080 polypeptide and the comparison
amino acid sequence of interest (i.e., the sequence against which
the PRO20080 polypeptide of interest is being compared which may be
a PRO20080 variant polypeptide) as determined by WU-BLAST-2 by (b)
the total number of amino acid residues of the PRO20080 polypeptide
of interest. For example, in the statement "a polypeptide
comprising an the amino acid sequence A which has or having at
least 80% amino acid sequence identity to the amino acid sequence
B", the amino acid sequence A is die comparison amino acid sequence
of interest and the amino acid sequence B is the amino acid
sequence of the PRO20080 polypeptide of interest.
[0086] Percent amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlni.nih.gov or otherwise obtained from the
National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses
several search parameters, wherein all of those search parameters
are set to default values including, for example, unmask=yes,
strand=all, expected occurrences=10, minimum low complexity
length=15/5, multi-pass e-value=0.01, constant for multi-pass=25,
dropoff for final gapped alignment=25 and scoring
matrix=BLOSUM62.
[0087] In situations where NCBI-BLAST2 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 NCBI-BLAST2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A.
[0088] "PRO20080 variant polynucleotide" or "PRO20080 variant
nucleic acid sequence" means a nucleic acid molecule which encodes
an active PRO20080 polypeptide as defined below and which has at
least about 80% nucleic acid sequence identity with a nucleotide
acid sequence encoding a full-length native sequence PRO20080
polypeptide sequence as disclosed herein, a full-length native
sequence PRO20080 polypeptide sequence lacking the signal peptide
as disclosed herein, an extracellular domain of a PRO20080
polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-length PRO20080 polypeptide
sequence as disclosed herein. Ordinarily, a PRO20080 variant
polynucleotide will have 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 with a nucleic acid sequence encoding a full-length native
sequence PRO20080 polypeptide sequence as disclosed herein, a
full-length native sequence PRO20080 polypeptide sequence lacking
the signal peptide as disclosed herein, an extracellular domain of
a PRO20080 polypeptide, with or without the signal sequence, as
disclosed herein or any other fragment of a full-length PRO20080
polypeptide sequence as disclosed herein. Variants do not encompass
the native nucleotide sequence.
[0089] Ordinarily, PRO20080 variant polynucleotides are at least
about 30 nucleotides in length, alternatively at least about 60
nucleotides in length, alternatively at least about 90 nucleotides
in length, alternatively at least about 120 nucleotides in length,
alternatively at least about 150 nucleotides in length,
alternatively at least about 180 nucleotides in length,
alternatively at least about 210 nucleotides in length,
alternatively at least about 240 nucleotides in length,
alternatively at least about 270 nucleotides in length,
alternatively at least about 300 nucleotides in length,
alternatively at least about 450 nucleotides in length,
alternatively at least about 600 nucleotides in length,
alternatively at least about 900 nucleotides in length, or
more.
[0090] "Percent (%) nucleic acid sequence identity" with respect to
PRO20080-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 PRO20080 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 call 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.
[0091] 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 "PRO20080-DNA", wherein "PRO20080-DNA"
represents a hypothetical PRO20080-encoding nucleic acid sequence
of interest, "Comparison DNA" represents the nucleotide sequence of
a nucleic acid molecule against which the "PRO20080-DNA" nucleic
acid molecule of interest is being compared, and "N", "L" and "V"
each represent different hypothetical nucleotides.
[0092] Unless specifically stated otherwise, all % nucleic acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program. However, % nucleic acid sequence identity values may also
be obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid
sequence identity value is determined by dividing (a) the number of
matching identical nucleotides between the nucleic acid sequence of
the PRO20080 polypeptide-encoding nucleic acid molecule of interest
having a sequence derived from the native sequence PRO20080
polypeptide-encoding nucleic acid and the comparison nucleic acid
molecule of interest (i.e., the sequence against which the PRO20080
polypeptide-encoding nucleic acid molecule of interest is being
compared which may be a variant PRO20080 polynucleotide) as
determined by WU-BLAST-2 by (b) the total number of nucleotides of
the PRO20080 polypeptide-encoding nucleic acid molecule of
interest. For example, in the statement "an isolated nucleic acid
molecule comprising a nucleic acid sequence A which has or having
at least 80% nucleic acid sequence identity to the nucleic acid
sequence B", the nucleic acid sequence A is the comparison nucleic
acid molecule of interest and the nucleic acid sequence B is the
nucleic acid sequence of the PRO20080 polypeptide-encoding nucleic
acid molecule of interest.
[0093] Percent nucleic acid sequence identity may also be
determined using the sequence comparison program NBI-BLAST2
(Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The
NCBI-BLAST2 sequence comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0094] In situations where NCBI-BLAST2 is employed for sequence
comparisons, the % nucleic acid sequence identity of a given
nucleic acid sequence C to, with, or against a given nucleic acid
sequence D (which can alternatively be phrased as a given nucleic
acid sequence C that has or comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program NCBI-BLAST2 in that program's
alignment of C and D, and where Z is the total number of
nucleotides in D. It will be appreciated that where the length of
nucleic acid sequence C is not equal to the length of nucleic acid
sequence D, the % nucleic acid sequence identity of C to D will not
equal the % nucleic acid sequence identity of D) to C.
[0095] In other embodiments, PRO20080 variant polynucleotides are
nucleic acid molecules that encode an active PRO20080 polypeptide
and which are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to nucleotide sequences encoding
a full-length PRO20080 polypeptide as disclosed herein. PRO20080
variant polypeptides may be those that are encoded by a PRO20080
variant polynucleotide.
[0096] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the PRO20080
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0097] An "isolated" PRO20080 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.
[0098] 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.
[0099] 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.
[0100] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-PRO020080 monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO20080 antibody compositions with polyepitopic
specificity, single chain anti-PRO20080 antibodies, and fragments
of anti-PRO20080 antibodies (see below). The term "monoclonal
antibody" as used herein refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally-occurring mutations that may be
present in minor amounts.
[0101] "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).
[0102] "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), 501-M 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.
[0103] "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.
[0104] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a PRO20080 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).
[0105] As used herein, the term "immunuoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0106] "Active" or "activity" for the purposes herein refers to
form(s) of a PRO20080 polypeptide which retain a biological and/or
an immunological activity of native or naturally-occurring
PRO20080, wherein "biological" activity refers to a biological
function (either inhibitory or stimulatory) caused by a native or
naturally-occurring PRO20080 other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring PRO20080 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 PRO20080.
[0107] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native PRO20080 polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native PRO20080 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 PRO20080
polypeptides, peptides, antisense oligonucleotides, small organic
molecules, etc. Methods for identifying agonists or antagonists of
a PRO20080 polypeptide may comprise contacting a PRO20080
polypeptide with a candidate agonist or antagonist molecule and
measuring a detectable change in one or more biological activities
normally associated with the PRO20080 polypeptide.
[0108] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whole the disorder is to be prevented.
[0109] "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.
[0110] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0111] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the
mammal is human.
[0112] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0113] "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..
[0114] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0115] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0116] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0117] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0118] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa and lambda, based on the amino acid sequences
of their constant domains.
[0119] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0120] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0121] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0122] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassic 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.
[0123] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope.
[0124] 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.
[0125] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0126] 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 PRO20080 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.
[0127] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0128] 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.
[0129] 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.
[0130] Examples of immune-related and inflammatory diseases, some
of which are immune or T cell mediated, which can be treated
according to the invention 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,
flashimoto'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,
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.
[0131] The term "effective amount" is a concentration or amount of
a PRO20080 polypeptide and/or agonist/antagonist which results in
achieving a particular stated purpose. An "effective amount" of a
PRO20080 polypeptide or agonist or antagonist thereof may be
determined empirically. Furthermore, a "therapeutically effective
amount" is a concentration or amount of a PRO20080 polypeptide
and/or agonist/antagonist which is effective for achieving a stated
therapeutic effect. This amount may also be determined
empirically.
[0132] 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., I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.186), chemotherapeutic agents, and toxins such as
enzymatically active toxins of bacterial, fungal, plant or animal
origin, or fragments thereof.
[0133] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers
Squibb Oncology, Princeton, N.J.), and doxetaxel (Taxotere,
Rhone-Poulenc Rorer, Antony, France), toxotere, methotrexate,
cisplatin, melphalan, vinblastine, bleomycin, etoposide,
ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,
carboplatin, teniposide, daunomycin, caminomycin, aminopterin,
dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.
4,675,187), melphalan and other related nitrogen mustards. Also
included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
[0134] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
cancer cell overexpressing any of the genes identified herein,
either in vitro or in vivo. Thus, the growth inhibitory agent is
one which significantly reduces the percentage of cells
overexpressing such genes 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), taxol, and topo 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, oncogens, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995),
especially p. 13.
[0135] 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-1.alpha., 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.
[0136] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0137] As used herein, the term "inflammatory cells" designates
cells that enhance the inflammatory response such as mononuclear
cells, cosinophils, macrophages, and polymorphonuclear neutrophils
(PMN).
[0138] As used herein, the term "delayed type hypersensitivity" or
"DTH" is an immune reaction caused by an antigen and is mediated by
the cellular component of the immune system, specifically, memory T
cells. The hallmark DTH response is induration, swelling, edema and
monocytic infiltration into the site of the lesion within 24 to 72
hours. DTH has been classified at Type IV hypersensitivity by
Coombs and Gell [Gell et al., (1968) Clinical Aspects in
Immunology. Blackwell, Oxford]
TABLE-US-00001 TABLE 2 PRO20080 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 PRO20080 polypeptide) = 5 divided by 15 = 33.3%
TABLE-US-00002 TABLE 3 PRO20080 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 PRO20080 polypeptide) = 5 divided by 10 =
50%
TABLE-US-00003 TABLE 4 PRO20080- NNNNNNNNNNNNNN (Length = 14
nucleotides) DNA 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 PRO20080-DNA nucleic acid sequence) = 6 divided
by 14 = 42.9%
TABLE-US-00004 TABLE 5 PRO20080-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
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 PRO20080-DNA nucleic acid sequence) = 4 divided by 12 =
33.3%
[0139] II. Compositions and Methods of the Invention
[0140] A. Full-Length PRO20080 Polypeptides
[0141] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO20080 polypeptides. In particular, cDNAs
encoding various PRO20080 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 PRO20080 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 PRO20080, will be
referred to as "PRO20080/number", regardless of their origin or
mode of preparation.
[0142] 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 PRO20080
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.
[0143] B. PRO20080 Polypeptide Variants
[0144] In addition to the full-length native sequence PRO20080
polypeptides described herein, it is contemplated that PRO20080
variants can be prepared. PRO20080 variants can be prepared by
introducing appropriate nucleotide changes into the PRO20080 DNA,
and/or by synthesis of the desired PRO20080 polypeptide. Those
skilled in the art will appreciate that amino acid changes may
alter post-translational processes of the PRO20080, such as
changing the number or position of glycosylation sites or altering
the membrane anchoring characteristics.
[0145] Variations in the native full-length sequence PRO20080 or in
various domains of the PRO20080 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 PRO20080 that results in a change in the amino acid sequence of
the PRO20080 as compared with the native sequence PRO20080.
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
PRO20080. 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
PRO20080 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.
[0146] PRO20080 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
PRO20080 polypeptide.
[0147] PRO20080 fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
PRO20080 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, PRO20080 polypeptide fragments share at
least one biological and/or immunological activity with the native
PRO20080 polypeptide disclosed herein.
[0148] In particular embodiments, 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 introduced and the products
screened.
TABLE-US-00005 TABLE 6 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C)
ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His
(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu
norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K)
arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile;
ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp
(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu;
met; phe; leu ala; norleucine
[0149] Substantial modifications in function or immunological
identity of the PRO20080 polypeptide are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: cys, srr, thr; (3) acidic: asp, glu; (4) basic: asn,
gin, his, lys, arg; (5) residues that influence chain orientation:
gly, pro; and (6) aromatic: trp, tyr, phe.
[0150] 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.
[0151] 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 PRO20080 variant DNA.
[0152] 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.
[0153] C. Modifications of PRO20080
[0154] Covalent modifications of PRO20080 are included within the
scope of this invention. One type of covalent modification includes
reacting targeted amino acid residues of a PRO20080 polypeptide
with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the
PRO20080. Derivatization with bifunctional agents is useful, for
instance, for crosslinking PRO20080 to a water-insoluble support
matrix or surface for use in the method for purifying anti-PRO20080
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.
[0155] 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.
[0156] Another type of covalent modification of the PRO20080
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 PRO20080 (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
PRO20080. 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.
[0157] Addition of glycosylation sites to the PRO20080 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 PRO20080 (for O-linked glycosylation sites). The
PRO20080 amino acid sequence may optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding
the PRO20080 polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids.
[0158] Another means of increasing the number of carbohydrate
moieties on the PRO20080 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).
[0159] Removal of carbohydrate moieties present on the PRO20080
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).
[0160] Another type of covalent modification of PRO20080 comprises
linking the PRO20080 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. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0161] The PRO20080 of the present invention may also be modified
in a way to form a chimeric molecule comprising PRO20080 fused to
another, heterologous polypeptide or amino acid sequence.
[0162] In one embodiment, such a chimeric molecule comprises a
fusion of the PRO20080 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 PRO20080. The presence of such epitope-tagged forms of the
PR020080 can be detected using an antibody against the tag
polypeptide. Also, provision of the epitope tag enables the
PRO20080 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)].
[0163] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the PRO20080 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 PRO20080 polypeptide in
place of at least one variable region within an lg molecule. In a
particularly preferred embodiment, the immunoglobulin fusion
includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3
regions of an IgG1 molecule. For the production of immunoglobulin
fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0164] D. Preparation of PRO20080
[0165] The description below relates primarily to production of
PRO20080 by culturing cells transformed or transfected with a
vector containing PRO20080 nucleic acid. It is, of course,
contemplated that alternative methods, which are well known in the
art, may be employed to prepare PRO20080. For instance, the
PRO20080 sequence, or portions thereof, may be produced by direct
peptide synthesis using solid-phase techniques [see, e.g., Stewart
et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San
Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,
85:2149-2154 (1963)]. In vitro protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be accomplished, for instance, using an Applied Biosystems Peptide
Synthesizer (Foster City, Calif.) using manufacturer's
instructions. Various portions of the PRO20080 may be chemically
synthesized separately and combined using chemical or enzymatic
methods to produce the full-length PRO20080.
1. Isolation of DNA Encoding PRO20080
[0166] DNA encoding PRO20080 may be obtained from a cDNA library
prepared from tissue believed to possess the PRO20080 mRNA and to
express it at a detectable level. Accordingly, human PRO20080 DNA
can be conveniently obtained from a cDNA library prepared from
human tissue, such as described in the Examples. The
PRO20080-encoding gene may also be obtained from a genomic library
or by known synthetic procedures (e.g., automated nucleic acid
synthesis).
[0167] Libraries can be screened with probes (such as antibodies to
the PRO20080 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 PRO20080 is to use PCR methodology
[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
[0168] 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.
[0169] 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.
[0170] 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.
2. Selection and Transformation of Host Cells
[0171] Host cells are transfected or transformed with expression or
cloning vectors described herein for PRO20080 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.
[0172] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0173] 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, Etivinia, Klebsiella, Proteus, Salmonella,
e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans,
and Shigella, as well as Bacilli such as B. subtillis 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 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0174] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for PRO20080-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 (ARCC 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, Tolypocladium (WO 91/00357 published 10
Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et
al., Biochem. Biophys. Res. Commun., 112:284-289 [1983); Tilburn et
al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci.
USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J.,
4:475-479 [1985]). Methylotropic yeasts are suitable herein and
include, but are not limited to, yeast capable of growth on
methanol selected from the genera consisting of Hansenula, Candida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A
list of specific species that are exemplary of this class of yeasts
may be found in C. Anthony, The Biochemistry of Methylotrophs, 269
(1982).
[0175] Suitable host cells for the expression of glycosylated
PRO20080 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.
3. Selection and Use of a Replicable Vector
[0176] The nucleic acid (e.g., cDNA or genomic DNA) encoding
PRO20080 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.
[0177] The PRO20080 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
PRO20080-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.
[0178] 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.
[0179] 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.
[0180] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the PRO20080-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)].
[0181] Expression and cloning vectors usually contain a promoter
operably linked to the PRO20080-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 PRO20080.
[0182] 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.
[0183] 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.
[0184] PRO20080 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.
[0185] Transcription of a DNA encoding the PRO20080 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 PRO20080 coding sequence, but is preferably located at a
site 5' from the promoter.
[0186] 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
PRO20080.
[0187] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of PRO20080 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.
4. Detecting Gene Amplification/Expression
[0188] 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.
[0189] 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 PRO20080 polypeptide or against a synthetic
peptide based on the DNA sequences provided herein or against
exogenous sequence fused to PRO20080 DNA and encoding a specific
antibody epitope.
5. Purification of Polypeptide
[0190] Forms of PRO20080 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
PRO20080 can be disrupted by various physical or chemical means,
such as freeze-thaw cycling, sonication, mechanical disruption, or
cell lysing agents.
[0191] It may be desired to purify PRO20080 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 PRO20080.
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
PRO20080 produced.
[0192] E. Tissue Distribution
[0193] The location of tissues expressing the PRO20080 can be
identified by determining mRNA expression in various human tissues.
The location of such genes provides information about which tissues
are most likely to be affected by the stimulating and inhibiting
activities of the PRO20080 polypeptides. The location of a gene in
a specific tissue also provides sample tissue for the activity
blocking assays discussed below.
[0194] As noted before, gene expression in various tissues may be
measured by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad.
Sci. USA, 77:5201-5205 119801), 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.
[0195] Gene expression in various tissues, alternatively, may be
measured by immunological methods, such as immunohistochemical
staining of 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 of a PRO20080 polypeptide or
against a synthetic peptide based on the DNA sequences encoding the
PRO20080 polypeptide or against an exogenous sequence fused to a
DNA encoding a PRO20080 polypeptide and encoding a specific
antibody epitope. General techniques for generating antibodies, and
special protocols for Northern blotting and in situ hybridization
are provided below.
[0196] F. Antibody Binding Studies
[0197] The activity of the PRO20080 polypeptides can be further
verified by antibody binding studies, in which the ability of
anti-PRO20080 antibodies to inhibit the effect of the PRO20080
polypeptides, respectively, on tissue cells is tested. Exemplary
antibodies include polyclonal, monoclonal, humanized, bispecific,
and heteroconjugate antibodies, the preparation of which will be
described hereinbelow.
[0198] Antibody binding studies may be carried out in any known
assay method, such as competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc., 1987).
[0199] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of target protein in the
test sample is inversely proportional to the amount of standard
that becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies preferably
are insolubilized before or after the competition, so that the
standard and analyte that are bound to the antibodies may
conveniently be separated from the standard and analyte which
remain unbound.
[0200] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three-part complex. See, e.g.,
U.S. Pat. No. 4,376,110. The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0201] For immunohistochemistry, the tissue sample may be fresh or
frozen or may be embedded in paraffin and fixed with a preservative
such as formalin, for example.
[0202] G. Cell-Based Assays
[0203] Cell-based assays and animal models for immune related
diseases can be used to further understand the relationship between
the genes and polypeptides identified herein and the development
and pathogenesis of immune related disease.
[0204] In a different approach, cells of a cell type known to be
involved in a particular immune related disease are transfected
with the cDNAs described herein, and the ability of these cDNAs to
stimulate or inhibit immune function is analyzed. Suitable cells
can be transfected with the desired gene, and monitored for immune
function activity. Such transfected cell lines can then be used to
test the ability of poly- or monoclonal antibodies or antibody
compositions to inhibit or stimulate immune function, for example
to modulate T-cell proliferation or inflammatory cell infiltration.
Cells transfected with the coding sequences of the genes identified
herein can further be used to identify drug candidates for the
treatment of immune related diseases.
[0205] In addition, primary cultures derived from transgenic
animals (as described below) can be used in the cell-based assays
herein, although stable cell lines are preferred. Techniques to
derive continuous cell lines from transgenic animals are well known
in the art (see, e.g., Small et al., Mol. Cell. Biol. 5: 642-648
[1985]).
[0206] One suitable cell based assay is the mixed lymphocyte
reaction (MLR). Current Protocols in Immunology, unit 3.12; edited
by J E Coligan, A M Kruisbeek, D H Marglies, E M Shevach, W
Strober, National Institutes of Health, Published by John Wiley
& Sons, Inc. In this assay, the ability of a test compound to
stimulate or inhibit the proliferation of activated T cells is
assayed. A suspension of responder T cells is cultured with
allogeneic stimulator cells and the proliferation of T-cells is
measured by uptake of tritiated thymidine. This assay is a general
measure of T cell reactivity. Since the majority of T cells respond
to and produce IL-2 upon activation, differences in responsiveness
in this assay in part reflect differences in IL-2 production by the
responding cells. The MLR results can be verified by a standard
lymphokine (IL-2) detection assay. Current Protocols in Immunology,
above, 3.15, 6.3.
[0207] A proliferative T cell response in an MLR assay may be due
to direct mitogenic properties of an assayed molecule or to
external antigen induced activation. Additional verification of the
T cell stimulatory activity of the PRO20080 polypeptides can be
obtained by a costimulation assay. T cell activation requires an
antigen specific signal mediated through the T-cell receptor (TCR)
and a costimulatory signal mediated through a second ligand binding
interaction, for example, the B7 (CD80, CD86)/CD28 binding
interaction. CD28 crosslinking increases lymphokine secretion by
activated T cells. T cell activation has both negative and positive
controls through the binding of ligands which have a negative or
positive effect. CD28 and CTLA-4 are related glycoproteins in the
Ig superfamily which bind to B7. CD28 binding to B7 has a positive
costimulation effect of T cell activation; conversely, CTLA-4
binding to B7 has a T cell deactivating effect. Chambers, C. A. and
Allison, J. P., Curr. Opin. Immunol. (1997) 9:396. Schwartz, R. H.,
Cell (1992) 71:1065; Linsey, P. S, and Ledbetter, J. A., Annu. Rev.
Immunol. (1993) 11:191; June, C. H. et al, Immunol. Today (1994)
15:321; Jenkins, M. K., Immunity (1994) 1:405. In a costimulation
assay, the PRO20080 polypeptides are assayed for T cell
costimulatory or inhibitory activity.
[0208] PRO20080 polypeptides, as well as other compounds of the
invention, which are stimulators (costimulators) of r cell
proliferation and agonists, e.g., agonist antibodies, thereto as
determined by MLR and costimulation assays, for example, are useful
in treating immune related diseases characterized by poor,
suboptimal or inadequate immune function. These diseases are
treated by stimulating the proliferation and activation of T cells
(and r cell mediated immunity) and enhancing the immune response in
a mammal through administration of a stimulatory compound, such as
the stimulating PRO20080 polypeptides. The stimulating polypeptide
may, for example, be a PRO20080 polypeptide or an agonist antibody
thereof.
[0209] Direct use of a stimulating compound as in the invention has
been validated in experiments with 4-IBB glycoprotein, a member of
the tumor necrosis factor receptor family, which binds to a ligand
(4-IBBL) expressed on primed T cells and signals T cell activation
and growth. Alderson, M. E. et al., J. Immunol. (1994) 24:2219.
[0210] The use of an agonist stimulating compound has also been
validated experimentally. Activation of 4-IBB by treatment with an
agonist anti-4-IBB antibody enhances eradication of tumors.
Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. (1998)
18:1. Immunoadjuvant therapy for treatment of tumors, described in
more detail below, is another example of the use of the stimulating
compounds of the invention.
[0211] An immune stimulating or enhancing effect can also be
achieved by antagonizing or blocking the activity of a PRO20080
which has been found to be inhibiting in the MLR assay. Negating
the inhibitory activity of the compound produces a net stimulatory
effect. Suitable antagonists/blocking compounds are antibodies or
fragments thereof which recognize and bind to the inhibitory
protein, thereby blocking the effective interaction of the protein
with its receptor and inhibiting signaling through the receptor.
This effect has been validated in experiments using anti-CTLA-4
antibodies which enhance T cell proliferation, presumably by
removal of the inhibitory signal caused by CTLA-4 binding. Walunas,
.degree. F. L. et al, Immunity (1994) 1:405.
[0212] On the other hand, PRO20080 polypeptides, as well as other
compounds of the invention, which are direct inhibitors of T cell
proliferation/activation or lymphokine secretion, can be directly
used to suppress the immune response. These compounds are useful to
reduce the degree of the immune response and to treat immune
related diseases characterized by a hyperactive, superoptimal, or
autoimmune response. This use of the compounds of the invention has
been validated by the experiments described above in which CTLA-4
binding to receptor B7 deactivates T cells. The direct inhibitory
compounds of the invention function in an analogous manner. The use
of compound which suppress vascular permeability would be expected
to reduce inflammation. Such uses would be beneficial in treating
conditions associated with excessive inflammation.
[0213] Alternatively, compounds, e.g., antibodies, which bind to
stimulating PRO20080 polypeptides and block the stimulating effect
of these molecules produce a net inhibitory effect and can be used
to suppress the T cell mediated immune response by inhibiting T
cell proliferation/activation and/or lymphokine secretion. Blocking
the stimulating effect of the polypeptides suppresses the immune
response of the mammal. This use has been validated in experiments
using an anti-IL2 antibody. In these experiments, the antibody
binds to IL2 and blocks binding of IL2 to its receptor thereby
achieving a T cell inhibitory effect.
[0214] H. Animal Models
[0215] The results of the cell based in vitro assays can be further
verified using in vivo animal models and assays for T-cell
function. A variety of well known animal models can be used to
further understand the role of the genes identified herein in the
development and pathogenesis of immune related disease, and to test
the efficacy of candidate therapeutic agents, including antibodies,
and other antagonists of the native polypeptides, including small
molecule antagonists. The in vivo nature of such models makes them
predictive of responses in human patients. Animal models of immune
related diseases include both non-recombinant and recombinant
(transgenic) animals. Non-recombinant animal models include, for
example, rodent, e.g., murine models. Such models can be generated
by introducing cells into syngeneic mice using standard techniques,
e.g., subcutaneous injection, tail vein injection, spleen
implantation, intraperitoneal implantation, implantation under the
renal capsule, etc.
[0216] Graft-versus-host disease occurs when immunocompetent cells
are transplanted into immunosuppressed or tolerant patients. The
donor cells recognize and respond to host antigens. The response
can vary from life threatening severe inflammation to mild cases of
diarrhea and weight loss. Graft-versus-host disease models provide
a means of assessing T cell reactivity against MHC antigens and
minor transplant antigens. A suitable procedure is described in
detail in Current Protocols in Immunology, above, unit 4.3.
[0217] An animal model for skin allograft rejection is a means of
testing the ability of T cells to mediate in vivo tissue
destruction and a measure of their role in transplant rejection.
The most common and accepted models use murine tail-skin grafts.
Repeated experiments have shown that skin allograft rejection is
mediated by T cells, helper T cells and killer-effector T cells,
and not antibodies. Auchincloss, H. Jr. and Sachs, D. H.,
Fundamental Immunology, 2nd ed., W. E. Paul ed., Raven Press, NY,
1989, 889-992. A suitable procedure is described in detail in
Current Protocols in Immunology, above, unit 4.4. Other transplant
rejection models which can be used to test the compounds of the
invention are the allogeneic heart transplant models described by
Tanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et
al, J. Immunol. (1994) 4330-4338.
[0218] Animal models for delayed type hypersensitivity provides an
assay of cell mediated immune function as well. Delayed type
hypersensitivity reactions are a T cell mediated in vivo immune
response characterized by inflammation which does not reach a peak
until after a period of time has elapsed after challenge with an
antigen. These reactions also occur in tissue specific autoimmune
diseases such as multiple sclerosis (MS) and experimental
autoimmune encephalomyelitis (EAE, a model for MS). A suitable
procedure is described in detail in Current Protocols in
Immunology, above, unit 4.5.
[0219] EAE is a T cell mediated autoimmune disease characterized by
T cell and mononuclear cell inflammation and subsequent
demyelination of axons in the central nervous system. EAE is
generally considered to be a relevant animal model for MS in
humans. Bolton, C., Multiple Sclerosis (1995) 1:143. Both acute and
relapsing-remitting models have been developed. The compounds of
the invention can be tested for T cell stimulatory or inhibitory
activity against immune mediated demyelinating disease using the
protocol described in Current Protocols in Immunology, above, units
15.1 and 15.2. See also the models for myelin disease in which
oligodendrocytes or Schwann cells are grafted into the central
nervous system as described in Duncan, I. D. et al, Molec. Med.
Today (1997) 554-561.
[0220] Contact hypersensitivity is a simple delayed type
hypersensitivity in vivo assay of cell mediated immune function. In
this procedure, cutaneous exposure to exogenous haptens which gives
rise to a delayed type hypersensitivity reaction which is measured
and quantitated. Contact sensitivity involves an initial
sensitizing phase followed by an elicitation phase. The elicitation
phase occurs when the T lymphocytes encounter an antigen to which
they have had previous contact. Swelling and inflammation occur,
making this an excellent model of human allergic contact
dermatitis. A suitable procedure is described in detail in Current
Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach and W. Strober, John Wiley & Sons,
Inc., 1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun.
Today 19 (1): 37-44 (1998).
[0221] An animal model for arthritis is collagen-induced arthritis.
This model shares clinical, histological and immunological
characteristics of human autoimmune rheumatoid arthritis and is an
acceptable model for human autoimmune arthritis. Mouse and rat
models are characterized by synovitis, erosion of cartilage and
subchondral bone. The compounds of the invention can be tested for
activity against autoimmune arthritis using the protocols described
in Current Protocols in Immunology, above, units 15.5. See also the
model using a monoclonal antibody to CD18 and VLA-4 integrins
described in Issekutz, A. C. et al., Immunology (1996) 88:569.
[0222] A model of asthma has been described in which
antigen-induced airway hyper-reactivity, pulmonary eosinophilia and
inflammation are induced by sensitizing an animal with ovalbumin
and then challenging the animal with the same protein delivered by
aerosol. Several animal models (guinea pig, rat, non-human primate)
show symptoms similar to atopic asthma in humans upon challenge
with aerosol antigens. Murine models have many of the features of
human asthma. Suitable procedures to test the compounds of the
invention for activity and effectiveness in the treatment of asthma
are described by Wolyniec, W. W. et al, Am. J. Respir. Cell Mol.
Biol. (1998) 18:777 and the references cited therein.
[0223] Additionally, the compounds of the invention can be tested
on animal models for psoriasis like diseases. Evidence suggests a T
cell pathogenesis for psoriasis. The compounds of the invention can
be tested in the scid/scid mouse model described by Schon, M. P. et
al, Nat. Med. (1997) 3:183, in which the mice demonstrate
histopathologic skin lesions resembling psoriasis. Another suitable
model is the human skin/scid mouse chimera prepared as described by
Nickoloff, B. J. et al. Am. J. Path. (1995) 146:580.
[0224] Recombinant (transgenic) animal models can be engineered by
introducing the coding portion of the genes identified herein into
the genome of animals of interest, using standard techniques for
producing transgenic animals. Animals that can serve as a target
for transgenic manipulation include, without limitation, mice,
rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human
primates, e.g., baboons, chimpanzees and monkeys. Techniques known
in the art to introduce a transgene into such animals include
pronucleic microinjection (Hoppe and Wanger, U.S. Pat. No.
4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82,
6148-615 [1985]); gene targeting in embryonic stem cells (Thompson
et al., Cell 56; 313-321 [19891); electroporation of embryos (Lo,
Mol. Cel. Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer
(Lavitrano et al., Cell 57, 717-73 (1989]). For review, see, for
example, U.S. Pat. No. 4,736,866.
[0225] For the purpose of the present invention, transgenic animals
include those that carry the transgene only in part of their cells
("mosaic animals"). The transgene can be integrated either as a
single transgene, or in concatamers, e.g., head-to-head or
head-to-tail tandems. Selective introduction of a transgene into a
particular cell type is also possible by following, for example,
the technique of Lasko et al., Proc. Nail. Acad. Sci. USA 89,
6232-636 (1992).
[0226] The expression of the transgene in transgenic animals can be
monitored by standard techniques. For example, Southern blot
analysis or PCR amplification can be used to verify the integration
of the transgene. The level of mRNA expression can then be analyzed
using techniques such as in situ hybridization, Northern blot
analysis, PCR, or immunocytochemistry.
[0227] The animals may be further examined for signs of immune
disease pathology, for example by histological examination to
determine infiltration of immune cells into specific tissues.
Blocking experiments can also be performed in which the transgenic
animals are treated with the compounds of the invention to
determine the extent of the T cell proliferation stimulation or
inhibition of the compounds. In these experiments, blocking
antibodies which bind to the PRO20080 polypeptide, prepared as
described above, are administered to the animal and the effect on
immune function is determined.
[0228] Alternatively, "knock out" animals can be constructed which
have a defective or altered gene encoding a polypeptide identified
herein, as a result of homologous recombination between the
endogenous gene encoding the polypeptide and altered genomic DNA
encoding the same polypeptide introduced into an embryonic cell of
the animal. For example, cDNA encoding a particular polypeptide can
be used to clone genomic DNA encoding that polypeptide in
accordance with established techniques. A portion of the genomic
DNA encoding a particular 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 Capecehi, 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 polypeptide.
[0229] I. ImmunoAdjuvant Therapy
[0230] In one embodiment, the immunostimulating compounds of the
invention can be used in immunoadjuvant therapy for the treatment
of tumors (cancer). It is now well established that T cells
recognize human tumor specific antigens. One group of tumor
antigens, encoded by the MAGE, BAGE and GAGE families of gene, are
silent in all adult normal tissues, but are expressed in
significant amounts in tumors, such as melanomas, lung tumors, head
and neck tumors, and bladder carcinomas. DeSmet, C. et al., (1996)
Proc. Natl. Acad. Sci. USA, 93:7149. It has been shown that
costimulation of T cells induces tumor regression and an antitumor
response both in vitro and in vivo. Melero, I. et al., Nature
Medicine (1997) 3:682; Kwon, E. D. et al., Proc. Natl. Acad. Sci.
USA (1997) 94: 8099; Lynch, D. H. et al, Nature Medicine (1997)
3:625; Finn, O. J. and Lotze, M. T., J. Immunol. (1998) 21:114. The
stimulatory compounds of the invention can be administered as
adjuvants, alone or together with a growth regulating agent,
cytotoxic agent or chemotherapeutic agent, to stimulate T cell
proliferation/activation and an antitumor response to tumor
antigens. The growth regulating, cytotoxic, or chemotherapeutic
agent may be administered in conventional amounts using known
administration regimes. Immunostimulating activity by the compounds
of the invention allows reduced amounts of the growth regulating,
cytotoxic, or chemotherapeutic agents thereby potentially lowering
the toxicity to the patient.
[0231] J. Screening Assays for Drug Candidates
[0232] Screening assays for drug candidates are designed to
identify compounds that bind to or complex with the polypeptides
encoded by the genes identified herein or a biologically active
fragment thereof, or otherwise interfere with the interaction of
the encoded polypeptides with other cellular proteins. Such
screening assays will include assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable
for identifying small molecule drug candidates. Small molecules
contemplated include synthetic organic or inorganic compounds,
including peptides, preferably soluble peptides,
(poly)peptide-immunoglobulin fusions, 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. 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. All assays are common in that they
call for contacting the drug candidate with a polypeptide encoded
by a nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0233] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the polypeptide encoded by the gene
identified herein or the drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by
coating the solid surface with a solution of the polypeptide and
drying. Alternatively, an immobilized antibody, e.g., a monoclonal
antibody, specific for the 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 labelled antibody specifically binding the immobilized
complex.
[0234] If the candidate compound interacts with but does not bind
to a particular protein encoded by a gene identified herein, its
interaction with that protein can be assayed by methods well known
for detecting protein-protein interactions. Such assays include
traditional approaches, such as, cross-linking,
co-immunoprecipitation, and co-purification through gradients or
chromatographic columns. In addition, protein-protein interactions
can be monitored by using a yeast-based genetic system described by
Fields and co-workers [Fields and Song, Nature (London) 340,
245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88,
9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Natl.
Acad. Sci. USA 89, 5789-5793 (1991). Many transcriptional
activators, such as yeast GAL4, consist of two physically discrete
modular domains, one acting as the DNA-binding domain, while 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 P-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.
[0235] In order to find compounds that interfere with the
interaction of a gene identified herein and other intra- or
extracellular components can be tested, a reaction mixture is
usually 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 test 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 above. 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.
[0236] K. Compositions and Methods for the Treatment of Immune
Related Diseases
[0237] The compositions useful in the treatment of immune related
diseases include, without limitation, proteins, antibodies, small
organic molecules, peptides, phosphopeptides, antisense and
ribozyme molecules, triple helix molecules, etc. that inhibit or
stimulate immune function, for example, T cell
proliferation/activation, lymphokine release, or immune cell
infiltration.
[0238] For example, antisense RNA and RNA molecules act to directly
block the translation of mRNA by hybridizing to targeted mRNA and
preventing protein translation. 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.
[0239] 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).
[0240] 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.
[0241] These molecules can be identified by any or any combination
of the screening assays discussed above and/or by any other
screening techniques well known for those skilled in the art.
[0242] L. Anti-PRO20080 Antibodies
[0243] The present invention further provides anti-PRO20080
antibodies. Exemplary antibodies include polyclonal, monoclonal,
humanized, bispecific, and heteroconjugate antibodies.
[0244] 1. Polyclonal Antibodies
[0245] The anti-PRO20080 antibodies may comprise polyclonal
antibodies. Methods of preparing polyclonal antibodies are known to
the skilled artisan. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant. Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent
may include the PRO20080 polypeptide or a fusion protein thereof.
It may be useful to conjugate the immunizing agent to a protein
known to be immunogenic in the mammal being immunized. Examples of
such immunogenic proteins include but are not limited to keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0246] 2. Monoclonal Antibodies
[0247] The anti-PRO20080 antibodies may, alternatively, be
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or
other appropriate host animal, is typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable
of producing antibodies that will specifically bind to the
immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
[0248] The immunizing agent will typically include the PRO20080
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0249] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0250] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against PRO20080. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0251] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0252] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0253] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra] or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0254] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0255] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0256] 3. Human and Humanized Antibodies
[0257] The anti-PRO20080 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'), 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)].
[0258] 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.
[0259] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnolopy 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0260] The antibodies may also be affinity matured using known
selection and/or mutagenesis methods as described above. Preferred
affinity matured antibodies have an affinity which is five times,
more preferably 10 times, even more preferably 20 or 30 times
greater than the starting antibody (generally murine, humanized or
human) from which the matured antibody is prepared.
[0261] 4. Bispecific Antibodies
[0262] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the PRO20080, the other one is for any other
antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
[0263] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0264] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least pan of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0265] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0266] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab'), 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.
[0267] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175:217-225 (1992) describe the production of a fully
humanized bispecific antibody F(ab').sub.2 molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0268] Various technique for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0269] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0270] Exemplary bispecific antibodies may bind to two different
epitopes on a given PRO20080 polypeptide herein. Alternatively, an
anti-PRO20080 polypeptide arm may be combined with an arm which
binds to a triggering molecule on a leukocyte such as a T-cell
receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fe receptors for
IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32)
and Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms
to the cell expressing the particular PRO20080 polypeptide.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells which express a particular PRO20080 polypeptide. These
antibodies possess a PRO20080-binding arm and an arm which binds a
cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA,
DOTA, or TETA. Another bispecific antibody of interest binds the
PRO20080 polypeptide and further binds tissue factor (TF).
[0271] 5. Heteroconjugate Antibodies
[0272] 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.
[0273] 6. Effector Function Engineering
[0274] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design. 3: 219-230 (1989).
[0275] 7. Immunoconjugates
[0276] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0277] 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, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.151I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0278] 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.
[0279] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
[0280] 8. Immunoliposomes
[0281] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0282] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0283] M. Pharmaceutical Compositions
[0284] The active PRO20080 molecules of the invention (e.g.,
PRO20080 polypeptides, anti-PRO20080 antibodies, and/or variants of
each) as well as other molecules identified by the screening assays
disclosed above, can be administered for the treatment of immune
related diseases, in the form of pharmaceutical compositions.
[0285] Therapeutic formulations of the active PRO20080 molecule,
preferably a polypeptide or antibody of the invention, are prepared
for storage by mixing the active molecule having the desired degree
of purity with optional pharmaceutically 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 and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); 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, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0286] Compounds identified by the screening assays disclosed
herein can be formulated in an analogous manner, using standard
techniques well known in the art.
[0287] Lipofections or liposomes can also be used to deliver the
PRO20080 molecule into cells. Where antibody fragments are used,
the smallest inhibitory fragment which 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 which 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]).
[0288] 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 a cytotoxic agent, cytokine or growth
inhibitory agent. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0289] The active PRO20080 molecules 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
16th edition, Osol, A. Ed. (1980).
[0290] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0291] Sustained-release preparations or the PRO20080 molecules 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.
[0292] N. Methods of Treatment
[0293] It is contemplated that the polypeptides, antibodies and
other active compounds of the present invention may be used to
treat various immune related diseases and conditions, such as T
cell mediated diseases, including those characterized by
infiltration of inflammatory cells into a tissue, stimulation of
T-cell proliferation, inhibition of T-cell proliferation, increased
or decreased vascular permeability or the inhibition thereof.
[0294] Exemplary conditions or disorders to be treated with the
polypeptides, antibodies and other compounds of the invention,
include, but are not limited to systemic lupus erythematosis,
rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis,
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), demyclinating
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, transplantation associated diseases
including graft rejection and graft-versus-host-disease.
[0295] In systemic lupus erythematosus, the central mediator of
disease is the production of auto-reactive antibodies to self
proteins/tissues and the subsequent generation of immune-mediated
inflammation. Antibodies either directly or indirectly mediate
tissue injury. Though T lymphocytes have not been shown to be
directly involved in tissue damage, T lymphocytes are required for
the development of auto-reactive antibodies. The genesis of the
disease is thus T lymphocyte dependent. Multiple organs and systems
are affected clinically including kidney, lung, musculoskeletal
system, mucocutaneous, eye, central nervous system, cardiovascular
system, gastrointestinal tract, bone marrow and blood.
[0296] Rheumatoid arthritis (RA) is a chronic systemic autoimmune
inflammatory disease that mainly involves the synovial membrane of
multiple joints with resultant injury to the articular cartilage.
The pathogenesis is T lymphocyte dependent and is associated with
the production of rheumatoid factors, auto-antibodies directed
against self IgG, with the resultant formation of immune complexes
that attain high levels in joint fluid and blood. These complexes
in the joint may induce the marked infiltrate of lymphocytes and
monocytes into the synovium and subsequent marked synovial changes;
the joint space/fluid if infiltrated by similar cells with the
addition of numerous neutrophils. Tissues affected are primarily
the joints, often in symmetrical pattern. However, extra-articular
disease also occurs in two major forms. One form is the development
of extra-articular lesions with ongoing progressive joint disease
and typical lesions of pulmonary fibrosis, vasculitis, and
cutaneous ulcers. The second form of extra-articular disease is the
so called Felty's syndrome which occurs late in the RA disease
course, sometimes after joint disease has become quiescent, and
involves the presence of neutropenia, thrombocytopenia and
splenomegaly. This can be accompanied by vasculitis in multiple
organs with formations of infarcts, skin ulcers and gangrene.
Patients often also develop rheumatoid nodules in the subcutis
tissue overlying affected joints; the nodules late stage have
necrotic centers surrounded by a mixed inflammatory cell
infiltrate. Other manifestations which can occur in RA include:
pericarditis, pleuritis, coronary arteritis, intestitial
pneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca,
and rhematoid nodules.
[0297] Juvenile chronic arthritis is a chronic idiopathic
inflammatory disease which begins often at less than 16 years of
age. Its phenotype has some similarities to RA; some patients which
are rhematoid factor positive are classified as juvenile rheumatoid
arthritis. The disease is sub-classified into three major
categories: pauciarticular, polyarticular, and systemic. The
arthritis can be severe and is typically destructive and leads to
joint ankylosis and retarded growth. Other manifestations can
include chronic anterior uveitis and systemic amyloidosis.
[0298] Spondyloarthropathies are a group of disorders with some
common clinical features and the common association with the
expression of HLA-B27 gene product. The disorders include:
ankylosing sponylitis, Reiter's syndrome (reactive arthritis),
arthritis associated with inflammatory bowel disease, spondylitis
associated with psoriasis, juvenile onset spondyloarthropathy and
undifferentiated spondyloarthropathy. Distinguishing features
include sacroileitis with or without spondylitis; inflammatory
asymmetric arthritis; association with HLA-B27 (a serologically
defined allele of the HLA-B locus of class I MHC); ocular
inflammation, and absence of autoantibodies associated with other
rheumatoid disease. The cell most implicated as key to induction of
the disease is the CD8+ T lymphocyte, a cell which targets antigen
presented by class I MHC molecules. CD8+ T cells may react against
the class I MHC allele HLA-B27 as if it were a foreign peptide
expressed by MHC class 1 molecules. It has been hypothesized that
an epitope of HLA-B27 may mimic a bacterial or other microbial
antigenic epitope and thus induce a CD8+ T cells response.
[0299] Systemic sclerosis (scleroderma) has an unknown etiology. A
hallmark of the disease is induration of the skin; likely this is
induced by an active inflammatory process. Scleroderma can be
localized or systemic; vascular lesions are common and endothelial
cell injury in the microvasculature is an early and important event
in the development of systemic sclerosis; the vascular injury may
be immune mediated. An immunologic basis is implied by the presence
of mononuclear cell infiltrates in the cutaneous lesions and the
presence of anti-nuclear antibodies in many patients. ICAM-1 is
often upregulated on the cell surface of fibroblasts in skin
lesions suggesting that T cell interaction with these cells may
have a role in the pathogenesis of the disease. Other organs
involved include: the gastrointestinal tract: smooth muscle atrophy
and fibrosis resulting in abnormal peristalsis/motility; kidney:
concentric subendothelial intimal proliferation affecting small
arcuate and interlobular arteries with resultant reduced renal
cortical blood flow, results in proteinuria, azotemia and
hypertension; skeletal muscle: atrophy, interstitial fibrosis;
inflammation; lung: interstitial pneumonitis and interstitial
fibrosis; and heart: contraction band necrosis,
scarring/fibrosis.
[0300] Idiopathic inflammatory myopathies including
dermatomyositis, polymyositis and others are disorders of chronic
muscle inflammation of unknown etiology resulting in muscle
weakness. Muscle injury/inflammation is often symmetric and
progressive. Autoantibodies are associated with most forms. These
myositis-specific autoantibodies are directed against and inhibit
the function of components, proteins and RNA's, involved in protein
synthesis.
[0301] Sjogren's syndrome is due to immune-mediated inflammation
and subsequent functional destruction of the tear glands and
salivary glands. The disease can be associated with or accompanied
by inflammatory connective tissue diseases. The disease is
associated with autoantibody production against Ro and La antigens,
both of which are small RNA-protein complexes. Lesions result in
keratoconjunctivitis sicca, xerostomia, with other manifestations
or associations including bilary cirrhosis, peripheral or sensory
neuropathy, and palpable purpura.
[0302] Systemic vasculitis are diseases in which the primary lesion
is inflammation and subsequent damage to blood vessels which
results in ischemia/necrosis/degeneration to tissues supplied by
the affected vessels and eventual end-organ dysfunction in some
cases. Vasculitides can also occur as a secondary lesion or
sequelae to other immune-inflammatory mediated diseases such as
rheumatoid arthritis, systemic sclerosis, etc., particularly in
diseases also associated with the formation of immune complexes.
Diseases in the primary systemic vasculitis group include: systemic
necrotizing vasculitis: polyarteritis nodosa, allergic angitis and
granulomatosis, polyangiitis; Wegener's granulomatosis;
lymphomatoid granulomatosis; and giant cell arteritis.
Miscellaneous vasculitides include: mucocutaneous lymph node
syndrome (MLNS or Kawasaki's disease), isolated CNS vasculitis,
Behet's disease, thromboangiitis obliterans (Buerger's disease) and
cutaneous necrotizing venulitis. The pathogenic mechanism of most
of the types of vasculitis listed is believed to be primarily due
to the deposition of immunoglobulin complexes in the vessel wall
and subsequent induction of an inflammatory response either via
ADCC, complement activation, or both.
[0303] Sarcoidosis is a condition of unknown etiology which is
characterized by the presence of epithelioid granulomas in nearly
any tissue in the body; involvement of the lung is most common. The
pathogenesis involves the persistence of activated macrophages and
lymphoid cells at sites of the disease with subsequent chronic
sequelae resultant from the release of locally and systemically
active products released by these cell types.
[0304] Autoimmune hemolytic anemia including autoimmune hemolytic
anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuria
is a result of production of antibodies that react with antigens
expressed on the surface of red blood cells (and in some cases
other blood cells including platelets as well) and is a reflection
of the removal of those antibody coated cells via complement
mediated lysis and/or ADCC/Fc-receptor-mediated mechanisms.
[0305] In autoimmune thrombocytopenia including thrombocytopenic
purpura, and immune-mediated thrombocytopenia in other clinical
settings, platelet destruction/removal occurs as a result of either
antibody or complement attaching to platelets and subsequent
removal by complement lysis, ADCC or FC-receptor mediated
mechanisms.
[0306] Thyroiditis including Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, and atrophic
thyroiditis, are the result of an autoimmune response against
thyroid antigens with production of antibodies that react with
proteins present in and often specific for the thyroid gland.
Experimental models exist including spontaneous models: rats (BUF
and BB rats) and chickens (obese chicken strain); inducible models:
immunization of animals with either thyroglobulin, thyroid
microsomal antigen (thyroid peroxidase).
[0307] Type I diabetes mellitus or insulin-dependent diabetes is
the autoimmune destruction of pancreatic islet .E-backward. cells;
this destruction is mediated by auto-antibodies and auto-reactive T
cells. Antibodies to insulin or the insulin receptor can also
produce the phenotype of insulin-non-responsiveness.
[0308] Immune mediated renal diseases, including glomerulonephritis
and tubulointerstitial nephritis, are the result of antibody or T
lymphocyte mediated injury to renal tissue either directly as a
result of the production of autoreactive antibodies or T cells
against renal antigens or indirectly as a result of the deposition
of antibodies and/or immune complexes in the kidney that are
reactive against other, non-renal antigens. Thus other
immune-mediated diseases that result in the formation of
immune-complexes can also induce immune mediated renal disease as
an indirect sequelae. Both direct and indirect immune mechanisms
result in inflammatory response that produces/induces lesion
development in renal tissues with resultant organ function
impairment and in some cases progression to renal failure. Both
humoral and cellular immune mechanisms can be involved in the
pathogenesis of lesions.
[0309] Demyelinating diseases of the central and peripheral nervous
systems, including Multiple Sclerosis; idiopathic demyelinating
polyneuropathy or Guillain-Barre syndrome; and Chronic Inflammatory
Demyelinating Polyneuropathy, are believed to have an autoimmune
basis and result in nerve demyelination as a result of damage
caused to oligodendrocytes or to myelin directly. In MS there is
evidence to suggest that disease induction and progression is
dependent on T lymphocytes. Multiple Sclerosis is a demyelinating
disease that is T lymphocyte-dependent and has either a
relapsing-remitting course or a chronic progressive course. The
etiology is unknown; however, viral infections, genetic
predisposition, environment, and autoimmunity all contribute.
Lesions contain infiltrates of predominantly T lymphocyte mediated,
microglial cells and infiltrating macrophages: CD4+ T lymphocytes
are the predominant cell type at lesions. The mechanism of
oligodendrocyte cell death and subsequent demyelination is not
known but is likely T lymphocyte driven.
[0310] Inflammatory and Fibrotic Lung Disease, including
Eosinophilic Pneumonias; Idiopathic Pulmonary Fibrosis, and
Hypersensitivity Pneumonitis may involve a disregulated
immune-inflammatory response. Inhibition of that response would be
of therapeutic benefit.
[0311] Autoimmune or Immune-mediated Skin Disease including Bullous
Skin Diseases, Erythema Multiforme, and Contact Dermatitis are
mediated by auto-antibodies, the genesis of which is T
lymphocyte-dependent.
[0312] Psoriasis is a T lymphocyte-mediated inflammatory disease.
Lesions contain infiltrates of T lymphocytes, macrophages and
antigen processing cells, and some neutrophils.
[0313] Allergic diseases, including asthma; allergic rhinitis;
atopic dermatitis; food hypersensitivity; and urticaria are T
lymphocyte dependent. These diseases are predominantly mediated by
T lymphocyte induced inflammation, IgE mediated-inflammation or a
combination of both.
[0314] Transplantation associated diseases, including Graft
rejection and Graft-Versus-Host-Disease (GVHD) are T
lymphocyte-dependent: inhibition of T lymphocyte function is
ameliorative. Other diseases in which intervention of the immune
and/or inflammatory response have benefit are infectious disease
including but not limited to viral infection (including but not
limited to AIDS, hepatitis A, B, C, D, E and herpes) bacterial
infection, fungal infections, and protozoal and parasitic
infections (molecules (or derivatives/agonists) which stimulate T
cells can be utilized therapeutically to enhance the immune
response to infectious agents), diseases of immunodeficiency
(molecules/derivatives/agonists) which stimulate T cells can be
utilized therapeutically to enhance the immune response for
conditions of inherited, acquired, infectious induced (as in HIV
infection), or iatrogenic (i.e., as from chemotherapy)
immunodeficiency, and neoplasia.
[0315] It has been demonstrated that some human cancer patients
develop an antibody and/or T lymphocyte response to antigens on
neoplastic cells. It has also been shown in animal models of
neoplasia that enhancement of the immune response can result in
rejection or regression of that particular neoplasm. Molecules that
enhance the T lymphocyte response have utility in vivo in enhancing
the immune response against neoplasia. Molecules which enhance the
T lymphocyte proliferative response (or small molecule agonists or
antibodies that affected the same receptor in an agonistic fashion)
can be used therapeutically to treat cancer.
[0316] Additionally, inhibition of molecules with proinflammatory
properties may have therapeutic benefit in reperfusion injury;
stroke; myocardial infarction; atherosclerosis; acute lung injury;
hemorrhagic shock; burn; sepsis/septic shock; acute tubular
necrosis; endometriosis; degenerative joint disease and
pancreatis.
[0317] The compounds of the present invention, e.g., polypeptides
or antibodies, are administered to a mammal, preferably a human, in
accord with known methods, such as intravenous administration as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, topical, or
inhalation (intranasal, intrapulmonary) routes. Intravenous or
inhaled administration of polypeptides and antibodies is
preferred.
[0318] In immunoadjuvant therapy, other therapeutic regimens, such
administration of an anti-cancer agent, may be combined with the
administration of the proteins, antibodies or compounds of the
instant invention. For example, the patient to be treated with a
the immunoadjuvant of the invention may also receive an anti-cancer
agent (chemotherapeutic agent) or radiation therapy. Preparation
and dosing schedules for such chemotherapeutic agents may be used
according to manufacturers' instructions or as determined
empirically by the skilled practitioner. Preparation and dosing
schedules for such chemotherapy are also described in Chemotherapy
Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
(1992). The chemotherapeutic agent may precede, or follow
administration of the immunoadjuvant or may be given simultaneously
therewith. Additionally, an anti-estrogen compound such as
tamoxifen or an anti-progesterone such as onapristone (see, EP
616812) may be given in dosages known for such molecules.
[0319] It may be desirable to also administer antibodies against
other immune disease associated or tumor associated antigens, such
as antibodies which bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3,
ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in
addition, two or more antibodies binding the same or two or more
different antigens disclosed herein may be coadministered to the
patient. Sometimes, it may be beneficial to also administer one or
more cytokines to the patient.
[0320] For the treatment or reduction in the severity of immune
related disease, the appropriate dosage of an a compound of the
invention will depend on the type of disease to be treated, as
defined above, the severity and course of the disease, whether the
agent is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the compound, and the discretion of the attending physician. The
compound is suitably administered to the patient at one time or
over a series of treatments.
[0321] For example, depending on the type and severity of the
disease, about 1 .mu.g/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of
polypeptide or antibody is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A typical
daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or
more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0322] O. Articles of Manufacture
[0323] In another embodiment of the invention, an article of
manufacture containing materials (e.g., comprising a PRO20080
molecule) useful for the diagnosis or treatment of the disorders
described above is provided. The article of manufacture comprises a
container and an instruction. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. The containers
may be formed from a variety of materials such as glass or plastic.
The container holds a composition which is effective for diagnosing
or treating the condition and may have a sterile access port (for
example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). The
active agent in the composition is usually a polypeptide or an
antibody of the invention. An instruction or label on, or
associated with, the container indicates that the composition is
used for diagnosing or treating the condition of choice. The
article of manufacture may further comprise a second container
comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
use.
[0324] P. Diagnosis and Prognosis of Immune Related Disease
[0325] Cell surface proteins, such as proteins which are
overexpressed in certain immune related diseases, are excellent
targets for drug candidates or disease treatment. The same proteins
along with secreted proteins encoded by the genes amplified in
immune related disease states find additional use in the diagnosis
and prognosis of these diseases. For example, antibodies directed
against the protein products of genes amplified in multiple
sclerosis, rheumatoid arthritis, or another immune related disease,
can be used as diagnostics or prognostics.
[0326] For example, antibodies, including antibody fragments, can
be used to qualitatively or quantitatively detect the expression of
proteins encoded by amplified or overexpressed genes ("marker gene
products"). The antibody preferably is equipped with a detectable,
e.g., fluorescent label, and binding can be monitored by light
microscopy, flow cytometry, fluorimetry, or other techniques known
in the art. These techniques are particularly suitable, if the
overexpressed gene encodes a cell surface protein Such binding
assays are performed essentially as described above.
[0327] In situ detection of antibody binding to the marker gene
products can be performed, for example, by immunofluorescence or
immunoelectron microscopy. For this purpose, a histological
specimen is removed from the patient, and a labeled antibody is
applied to it, preferably by overlaying the antibody on a
biological sample. This procedure also allows for determining the
distribution of the marker gene product in the tissue examined. It
will be apparent for those skilled in the art that a wide variety
of histological methods are readily available for in situ
detection.
[0328] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0329] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0330] 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
Isolation of cDNA Clones Encoding a Human PRO20080
[0331] 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 prediction program GENSCAN, licensed 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.
[0332] A consensus sequence is herein designated DNA 145436.
[0333] Based on the DNA145436 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 PRO20080.
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.
[0334] PCR primers (forward and reverse) were synthesized:
TABLE-US-00006 forward PCR primer (SEQ ID NO:3)
5'-GAGAGAAGGTCAACTTCATCACTTGGCTTTTC-3' reverse PCR primer (SEQ ID
NO:4) 5'-CTGCAGGAAACTCCAGGGGAAGAGTTACT-3'
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA145436 sequence which had the
following nucleotide sequence
Hybridization Probe
TABLE-US-00007 [0335]
5'-CTTCACCCAGTCCTACTCCCTGCAACTCAGCAACCTGAAGATGGAAGACA-3' (SEQ ID
NO:5)
[0336] 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.
[0337] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length PRO20080
polypeptide (designated herein as DNA161000-2896 [FIG. 1, SEQ ID
NO: 1]) and the derived protein sequence for that PRO20080
polypeptide.
[0338] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 62-64 and a stop signal at nucleotide
positions 1058-1060 (FIG. 1, SEQ ID NO: 1). The predicted
polypeptide precursor is 332 amino acids long, has a calculated
molecular weight of approximately 37345 daltons and an estimated pI
of approximately 6.72. Analysis of the full-length PRO20080
sequence shown in FIG. 2 (SEQ ID NO:2) evidences the presence of a
variety of important polypeptide domains as shown in FIG. 2,
wherein the locations given for those important polypeptide domains
are approximate as described above. PRO20080 amino acid sequence
has 22% identity with CD84, 14% identity with SLAM and 11% identity
with 2B4.
[0339] DNA161000-2896 was then deposited under the terms of the
Budapest Treaty with the American Type Culture Collection, 10801
University Blvd., Manassas, Va. 20110-2209, USA (ATCC). Clone
DNA161000-2896 has been deposited with ATCC on Apr. 18, 2000 and is
assigned ATCC deposit number PTA-1731.
Example 2
Expression of PRO20080 in Multiple Human Tissues
[0340] Multiple tissue Northern Blots were obtained from Clontech
(Palo Alto, Calif.). These blots were hybridized with a probe
corresponding to the full length PRO20080 cDNA made by random
primer labeling with .sup.32P-.gamma.ATP label. Blots were washed
3.times. with 2.times.SSC/0.2% SDS and 1.times. with
0.2.times.SSC/0.1% SDS at 42 C. The blots were then exposed to
X-OMAT film with intensifying screens for overnight. The result is
shown in FIG. 3. This shows that PRO20080 receptor is expressed
highly in spleen and lymph node, with lower level of PRO20080
expression observed in thymus and bone marrow, with little
expression in fetal liver. No expression was detected in brain,
heart, colon or lung.
Example 3
Expression of PRO20080 in Multiple Human Cell Lines
[0341] Cell surface expression analysis of PRO20080 was performed
by washing 1.times.10.sup.6 cells in 1% FBS/PBS. An ALEXA-488
labeled monoclonal antibody raised against the extracellular domain
of PRO20080 was used to detect surface staining on cell lines at a
concentration of 1 ug per million cells. Cells were then washed
again and analyzed by flow cytometry. All of the T cell derived
lines such as Jurkat, MOLT3 and MOLT4 were positive for cell
surface staining of PRO20080. The B cell lines that tested positive
for PRO20080 expression were the Raji, Ramos and Daudi lines, with
no detection of PRO20080 in the remainder of the B cell lines. This
is shown in FIG. 4.
Example 4
Increased Expression of PRO20080 Upon Stimulation
[0342] Cell surface expression was detected by FACS analysis on
isolated human peripheral T cells, as shown in FIG. 5. Human T
cells were activated with plate-bound anti-CD3 antibody (coated at
0.1 ug/ml; Pharmingen) and anti-CD28 antibody (coated at 1 ug/ml;
Pharmingen). 0.5.times.10.sup.6 cells were plated per well. Both
resting and activated T cells were then analyzed for expression of
PRO20080 or CD69 at 24 and 48 hours by flow cytometry. PRO20080
expression was detected using the ALEXA-488 labeled monoclonal
antibody raised against the extracellular domain of PRO20080 as
described in Example 3. CD69, an activation marker, was detected
using by flow cytometry using a FITC-labeled antibody (Pharmingen).
Co-stimulation of isolated T cells with anti-CD3 and anti-CD28
antibody led to increased expression of PRO20080 on the cell
surface as determined by FACS analysis at 24 hours (data not shown)
and even greater expression at 48 hours post co-stimulation (FIG.
5a). High expression of PRO20080 on the surface of CD4+ CD45RO+
memory T cells without co-stimulation was also determined (FIG.
5b).
Example 5
Homotypic Binding of PRO20080
[0343] To determine if PRO20080 interacts with itself in a
PRO20080-PRO20080 association similar to SLAM, an ELISA assay was
performed. Purified PRO20080-Fc fusion protein was coated onto a
standard Nunc Maxisorb ELISA plate at a concentration of 2 .mu.g/ml
and increasing concentrations of biotinylated PRO20080 were
incubated and subsequently washed. Binding of the biotinylated
protein was detected using a streptavidin-HRP conjugate. It was
determined that the biotinlyated PRO20080 can associate with the
bound PRO20080 polypeptide and not with appropriate protein
controls (FIG. 6).
[0344] To determine if the ECD of PRO20080 can interact with a wild
type PRO20080 on the cell surface, COS7 cells were transfected with
wild type PRO20080 or human CD4, which contains an Ig domain, for
use as a control. Forty-eight hours after transfection, the
transfected cells washed and incubated with 5:g/ml purified
PRO20080 ECD-Fc fusion protein plus 5% normal goat serum. The
treated cells were washed and fixed in 4% paraformadehyde for 15
minutes and then probed with a biotinlyated goat anti-human Fc
antibody (Jackson Immunoresearch) at a dilution of 1:400. After a
30 minute incubation period, the fixed cells were washed and
subsequently probed with a 1:400 dilution of Cy3-conjugated
streptavidin label (Jackson Immunoresearch) for detection.
Fluorescence was detected only in COS7 cells transfected with
PRO20080, and not those COS7 cells transfected with CD 4, this
demonstrates that soluble PRO20080 can associate with cell-bound
PRO20080 in a specific manner, since no staining is observed on
cells transfected with CD4, a receptor also containing Ig domain
repeats. This is shown in FIG. 7.
[0345] Lastly, a BIAcore.RTM. assay was performed to determine if a
His-tagged PRO20080 would associate with itself. The association
rate can be determined by any method in which formation of the
complex may be observed as a function of time. In the BIAcore.RTM.
analysis, in which one measures the association of the protein to
an protein that has been immobilized on a biosensor surface
(reviewed by Rich & Myszka, Curr. Opin. Biotechnol. 11:54-61
(2000)). The homotypic interaction of His-tagged PRO20080 was
calculated using BIAcore evaluation software version 3, and found
to be a weak interaction, with a Kd of greater than 100:M. This was
a specific interaction as the His-tagged PRO20080 did not associate
with a CD40 control protein. The Kd determined as determined by
this assay is of the same order as the homotypic interaction of
SLAM previously reported as 200:M [Mavaddat et al., (2000) J Biol
Chem 275(36): 28100-28109].
[0346] Taken together, the conclusion from these assays is that
like SLAM, PRO20080 has self-interaction.
Example 6
PRO20080-CD3 Costimulation Induces T Cell Proliferation and
Increased INF-(Production
[0347] The crosslinking of SLAM and CD3 on T cells has a
co-stimulatory effect, which is to increase proliferation of the T
cells and the production of INF-(. To determine if PRO20080 has a
similar effect, human peripheral T cells were isolated and
co-stimulated with an anti-PRO20080 antibody and a suboptimal dose
of anti-CD3 antibody. Nunc Maxisorp 96-well plates were coated over
night with 0.1:g ml anti-CD3 antibody (Pharmingen), 1:g ml
anti-CD28 (Pharmingen), or 1:g ml anti-PRO20080 antibody generated
against the extracellular domain of PRO20080. PRO20080-FC fusion
protein was coated at 5 ug/ml. Human CD4+ T cells were purified
using anti-CD4 beads and AutoMacs sorting from whole blood. For the
costimulation assay 0.5.times.10.sup.6 T cells were plated per well
in triplicate. On the third day, post-costimulation, supernatant
was removed for cytokine analysis of IFN-.gamma. or 11-4 (by ELISA
or Luminex-(Upstate Biotechnology)) and the cells were pulsed for
16 hours with [3H]thymidine to measure proliferation. This
co-stimulation induced a substantial increase in proliferation
comparable with the increase in proliferation induced by the
co-stimulation of anti-CD3 and anti-CD28 treatment. Anti-PRO20080
antibody treatment alone did not induce proliferation. Anti-CD3
antibody treatment alone also did not induce proliferation. This is
shown in FIG. 8a.
[0348] INF-(production in the removed supernatant was measured, and
was found to have significantly increased over untreated cells or
cells treated with anti-PRO20080 antibody or anti-CD3 antibody
alone. The increase in INF-(production upon co-stimulation with
anti-PRO20080 antibody and anti-CD3 antibody was comparable to the
increase in INF-(production produced by co-stimulation with
anti-CD3 antibody and anti-CD28 antibody. Treatment with
anti-PRO20080 antibody alone did not induce any detectable increase
in INF-(production. This is shown in FIG. 8b.
[0349] Nonspecific induction of proliferation due to contaminants
was ruled out with the use of boiled anti-PRO20080 and PRO20080
preps. No T cell proliferation was observed under these conditions
(data not shown).
Example 7
PRO20080 Interacts with SAP
[0350] SLAM family members contain from two to four SAP binding
motifs of the consensus (TxYxxV/I), where x can be any amino acid.
SAP binds to non-phosphorylated and phosphorylated forms of SLAM,
but greater amounts of SAP binding is seen when SLAM is
phosphorylated. SLAM also binds the intracellular proteins SHP1 and
SHP2, as well as binding SAP [Castro et al., (1999) J. Immunol.
163(11): 5860-5870, Lewis et al., (2001) Clin Immunol 100(1):
15-23, Shlapatska et al., (2001) J. Immunol. 166(9): 5480-5487].
PRO20080 contains two SAP binding motifs in the intracellular
domain of the molecule (FIG. 2), and to determine if SAP, SHP1 and
SHP2 bound to PRO20080, 107 Jurkat T cells were treated with
pervanadate for 15 minutes at 37.degree. C. and lysed in RIPA
buffer with 1% NP-40 and protease and phosphatase inhibitors.
Lysates were pre-cleared with 10 .mu.g/ml IgG1 and protein G; then
PRO20080 was immunoprecipitated using 10 .mu.g of a mixture of IgG1
monoclonal antibodies raised against the extracellular domain of
PRO20080. All samples were then run on a 4-20% SDS-PAGE gel
(Novagen Madison, Wis.). Association of SAP was detected by Western
blotting using an antibody raised against full length SAP in rabbit
(Santa Cruz Biotechnology Santa Cruz, Calif.). SAP was detected
using 2 .mu.g/ml antibody diluted in 5% powdered milk. These
results of PRO20080 Western blotting are shown in FIG. 9a. There
was no PRO20080 association with SAP in untreated cells. PRO20080
did associate with SAP in pervanadate treated cells, implying a
phosphorylation-dependant interaction. SHP1 and SHP2 are expressed
in Jurkat cells, but no interaction with PRO20080 was detected
under any of the conditions tested. Therefore, PRO20080 interacts
with SAP, but not with SHP1 or SHP2, making the signal transduction
pathway of PRO20080 differ significantly from that of SLAM.
Example 8
PRO20080 Phosphorylation Upon Co-Stimulation
[0351] To examine the signaling events surrounding PRO20080 during
co-stimulation, a phosphorylation timecourse experiment was
performed. For the Jurkat timcourse, cells were first incubated
with anti-CD3 and anti-PRO20080 antibodies at 0.5 .mu.g per
10.sup.6 cells (Pharmingen). Cells were then washed, let to rest in
PBS, then stimulated with goat anti-mouse Fc antibody (Jackson
Immunoresearch) at 0.1 .mu.g per 10.sup.6 cells to crosslink the
receptors. Cross linking times began with a minimum 30 seconds to a
maximum of 10 minutes. It was found that PRO20080 was
phosphorylated within 30 seconds (FIG. 9b).
[0352] Phosphorylation of PRO20080 was also examined in isolated
human T cells. Human T cells were isolated by AutoMACS sorting of
CD4 positive cells. 400 million cells were used per condition. In
similar conditions used for the Jurkat cells, PRO20080 is
phosphorylated upon CD3 and PRO20080 cross linking, as shown by
immunoprecipitation of PRO20080 and subsequent western blotting
with an anti-phosphotyrosine antibody. In detecting for SAP, it was
found that SAP does not associate with PRO20080 in untreated human
T cells, but does associate with phosphorylated PRO20080. As in the
Jurkat cell line, SHP1 and SHP2 do not associate with PRO20080 even
though both SHP1 and SHP2 are expressed (FIG. 9c). These results
suggest a SAP dependant, but SHP1/SHP2 independent pathway,
different from SLAM.
Example 9
PRO20080 and Delayed Type Hypersensitive Response
[0353] As mentioned previously SLAM plays a role in Th1 cell
response. To determine if PRO20080 also has a role in Th1 cell
response, the murine homologue of PRO20080 was cloned, and used in
in vivo experiments to test for delayed type hypersenstivity (DTH).
For mouse studies CD4+ T cells were isolated from the spleen by
AutoMacs sorting, and 1, 5, and 10 ug ml of murine PRO20080 was
used for stimulation. Anti-mouse CD3 antibody and anti-mouse CD28
antibody were used at similar concentrations as for the human
assays (0.1 ug ml-l; 1 ug ml-l; Pharmingen). Cells were stimulated
for three days and a fraction of the supernatant was removed from
the wells to test for cytokine expression and cells were pulsed
with [3H]thymidine in the final 16 hours of culture to determine
proliferation levels. Similar results were found with
co-stimulation involving murine PRO20080-Fc fusion protein and
human PRO20080.
[0354] Mice were injected in the footpad with 100 .mu.g
keyhole-limpet hemocyanin (KLH) 1:1 in CFA. Mice were also injected
IP every other day with 100 .mu.g murine PRO20080-Fc or saline. On
day 9, draining lymph nodes were collected from the mice and cells
were incubated with KLH. On the third day, supernatant was removed
for cytokine analysis and the cells were pulsed with [3H]thymidine
to measure proliferation. This result is shown in FIG. 10a
(proliferation) and FIG. 10b (IFN-(increase). Lymphocytes from
murine PRO20080-Fc treated mice increased in proliferation in
response to KLH challenge. IFN-(production was also increased 2-4
fold.
[0355] The increase in IFN-(is proposed to modify T cell response
to a Th1 response, and possibly DTH. For DTH experiments, mice were
injected with 100 .mu.g of KLH (1:1 mixture with CFA) in the base
of the tail. Mice were injected IP with 100 .mu.g of the murine
PRO20080-Fc or saline on day 0, 2, 5, 6, and 7. On day 6 mice were
injected with 150 .mu.g of KLH in the left ear and saline in the
right ear (25 .mu.l volume). Ear swelling was measured on day 8, 48
hours after ear injection. The difference in size between the left
and right ear was plotted, and is shown in FIG. 10c. These results
suggest that the murine PRO20080 is involved in DTH, a Th1
response.
Example 10
Preparation of Antibodies that Bind PRO20080
[0356] This example illustrates preparation of monoclonal
antibodies which can specifically bind PRO20080. In this instance,
antibodies were made to the ECD of PRO20080, from about amino acid
13 to about amino acid 228 of FIG. 2 [SEQ ID NO:2].
[0357] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Selection of the immunogen can be made by the skilled artisan
without undue experimentation.
[0358] Mice, such as Balb/c, are immunized with the PRO20080
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-PRO20080 antibodies.
[0359] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO20080. 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.
[0360] The hybridoma cells will be screened in an ELISA for
reactivity against PRO20080. Determination of "positive" hybridoma
cells secreting the desired monoclonal antibodies against PRO20080
is within the skill in the art.
[0361] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO20080 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. These antibodies were useful in the
identification of PRO20080 in cell expression analysis, ELISA, and
Western blotting.
Example 11
Use of PRO20080 as a Hybridization Probe
[0362] The following method describes use of a nucleotide sequence
encoding PRO20080 as a hybridization probe.
[0363] DNA comprising the coding sequence of full-length or mature
PRO20080 as disclosed herein is employed as a probe to screen for
homologous DNAs (such as those encoding naturally-occurring
variants of PRO20080) in human tissue cDNA libraries or human
tissue genomic libraries.
[0364] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO20080-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.
[0365] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO20080 can then be
identified using standard techniques known in the art.
Example 12
Expression of PRO20080 in E. coli
[0366] This example illustrates preparation of an unglycosylated
form of PRO20080 by recombinant expression in E. coli.
[0367] The DNA sequence encoding PRO20080 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 PRO20080 coding region, lambda transcriptional
terminator, and an argU gene.
[0368] 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.
[0369] 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.
[0370] 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 PRO20080 protein can then be purified
using a metal chelating column under conditions that allow tight
binding of the protein.
[0371] PRO20080 may be expressed in E. coli in a poly-His tagged
form, using the following procedure. The DNA encoding PRO20080 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.2H2O, 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.
[0372] 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.
[0373] 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
RI/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.
[0374] Fractions containing the desired folded PRO20080 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.
[0375] Many of the PRO20080 polypeptides disclosed herein were
successfully expressed as described above.
Example 13
Expression of PRO20080 in Mammalian Cells
[0376] This example illustrates preparation of a potentially
glycosylated form of PRO20080 by recombinant expression in
mammalian cells.
[0377] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO20080 DNA
is ligated into pRK5 with selected restriction enzymes to allow
insertion of the PRO20080 DNA using ligation methods such as
described in Sambrook et al., supra. The resulting vector is called
pRK5-PRO20080.
[0378] 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-PRO20080 DNA is mixed with about 1 .mu.g DNA encoding
the VA RNA gene [Thimmappaya et al., Cell. 31:543 (1982)] and
dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA. 0.227 M
CaCl.sub.2. To this mixture is added, dropwise. 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0379] 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 PRO20080 polypeptide. The cultures containing
transfected cells may undergo further incubation (in serum free
medium) and the medium is tested in selected bioassays.
[0380] In an alternative technique, PRO20080 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-PRO20080 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 PRO20080 can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0381] In another embodiment, PRO20080 can be expressed in CHO
cells. The pRK5-PRO20080 can be transfected into CHO cells using
known reagents such as CaPO.sub.4 or DEAE-dextran. As described
above, the cell cultures can be incubated, and the medium replaced
with culture medium (alone) or medium containing a radiolabel such
as .sup.35S-methionine. After determining the presence of PRO20080
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 PRO20080 can then be concentrated and purified by any
selected method.
[0382] Epitope-tagged PRO20080 may also be expressed in host CHO
cells. The PRO20080 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 PRO20080 insert can then be subcloned
into a SV40 promoter/enhancer containing 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 promoter/enhancer containing vector. Labeling may be
performed, as described above, to verify expression. The culture
medium containing the expressed poly-His tagged PRO20080 can then
be concentrated and purified by any selected method, such as by
Ni.sup.2+-chelate affinity chromatography.
[0383] PRO20080 may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[0384] 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.
[0385] 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.
[0386] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect' (Quiagen), Dosper' or
Fugene' (Bochringer 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.
[0387] 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 mL of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered 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, pH is 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.
[0388] 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.
[0389] 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.
[0390] Many of the PRO20080 polypeptides disclosed herein were
successfully expressed as described above.
Example 14
Expression of PRO20080 in Yeast
[0391] The following method describes recombinant expression of
PRO20080 in yeast.
[0392] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO20080 from the
ADH2/GAPDH promoter. DNA encoding PRO20080 and the promoter is
inserted into suitable restriction enzyme sites in the selected
plasmid to direct intracellular expression of PRO20080. For
secretion, DNA encoding PRO20080 can be cloned into the selected
plasmid, together with DNA encoding the ADH2/GAPDH promoter, a
native PRO20080 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 PRO20080.
[0393] 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.
[0394] Recombinant PRO20080 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 PRO20080 may further
be purified using selected column chromatography resins.
[0395] Many of the PRO20080 polypeptides disclosed herein were
successfully expressed as described above.
Example 15
Expression of PRO20080 in Baculovirus-Infected Insect Cells
[0396] The following method describes recombinant expression of
PRO20080 in Baculovirus-infected insect cells.
[0397] The sequence coding for PRO20080 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 PRO20080 or the
desired portion of the coding sequence of PRO20080 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.
[0398] 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).
[0399] Expressed poly-his tagged PRO20080 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 or 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 PRO20080 are pooled and dialyzed against loading
buffer.
[0400] Alternatively, purification of the IgG tagged (or Fe tagged)
PRO20080 can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
[0401] Many of the PRO20080 polypeptides disclosed herein were
successfully expressed as described above.
Example 16
Purification of PRO20080 Polypeptides Using Specific Antibodies
[0402] Native or recombinant PRO20080 polypeptides may be purified
by a variety of standard techniques in the art of protein
purification. For example, pro-PRO20080 polypeptide, mature
PRO20080 polypeptide, or pre-PRO20080 polypeptide is purified by
immunoaffinity chromatography using antibodies specific for the
PRO20080 polypeptide of interest. In general, an immunoaffinity
column is constructed by covalently coupling the anti-PRO20080
polypeptide antibody to an activated chromatographic resin.
[0403] 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.
[0404] Such an immunoaffinity column is utilized in the
purification of PRO20080 polypeptide by preparing a fraction from
cells containing PRO20080 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 PRO20080 polypeptide containing a
signal sequence may be secreted in useful quantity into the medium
in which the cells are grown.
[0405] A soluble PRO20080 polypeptide-containing preparation is
passed over the immunoaffinity column, and the column is washed
under conditions that allow the preferential absorbance of PRO20080
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/PRO20080 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 PRO20080
polypeptide is collected.
Example 17
Drug Screening
[0406] This invention is particularly useful for screening
compounds by using PRO20080 polypeptides or binding fragment
thereof in any of a variety of drug screening techniques. The
PRO20080 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 PRO20080
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
PRO20080 polypeptide or a fragment and the agent being tested.
Alternatively, one can examine the diminution in complex formation
between the PRO20080 polypeptide and its target cell or target
receptors caused by the agent being tested.
[0407] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO20080
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an PRO20080 polypeptide or fragment
thereof and assaying (I) for the presence of a complex between the
agent and the PRO20080 polypeptide or fragment, or (ii) for the
presence of a complex between the PRO20080 polypeptide or fragment
and the cell, by methods well known in the art. In such competitive
binding assays, the PRO20080 polypeptide or fragment is typically
labeled. After suitable incubation, free PRO20080 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 PRO20080 polypeptide or to
interfere with the PRO20080 polypeptide/cell complex.
[0408] 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 PRO20080 polypeptide, the peptide test compounds are reacted
with PRO20080 polypeptide and washed. Bound PRO20080 polypeptide is
detected by methods well known in the art. Purified PRO20080
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.
[0409] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO20080 polypeptide specifically compete with a test
compound for binding to PRO20080 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
PRO20080 polypeptide.
Example 18
Rational Drug Design
[0410] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a
PRO20080 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 PRO20080 polypeptide or which enhance or
interfere with the function of the PRO20080 polypeptide in vivo
(c.f., Hodgson, Bio/Technology, 9: 19-21 (1991)).
[0411] In one approach, the three-dimensional structure of the
PRO20080 polypeptide, or of a PRO20080 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 PRO20080 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 PRO20080 polypeptide may be gained
by modeling based on the structure of homologous proteins. In both
cases, relevant structural information is used to design analogous
PRO20080 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).
[0412] 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.
[0413] By virtue of the present invention, sufficient amounts of
the PRO20080 polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the PRO20080 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.
[0414] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
511868DNAHomo Sapien 1cagaagggca aaaacattga ctgcctcaag gtctcaagca
ccagtcttca 50ccgcggaaag catgttgtgg ctgttccaat cgctcctgtt tgtcttctgc
100tttggcccag ggaatgtagt ttcacaaagc agcttaaccc cattgatggt
150gaacgggatt ctgggggagt cagtaactct tcccctggag tttcctgcag
200gagagaaggt caacttcatc acttggcttt tcaatgaaac atctcttgcc
250ttcatagtac cccatgaaac caaaagtcca gaaatccacg tgactaatcc
300gaaacaggga aagcgactga acttcaccca gtcctactcc ctgcaactca
350gcaacctgaa gatggaagac acaggctctt acagagccca gatatccaca
400aagacctctg caaagctgtc cagttacact ctgaggatat taagacaact
450gaggaacata caagttacca atcacagtca gctatttcag aatatgacct
500gtgagctcca tctgacttgc tctgtggagg atgcagatga caatgtctca
550ttcagatggg aggccttggg aaacacactt tcaagtcagc caaacctcac
600tgtctcctgg gaccccagga tttccagtga acaggactac acctgcatag
650cagagaatgc tgtcagtaat ttatccttct ctgtctctgc ccagaagctt
700tgcgaagatg ttaaaattca atatacagat accaaaatga ttctgtttat
750ggtttctggg atatgcatag tcttcggttt catcatactg ctgttacttg
800ttttgaggaa aagaagagat tccctatctt tgtctactca gcgaacacag
850ggccccgcag agtccgcaag gaacctagag tatgtttcag tgtctccaac
900gaacaacact gtgtatgctt cagtcactca ttcaaacagg gaaacagaaa
950tctggacacc tagagaaaat gatactatca caatttactc cacaattaat
1000cattccaaag agagtaaacc cactttttcc agggcaactg cccttgacaa
1050tgtcgtgtaa gttgctgaaa ggcctcagag gaattcggga atgacacgtc
1100ttctgatccc atgagacaga acaaagaaca ggaagcttgg ttcctgttgt
1150tcctggcaac agaatttgaa tatctaggat aggatgatca cctccagtcc
1200ttcggactta aacctgccta cctgagtcaa acacctaagg ataacatcat
1250ttccagcatg tggttcaaat aatattttcc aatccacttc aggccaaaac
1300atgctaaaga taacacacca gcacattgac tctctctttg ataactaagc
1350aaatggaatt atggttgaca gagagtttat gatccagaag acaaccactt
1400ctctcctttt agaaagcagc aggattgact tattgagaaa taatgcagtg
1450tgttggttac atgtgtagtc tctggagttg gatgggccca tcctgataca
1500agttgagcat cccttgtctg aaatgcttgg gattagaaat gtttcagatt
1550tcaatttttt ttcagatttt ggaatatttg cattatattt agcggttgag
1600tatccaaatc caaaaatcca aaattcaaaa tgctccaata agcatttccc
1650ttgagtttca ttgatgtcga tgcagtgctc aaaatctcag attttggagc
1700aatttggata ttggattttt ggatttggga tgctcaactt gtacaatgtt
1750tattagacac atctcctggg acatactgcc taaccttttg gagccttagt
1800ctcccagact gaaaaaggaa gaggatggta ttacatcagc tccattgttt
1850gagccaagaa tctaagtc 18682332PRTHomo Sapien 2Met Leu Trp Leu Phe
Gln Ser Leu Leu Phe Val Phe Cys Phe Gly 1 5 10 15Pro Gly Asn Val
Val Ser Gln Ser Ser Leu Thr Pro Leu Met Val 20 25 30Asn Gly Ile Leu
Gly Glu Ser Val Thr Leu Pro Leu Glu Phe Pro 35 40 45Ala Gly Glu Lys
Val Asn Phe Ile Thr Trp Leu Phe Asn Glu Thr 50 55 60Ser Leu Ala Phe
Ile Val Pro His Glu Thr Lys Ser Pro Glu Ile 65 70 75His Val Thr Asn
Pro Lys Gln Gly Lys Arg Leu Asn Phe Thr Gln 80 85 90Ser Tyr Ser Leu
Gln Leu Ser Asn Leu Lys Met Glu Asp Thr Gly 95 100 105Ser Tyr Arg
Ala Gln Ile Ser Thr Lys Thr Ser Ala Lys Leu Ser 110 115 120Ser Tyr
Thr Leu Arg Ile Leu Arg Gln Leu Arg Asn Ile Gln Val 125 130 135Thr
Asn His Ser Gln Leu Phe Gln Asn Met Thr Cys Glu Leu His 140 145
150Leu Thr Cys Ser Val Glu Asp Ala Asp Asp Asn Val Ser Phe Arg 155
160 165Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln Pro Asn Leu Thr
170 175 180Val Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp Tyr Thr
Cys 185 190 195Ile Ala Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val
Ser Ala 200 205 210Gln Lys Leu Cys Glu Asp Val Lys Ile Gln Tyr Thr
Asp Thr Lys 215 220 225Met Ile Leu Phe Met Val Ser Gly Ile Cys Ile
Val Phe Gly Phe 230 235 240Ile Ile Leu Leu Leu Leu Val Leu Arg Lys
Arg Arg Asp Ser Leu 245 250 255Ser Leu Ser Thr Gln Arg Thr Gln Gly
Pro Ala Glu Ser Ala Arg 260 265 270Asn Leu Glu Tyr Val Ser Val Ser
Pro Thr Asn Asn Thr Val Tyr 275 280 285Ala Ser Val Thr His Ser Asn
Arg Glu Thr Glu Ile Trp Thr Pro 290 295 300Arg Glu Asn Asp Thr Ile
Thr Ile Tyr Ser Thr Ile Asn His Ser 305 310 315Lys Glu Ser Lys Pro
Thr Phe Ser Arg Ala Thr Ala Leu Asp Asn 320 325 330Val
Val332DNAArtificial SequenceSynthetic oligonucleotide probe
3gagagaaggt caacttcatc acttggcttt tc 32429DNAArtificial
SequenceSynthetic oligonucleotide probe 4ctgcaggaaa ctccagggga
agagttact 29550DNAArtificial SequenceSynthetic oligonucleotide
probe 5cttcacccag tcctactccc tgcaactcag caacctgaag atggaagaca
50
* * * * *
References