U.S. patent application number 11/601407 was filed with the patent office on 2007-03-29 for novel human cell surface protein with immunoglobulin folds, bgs-19.
This patent application is currently assigned to Bristol-Myers Squibb Company. Invention is credited to Jian Chen, John N. Feder, Liana M. Lee, Shujian Wu.
Application Number | 20070071743 11/601407 |
Document ID | / |
Family ID | 28675487 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070071743 |
Kind Code |
A1 |
Lee; Liana M. ; et
al. |
March 29, 2007 |
Novel human cell surface protein with immunoglobulin folds,
BGS-19
Abstract
The present invention provides novel polynucleotides encoding
BGS-19 polypeptides, fragments and homologues thereof. Also
provided are vectors, host cells, antibodies, and recombinant and
synthetic methods for producing said polypeptides. The invention
further relates to diagnostic and therapeutic methods for applying
the novel BGS-19 polypeptides to the diagnosis, treatment, and/or
prevention of various diseases and/or disorders related to these
polypeptides. The invention further relates to screening methods
for identifying agonists and antagonists of the polynucleotides and
polypeptides of the present invention.
Inventors: |
Lee; Liana M.; (San
Francisco, CA) ; Feder; John N.; (Belle Mead, NJ)
; Wu; Shujian; (Langhorne, PA) ; Chen; Jian;
(Princeton, NJ) |
Correspondence
Address: |
LOUIS J. WILLE;BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Assignee: |
Bristol-Myers Squibb
Company
|
Family ID: |
28675487 |
Appl. No.: |
11/601407 |
Filed: |
November 17, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10403938 |
Mar 28, 2003 |
|
|
|
11601407 |
Nov 17, 2006 |
|
|
|
60368422 |
Mar 28, 2002 |
|
|
|
Current U.S.
Class: |
424/131.1 ;
435/320.1; 435/325; 435/327; 435/69.1; 530/350; 530/387.2;
530/388.22; 536/23.53 |
Current CPC
Class: |
C07K 14/70503 20130101;
C07K 14/705 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/131.1 ;
536/023.53; 530/388.22; 530/387.2; 530/350; 435/069.1; 435/320.1;
435/325; 435/327 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 5/06 20060101 C12N005/06; C07K 14/705 20060101
C07K014/705; C07K 16/42 20060101 C07K016/42 |
Claims
1-20. (canceled)
21. An isolated polypeptide comprising a polypeptide sequence
selected from the group consisting of: (a) an isolated polypeptide
comprising amino acids 1 to 385 of SEQ ID NO:2; (b) an isolated
polypeptide comprising amino acids 2 to 385 of SEQ ID NO:2; (c) an
isolated polypeptide comprising amino acids 16 to 385 of SEQ ID
NO:2 (d) an isolated polypeptide comprising the polypeptide
sequence encoded by nucleotides 140 to 1294 of SEQ ID NO:1; (e) an
isolated polypeptide comprising the polypeptide sequence encoded by
nucleotides 143 to 1294 of SEQ ID NO:1; and (f) an isolated
polypeptide comprising the polypeptide sequence encoded by
nucleotides 185 to 1294 of SEQ ID NO:1.
22. The isolated polypeptide of claim 21, wherein said polypeptide
is (a).
23. The isolated polypeptide of claim 21, wherein said polypeptide
is (b).
24. The isolated polypeptide of claim 21, wherein said polypeptide
is (c).
25. The isolated polypeptide of claim 21, wherein said polypeptide
is (d).
26. The isolated polypeptide of claim 21, wherein said polypeptide
is (e).
27. The isolated polypeptide of claim 21, wherein said polypeptide
is (f).
28. An isolated polypeptide produced by a method comprising: (a)
culturing an isolated recombinant host cell comprising a vector
that comprises the coding region encoding the polypeptide of claim
21 under conditions such that the polypeptide of claim 21 is
expressed; and (b) recovering said polypeptide.
29. The isolated polypeptide of claim 21 further comprising a
heterologous polypeptide sequence.
30. The isolated polypeptide of claim 40 wherein said heterologous
polypeptide is the Fc domain of immunoglobulin.
31. An isolated polypeptide comprising the polypeptide encoded by
the BGS-19 cDNA clone contained in ATCC Deposit No. PTA-3949.
Description
[0001] This application claims benefit to provisional application
U.S. Ser. No. 60/368,422 filed Mar. 28, 2002, under 35 U.S.C.
119(e). The entire teachings of the referenced application are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to BGS-19, a novel human cell
surface receptor in the immunoglobulin superfamily ("IgSF"). In
particular, the invention relates to BGS-19 polynucleotides,
polypeptides, agonists, antagonists, and variants thereof. The
invention also relates to pharmaceutical compositions comprising
BGS-19 polynucleotides, polypeptides, agonists or antagonists.
[0003] The invention also relates to methods for detecting,
identifying and measuring compounds that bind, inhibit and/or
activate BGS-19 polynucleotides and polypeptides. The invention
also relates to methods for measuring the amount or degree of such
binding. The invention also relates to methods of synthesizing the
compositions of the invention.
[0004] The invention also relates to methods for preventing or
treating disorders related to BGS-19 (e.g., cancer, immune-related
disorders, developmental disorders), comprising administering to a
patient in need of such treatment a composition of the invention.
The invention also relates to methods for diagnosing disorders
related to BGS-19. The invention also relates to methods for
monitoring the progression of disorders, and methods for assessing
treatment efficacy.
BACKGROUND
[0005] Many identified cell-surface antigens and receptors are
members of the immunoglobulin superfamily ("IgSF"). IgSF proteins
are characterized by one or more disulfide-linked loops formed
between a highly conserved and properly spaced pair of cysteine
residues, which organizes two .beta. sheets composed of seven or
nine antiparallel .beta.-strands. These loops, which are referred
to as immunoglobulin-like domains, are classified as variable or
constant immunoglobulin-type domains. The variable (or V-type
domains) polynucleotiderally possess disulfide loops with cysteine
residues spaced by 65-75 amino acids and thus accommodate nine
antiparallel .beta.-strands whereas the constant (or C-type)
domains typically exhibit intercysteine distances of 35-55
residues, and thus accommodate only seven antiparallel
.beta.-strands. Although some IgSF members contain multiple domains
of a single type (e.g., NCAM which has five C2-type domains), most
members possess either a single immunoglobulin-type domain or a
mixture of both V-type and C-type domains (Williams and Barclay,
1988 "The immunoglobulin superfamily--domains for cell surface
recognition", Annu Rev Immunol. 6:381-405).
[0006] IgSF proteins are known to function as antigen receptors,
cytokine receptors, receptors for cell-surface molecules (e.g.,
other IgSF proteins, adhesion molecules), and as
counter-receptors.
SUMMARY OF THE INVENTION
[0007] The present invention relates to BGS-19 polynucleotides,
polypeptides, antagonists, agonists, and variants thereof, and
optionally, a pharmaceutically acceptable carrier. Accordingly, the
present invention provides compositions comprising agonists or
antagonists of a BGS-19 polynucleotide, BGS-19 polypeptide, or of
complexes comprising a BGS-19 polynucleotide or BGS-19 polypeptide.
Compositions comprising activators and inhibitors of such agonists
and antagonists are also encompassed by the present invention.
[0008] The present invention also relates to vectors, recombinant
cells, and transgenic animals expressing a BGS-19 polynucleotide,
polypeptide, antagonist, agonist, or a variant thereof. In
particular embodiments, the vectors comprise a polynucleotide
sequence of the invention. In other embodiments, the
polynucleotidetically engineered host cells comprise a
polynucleotide sequence of the invention. In other embodiments, the
transgenic animals comprise a polynucleotide sequence of the
invention. In further embodiments of the above, the polynucleotide
sequence is operatively associated with a regulatory element that
directs the expression of the polynucleotide sequence. In a
specific embodiment, the regulatory element is a tissue-specific
promoter, inducible promoter, non-inducible promoter, enhancer or
operator.
[0009] The present invention also relates to screening assays
particularly useful in drug discovery efforts. Thus, the invention
provides methods for screening for compounds that bind and/or
modulate a BGS-19 polynucleotide or polypeptide. Accordingly, in
one embodiment, the invention provides a method for detecting an
analyte that binds a BGS-19 polypeptide comprising the steps of
contacting the BGS-19 polypeptide, or a variant thereof, with an
analyte under conditions that allow the BGS-19 polypeptide to be
bound by the analyte, and detecting binding of the BGS-19
polypeptide to the analyte, wherein detection of binding indicates
presence of an analyte that binds the BGS-19 polypeptide. In
particular embodiments, such methods can be used to detect and
identify compounds that bind or affect the pharmacokinetics (e.g.,
catalytic activity) of a polypeptide of the invention.
[0010] In particular embodiments, the analyte is a protein.
Accordingly, in one embodiments, the present invention provides a
method for identifying a BGS-19-binding protein comprising the
steps of contacting a BGS-19 polypeptide, or a variant thereof,
with an array comprising a plurality of proteins, and detecting
binding of the BGS-19 polypeptide to a protein on the array,
wherein detection of binding indicates presence of a BGS-19-binding
protein.
[0011] The present invention also relates to methods for detecting
an analyte that binds a BGS-19 polynucleotide comprising the steps
of contacting the BGS-19 polynucleotide, or a variant thereof, with
an analyte under conditions that allow the BGS-19 polynucleotide to
be bound by the analyte, and detecting binding of the BGS-19
polynucleotide to the analyte, wherein detection of binding
indicates presence of an analyte that binds the BGS-19
polynucleotide. In particular embodiments, such methods can be used
to detect and identify compounds that modulate transcription or
translation of a BGS-19 polynucleotide product.
[0012] In particular embodiments, the present invention provides
methods for detecting and identifying proteins that bind BGS-19 DNA
sequences, such proteins including, but not limited to, proteins
that affect DNA conformation and proteins that modulate
transcriptional activity (e.g., transcription factors, proteins
that bind enhancers). In particular embodiments, the present
invention provides methods for detecting and identifying factors
that bind BGS-19 RNA sequences, such factors including, but not
limited to, proteins, steroid hormones, or other small molecules.
In further embodiments, the BGS-19 RNA-binding factors modulate
translational efficacy and/or affect RNA stability.
[0013] The present invention also relates to methods for detecting
and identifying BGS-19 agonists, antagonists, as well as activators
and inhibitors thereof. In specific non-limiting embodiments,
BGS-19 agonists, BGS-19 antagonists, inhibitors of BGS-19 agonists,
activators of BGS-19 agonists, inhibitors of BGS-19 antagonists and
activators of BGS-19 antagonists are small molecules (i.e., less
than 500 daltons) that bind a BGS-19 polynucleotide or BGS-19
polypeptide of the invention.
[0014] The present invention also relates to methods for screening
for proteins that bind specific domains of a BGS-19 polypeptide,
wherein the domains exhibit a biological activity. In one
embodiment, the invention provides a method for identifying a
protein having a SH2 domain comprising the steps of contacting a
SH2-binding domain of a BGS-19 polypeptide, or a variant thereof,
with an analyte under conditions that allow the SH2-binding domain
to be bound by the analyte, and detecting binding of the
SH2-binding domain to the analyte, wherein detection of binding
indicates the presence of a protein having a SH2 domain. In a
further embodiment, the SH2-binding domain of the BGS-19 protein
comprises an immunotyrosine-based inhibition motif ("ITIM").
[0015] The present invention also relates to methods for
synthesizing a BGS-19 polynucleotide, polypeptide, antagonist,
agonist, or a variant thereof, comprising the steps of culturing a
recombinant cell comprising a BGS-19 polynucleotide, or a variant
thereof, under conditions that allow the BGS-19 polynucleotide to
be expressed by the cell, and optionally, isolating the expressed
BGS-19 polynucleotide.
[0016] The present invention also relates to methods for preventing
or treating a BGS-19-related disorder (e.g., cancer), comprising
administering to a subject in need thereof an effective amount of a
BGS-19 polynucleotide, polypeptide, antagonist, agonist, inhibitor
of a BGS-19 agonist, activator of a BGS-19 agonist, inhibitor of a
BGS-19 antagonist, activator of a BGS-19 antagonist, or a variant
thereof, and optionally, a pharmaceutically acceptable carrier. In
one embodiment, the method comprises administering an expression
vector that expresses a BGS-19 polynucleotide, polypeptide,
antagonist or agonist. In another embodiment, the method comprises
administering a recombinant cell that expresses a BGS-19
polynucleotide, polypeptide, antagonist or agonist. In particular
embodiments, the BGS-19-related disorder is cancer, an
immune-related disorder, or a developmental disorder.
[0017] The present invention also relates to methods for
diagnosing, staging, determining a prognosis of, or monitoring the
progression of a BGS-19-related disorder, comprising determining a
level of BGS-19 polynucleotide or BGS-19 polypeptide expression in
a biological sample.
[0018] Accordingly, the present invention relates to methods for
diagnosing a BGS-19-related disorder comprising detecting in a
biological sample from a subject a BGS-19 polynucleotide or BGS-19
polypeptide. In one embodiment, the method comprises the steps of
contacting a compound that binds a BGS-19 polypeptide with a
patient sample, suspected of containing the BGS-19 polypeptide,
under conditions that allow the BGS-19 polypeptide to be bound by
the compound, and detecting or measuring binding of the compound to
the BGS-19 polypeptide, wherein detection or measurement of binding
indicates presence or amount, respectively, of the BGS-19
polypeptide, and wherein the BGS-19-related disorder is determined
to be present when the presence or amount of detected BGS-19
polypeptide differs from a control value representing the amount of
BGS-19 polypeptide present in an analogous sample from a subject
not having the BGS-19-related disorder.
[0019] The present invention also relates to methods for
establishing a prognosis for a BGS-19-related disorder comprising
the steps of contacting a compound that binds a BGS-19 polypeptide
with a patient sample, suspected of containing the BGS-19
polypeptide, under conditions that allow the BGS-19 polypeptide to
be bound by the compound, and detecting or measuring binding of the
compound to the BGS-19 polypeptide, wherein detection or
measurement of binding indicates presence or amount, respectively,
of the BGS-19 polypeptide, and wherein the stage of the
BGS-19-related disorder is determined when the presence or amount
of detected BGS-19 polypeptide is compared with the amount of
BGS-19 polypeptide present in an analogous sample from a subject
having a particular stage of the BGS-19-related disorder.
[0020] The present invention also relates to methods for inhibiting
natural killer cell cytotoxic activity comprising the steps of
contacting a BGS-19 polynucleotide or BGS-19 polypeptide with a
natural killer cell, and measuring cytotoxic activity of the
natural killer cell.
[0021] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2, in addition to, its encoding nucleic acid,
or a modulator thereof, wherein the medical condition is ovarian
cancer or related proliferative condition of the ovary.
[0022] The invention further relates to a method of diagnosing a
pathological condition or a susceptibility to a pathological
condition in a subject comprising the steps of (a) determining the
presence or amount of expression of the polypeptide of SEQ ID NO:2
in a biological sample; (b) and diagnosing a pathological condition
or a susceptibility to a pathological condition based on the
presence or amount of expression of the polypeptide relative to a
control, wherein said condition is a member of the group consisting
of ovarian cancer or related proliferative condition of the
ovary.
[0023] The present invention also relates to drug delivery means
and therapeutic regimens for the compositions of the invention. In
particular embodiments, the compositions of the invention are
administered to a subject by polynucleotide therapy. In other
embodiments, the compositions are administered to a subject in a
combination therapy regimen.
[0024] The present invention also relates to kits comprising a
BGS-19 polynucleotide, polypeptide, antagonist, agonist, inhibitor
of a BGS-19 agonist, and/or inhibitor of a BGS-19 antagonist, and
optionally, detection means to detect and/or measure binding
interactions of such compounds.
[0025] The present invention also relates to an isolated
polynucleotide consisting of the nucleotide sequence of the human
BGS-19 polynucleotide.
[0026] The present invention also relates to an isolated
polynucleotide consisting of the coding region of the human BGS-19
polynucleotide.
[0027] The present invention also relates to an isolated
polynucleotide consisting of a portion of the human BGS-19
polynucleotide consisting of at least 8 bases, specifically
excluding Genbank Accession Nos. gi|BI518708, gi|BF308356,
gi|BF205116, gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0028] The present invention also relates to an isolated
polynucleotide consisting of a nucleotide sequence complementary to
a portion of the human BGS-19 polynucleotide.
[0029] The present invention also relates to an isolated
polynucleotide consisting of a nucleotide sequence that hybridizes
to a nucleotide sequence complementary to the coding region of the
human BGS-19 polynucleotide.
[0030] The present invention also relates to an isolated
polynucleotide consisting of a nucleotide sequence that hybridizes
to the nucleotide sequence of a human BGS-19 mRNA.
[0031] The invention further relates to an isolated nucleic acid
molecule of SEQ ID NO:1, wherein the nucleotide sequence comprises
sequential nucleotide deletions from either the C-terminus or the
N-terminus. Such N-terminus or C-terminus deletions of a
polypeptide of the present invention may, in fact, result in a
significant increase in one or more of the biological activities of
the polypeptide(s). For example, biological activity of many
polypeptides are governed by the presence of regulatory domains at
either one or both termini. Such regulatory domains effectively
inhibit the biological activity of such polypeptides in lieu of an
activation event (e.g., binding to a cognate ligand or receptor,
phosphorylation, proteolytic processing, etc.). Thus, by
eliminating the regulatory domain of a polypeptide, the polypeptide
may effectively be rendered biologically active in the absence of
an activation event.
[0032] The present invention also relates to an isolated
polynucleotide consisting of a nucleotide sequence encoding a
fragment of the human BGS-19 protein, wherein said fragment
displays one or more functional activities specifically excluding
Genbank Accession Nos. gi|BI518708, gi|BF308356, gi|BF205116,
gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0033] The present invention also relates to an isolated
polynucleotide consisting of a nucleotide sequence encoding a
domain of a human BGS-19 polypeptide, wherein said domain is
selected from the group consisting of a signal sequence,
transmembrane domain, extracellular domain, cytoplasmic domain,
glycosylation site, phosphorylation site, ITIM domain, and
immunoglobulin domain specifically excluding Genbank Accession Nos.
gi|BI518708, gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221,
and/or gi|AA341128.
[0034] The present invention also relates to an isolated
polynucleotide consisting of a fragment of the human BGS-19
polynucleotide, wherein the polynucleotide sequence encodes a
chimeric protein.
[0035] The present invention also relates to the polynucleotide of
SEQ ID NO:1 and consisting of 10 to 50 bases specifically excluding
Genbank Accession Nos. gi|BI518708, gi|BF308356, gi|BF205116,
gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0036] The present invention also relates to the polynucleotide of
SEQ ID NO:1 and consisting of 15 to 100 bases specifically
excluding Genbank Accession Nos. gi|BI518708, gi|BF308356,
gi|BF205116, gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0037] The present invention also relates to the polynucleotide of
SEQ ID NO:1 and consisting of 100 to 1000 bases specifically
excluding Genbank Accession Nos. gi|BI518708, gi|BF308356,
gi|BF205116, gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0038] The present invention also relates to an isolated RNA
encoding a human BGS-19 polypeptide.
[0039] The present invention also relates to a purified human
BGS-19 polypeptide.
[0040] The present invention also relates to a purified human
BGS-19 polypeptide consisting of the amino acid sequence
substantially as set forth in SEQ ID NO:2.
[0041] The present invention also relates to a purified fragment of
a human BGS-19 polypeptide consisting of a domain of a human BGS-19
polypeptide, wherein said domain is selected from the group
consisting of a signal sequence, transmembrane domain,
extracellular domain, cytoplasmic domain, glycosylation site,
phosphorylation site, IMAN domain, and immunoglobulin domain
specifically excluding Genbank Accession Nos. gi|BI518708,
gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221, and/or
gi|AA341128.
[0042] The present invention also relates to a polypeptide
consisting of an amino acid sequence that has at least 60% identity
to a purified fragment of a human BGS-19 polypeptide consisting of
a domain of a human BGS-19 polypeptide, wherein said domain is
selected from the group consisting of a signal sequence,
transmembrane domain, extracellular domain, cytoplasmic domain,
glycosylation site, phosphorylation site, IMAN domain, and
immunoglobulin domain, wherein the percent identity is determined
over an amino acid sequence of identical size to the domain
specifically excluding Genbank Accession Nos. gi|BI518708,
gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221, and/or
gi|AA341128.
[0043] The present invention also relates to a polypeptide
consisting of an amino acid sequence that has at least 90% identity
to a purified fragment of a human BGS-19 polypeptide consisting of
a domain of a human BGS-19 polypeptide, wherein said domain is
selected from the group consisting of a signal sequence,
transmembrane domain, extracellular domain, cytoplasmic domain,
glycosylation site, phosphorylation site, IMAN domain, and
immunoglobulin domain, wherein the percent identity is determined
over an amino acid sequence of identical size to the domain
specifically excluding Genbank Accession Nos. gi|BI518708,
gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221, and/or
gi|AA341128.
[0044] The present invention also relates to a chimeric protein
consisting of an amino acid sequence that has at least 90% identity
to a purified fragment of a human BGS-19 polypeptide consisting of
a domain of a human BGS-19 polypeptide, wherein said domain is
selected from the group consisting of a signal sequence,
transmembrane domain, extracellular domain, cytoplasmic domain,
glycosylation site, phosphorylation site, IMAN domain, and
immunoglobulin domain, wherein the percent identity is determined
over an amino acid sequence of identical size to the domain
specifically excluding Genbank Accession Nos. gi|BI518708,
gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221, and/or
gi|AA341128, and further consisting of a first polypeptide of at
least six amino acids fused, via a covalent bond, to second
polypeptide.
[0045] The present invention also relates to an antibody that
immunospecifically binds to a human BGS-19 polypeptide.
[0046] The present invention also relates to a compound consisting
of a fragment of an antibody that immunospecifically binds to a
human BGS-19 polypeptide.
[0047] The present invention also relates to an expression vector
containing of a BGS-19 polynucleotide.
[0048] The present invention also relates to a recombinant cell
containing a recombinant BGS-19 polynucleotide.
[0049] The present invention also relates to a transgenic non-human
animal, wherein a BGS-19 polynucleotide is expressed as a
transpolynucleotide.
[0050] The present invention also relates to pharmaceutical
composition comprising a therapeutically effective amount of a
BGS-19 polynucleotide, and a pharmaceutically acceptable
carrier.
[0051] The present invention also relates to a pharmaceutical
composition comprising a therapeutically effective amount of a
BGS-19 polypeptide, and a pharmaceutically acceptable carrier.
[0052] The present invention also relates to a pharmaceutical
composition comprising a therapeutically effective amount of an
antibody that immunospecifically binds to a BGS-19 polypeptide, and
a pharmaceutically acceptable carrier.
[0053] The present invention also relates to a method for
synthesizing a BGS-19 polypeptide comprising the steps of: (a)
culturing a recombinant cell containing a BGS-19 polynucleotide
under conditions that allow the BGS-19 polypeptide to be expressed
by the cell; and (b) isolating the expressed BGS-19 polypeptide.
The present invention also relates to the product produced by this
process.
[0054] The present invention also relates to a method for
preventing or treating a BGS-19-related disorder, the method
comprising administering to a subject in need thereof an amount of
the pharmaceutical composition of the present invention effective
for preventing or treating the BGS-19-related disorder.
[0055] The present invention also relates to a method for
preventing or treating a BGS-19-related disorder, the method
comprising administering to a subject in need thereof an amount of
the expression vector of the present invention effective for
preventing or treating the BGS-19-related disorder.
[0056] The present invention also relates to a method for
diagnosing a BGS-19-related disorder in a subject comprising the
steps of: (a) contacting a BGS-19 antibody with a sample, suspected
of containing a BGS-19 polypeptide, from the subject under
conditions that allow the BGS-19 polypeptide to be bound by the
BGS-19 antibody; and (b) detecting or measuring binding of the
BGS-19 antibody to the BGS-19 polypeptide; wherein detection or
measurement of binding indicates presence or amount, respectively,
of the BGS-19 polypeptide; and wherein the BGS-19-related disorder
is determined to be present when the presence or amount of detected
BGS-19 polypeptide differs from a control value representing the
amount of BGS-19 polypeptide present in an analogous sample from a
subject not having the BGS-19-related disorder.
[0057] The present invention also relates to a method for staging a
BGS-19-related disorder in a subject comprising the steps of: (a)
contacting a BGS-19 antibody with a sample, suspected of containing
a BGS-19 polypeptide, from the subject under conditions that allow
the BGS-19 polypeptide to be bound by the BGS-19 antibody; and (b)
detecting or measuring binding of the BGS-19 antibody to the BGS-19
polypeptide; wherein detection or measurement of binding indicates
presence or amount, respectively, of the BGS-19 polypeptide; and
wherein the stage of a BGS-19-related disorder in a subject is
determined when the presence or amount of detected BGS-19
polypeptide is compared with the amount of BGS-19 polypeptide
present in an analogous sample from a subject having a particular
stage of a BGS-19-related disorder.
[0058] The present invention also relates to a method for
identifying an analyte that binds a BGS-19 polypeptide comprising
the steps of: (a) contacting the BGS-19 polypeptide with an analyte
under conditions that allow the BGS-19 polypeptide to be bound by
the analyte; and (b) detecting binding of the BGS-19 polypeptide to
the analyte; wherein detection of binding indicates presence of an
analyte that binds the BGS-19 polypeptide.
[0059] The present invention also relates to a method for
identifying a protein that binds a BGS-19 polypeptide comprising
the steps of: (a) contacting the BGS-19 polypeptide with a
positionally addressable array comprising a plurality of proteins,
with each protein being at a different position on a solid support;
and (b) detecting binding of the BGS-19 polypeptide to a protein on
the array; wherein detection of binding indicates presence of a
protein that binds the BGS-19 polypeptide.
[0060] The present invention also relates to a method for
identifying a polypeptide having a SH2 domain comprising the steps
of: (a) contacting a SH2-binding domain of the BGS-19 protein with
an analyte under conditions that allow the SH2-binding domain to be
bound by the analyte; and (b) detecting binding of the SH2-binding
domain to the analyte; wherein detection of binding indicates a
polypeptide having a SH2 domain.
[0061] The present invention also relates to a method for
identifying an inhibitor of natural killer cell cytotoxic activity
comprising the steps of: (a) contacting a BGS-19 polypeptide with a
natural killer cell; and (b) measuring cytotoxic activity of a
natural killer cell, wherein inhibition of cytotoxic activity
indicates the presence of an inhibitor of natural killer cell
cytotoxic activity.
[0062] The present invention also relates to a kit comprising: (a)
in a first container, a purified BGS-19 polypeptide; (b) in a
second container, a first compound that binds the polypeptide; and
(c) a solid support having a second, different compound attached
thereto; wherein the second compound binds the polypeptide when the
polypeptide is bound to the first compound. The present invention
also relates to a kit further comprising a detection means to
detect the first compound when bound to the polypeptide.
DEFINITIONS
[0063] As used herein, the phrase "BGS-19 polynucleotide", "BGS-19
nucleic acid", "polynucleotide of the invention", or "nucleic acid
of the invention" refers to a polynucleotide derived from the
nucleotide sequence of SEQ ID NO:1 and depicted in FIGS. 1A-C, a
complementary sequence thereof, and variants thereof. BGS-19
polynucleotides include, but are not limited to, DNA molecules
(e.g., cDNA, genomic DNA), RNA molecules (e.g., hnRNA, pre-mRNA,
mRNA), and variants thereof. BGS-19 polynucleotides also encompass
sense and antisense sequences, as well as single-stranded and
double-stranded molecules. BGS-19 polynucleotides preferably are
polynucleotides of at least fifteen nucleic acids in length.
[0064] As used herein, the phrase "BGS-19 polypeptide" or
"polypeptide of the invention" refers to a polypeptide encoded by a
BGS-19 polynucleotide, and variants thereof. BGS-19 polypeptides
encompass proteins and peptides, and preferably are of at least six
amino acids in length.
[0065] As used herein, a "naturally occurring" polynucleotide
refers to an RNA or DNA molecule having a nucleotide sequence that
occurs in nature (e.g., encodes a natural protein).
[0066] As used herein, the term "variant" or "variants" refers to,
where appropriate, variations of the nucleic acid or amino acid
sequence of BGS-19 molecules such as, but not limited to, homologs,
analogs, derivatives, fragments, hybrids, mimetics,
conpolynucleotiders, and nucleotide and amino acid substitutions,
additions, deletions, or other chemical modifications.
[0067] As used herein, the term "fragment" or "fragments" as used
herein refers to a polypeptide having an amino acid sequence of at
least 10 contiguous amino acid residues, at least 15 contiguous
amino acid residues, at least 20 contiguous amino acid residues, at
least 25 contiguous amino acid residues, or at least 30 contiguous
amino acid residues of the amino acid sequence of a BGS-19
polypeptide. Polypeptide fragments may also be at least 40
contiguous amino acid residues, at least 50 contiguous amino acid
residues, at least 70 contiguous amino acid residues, at least 80
contiguous amino acid residues, at least 90 contiguous amino acid
residues, at least 100 contiguous amino acid residues, at least 125
contiguous amino acid residues, at least 150 contiguous amino acid
residues, at least 200 contiguous amino acid residues, at least 250
contiguous amino acid residues, or at least 300 contiguous amino
acid residues, at least 350 contiguous amino acid residues.
Preferably such fragments retain the biological activity of the
native full-length polypeptide.
[0068] In a preferred embodiment, the functional activity displayed
by a polypeptide encoded by a polynucleotide fragment of the
invention may be one or more biological activities typically
associated with the full-length polypeptide of the invention.
Illustrative of these biological activities includes the fragments
ability to bind to at least one of the same antibodies which bind
to the full-length protein, the fragments ability to interact with
at lease one of the same proteins which bind to the full-length,
the fragments ability to elicit at least one of the same immune
responses as the full-length protein (i.e., to cause the immune
system to create antibodies specific to the same epitope, etc.),
the fragments ability to bind to at least one of the same
polynucleotides as the full-length protein, the fragments ability
to bind to a receptor of the full-length protein, the fragments
ability to bind to a ligand of the full-length protein, and the
fragments ability to multimerize with the full-length protein.
However, the skilled artisan would appreciate that some fragments
may have biological activities which are desirable and directly
inapposite to the biological activity of the full-length protein.
The functional activity of polypeptides of the invention, including
fragments, variants, derivatives, and analogs thereof can be
determined by numerous methods available to the skilled artisan,
some of which are described elsewhere herein.
[0069] As used herein, the phrase "isolated polynucleotide" refers
to a polynucleotide which is separated from other polynucleotides
which are present in the natural source of the polynucleotide.
Preferably, an isolated polynucleotide is free of sequences
(preferably protein encoding sequences) which naturally flank the
polynucleotide (i.e., sequences located at the 5' and 3' ends of
the polynucleotide) in the genomic DNA of the organism from which
the polynucleotide is derived. In other embodiments, the isolated
polynucleotide is free of intron sequences. For example, in various
embodiments, the isolated polynucleotide can contain less than
about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide
sequences which naturally flank the polynucleotide in genomic DNA
of the cell from which the polynucleotide is derived. Moreover, an
isolated polynucleotide, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In one embodiment, the polynucleotides of the invention comprise a
contiguous open reading frame encoding a polypeptide of the
invention. As used herein, an isolated polynucleotide does not
include an isolated chromosome, and does not include the poly(A)
tail of an mRNA, if present.
[0070] As used herein, the phrase "isolated polypeptide" refers to
a polypeptide that is substantially free of cellular material or
other contaminating proteins from the cell or tissue source from
which the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
polypeptide, or fragment thereof is recombinantly produced, it is
also preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, 10%, or 5% of the volume of
the protein preparation. When the protein is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. Accordingly such preparations of the protein have
less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or compounds other than the polypeptide of interest. In
preferred embodiments, purified or isolated preparations will lack
any contaminating proteins from the same animal from which the
protein is normally produced, as can be accomplished by recombinant
expression of, for example, a human protein in a non-human
cell.
[0071] As will be appreciated by the skilled practitioner, should
the amino acid fragment comprise an antigenic epitope, for example,
biological function per se need not be maintained. The terms BGS-19
polypeptide and BGS-19 protein are used interchangeably herein to
refer to the encoded product of the BGS-19 nucleic acid sequence
according to the present invention.
[0072] It is another aspect of the present invention to provide
modulators of the BGS-19 protein and BGS-19 peptide targets which
can affect the function or activity of BGS-19 in a cell in which
BGS-19 function or activity is to be modulated or affected. In
addition, modulators of BGS-19 can affect downstream systems and
molecules that are regulated by, or which interact with, BGS-19 in
the cell. Modulators of BGS-19 include compounds, materials,
agents, drugs, and the like, that antagonize, inhibit, reduce,
block, suppress, diminish, decrease, or eliminate BGS-19 function
and/or activity. Such compounds, materials, agents, drugs and the
like can be collectively termed "antagonists". Alternatively,
modulators of BGS-19 include compounds, materials, agents, drugs,
and the like, that agonize, enhance, increase, augment, or amplify
BGS-19 function in a cell. Such compounds, materials, agents, drugs
and the like can be collectively termed "agonists".
[0073] As used herein the terms "modulate" or "modulates" refer to
an increase or decrease in the amount, quality or effect of a
particular activity, DNA, RNA, or protein. The definition of
"modulate" or "modulates" as used herein is meant to encompass
agonists and/or antagonists of a particular activity, DNA, RNA, or
protein.
[0074] As used herein, the term "vector" refers to a polynucleotide
capable of transporting another polynucleotide to which it has been
linked. One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome.
[0075] As used herein, the term "biological sample" refers to a
cell, tissue, organ, multicellular organism, biological fluid
(e.g., blood), or preparation (e.g., extract) thereof. A biological
sample can be derived, for example, from cells or tissue cultures
in vitro. A biological sample also can be derived from a living
organism or from a population of single cell organisms.
[0076] As used herein, the phrase "BGS-19-related disorder" refers
to a disease that involves regulation of the BGS-19 polynucleotide,
(e.g., diseases involving cells expressing BGS-19 polynucleotide,
particularly diseases involving above-normal or unregulated
expression of BGS-19 polynucleotide). BGS-19-related disorders
include, but are not limited to, cancer (e.g., adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus), immune-related disorders (e.g., acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,
gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections, trauma, X-linked
agammaglobinemia of Bruton, common variable immunodeficiency
("CVI"), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease
("SCID"), immunodeficiency with thrombocytopenia and eczema
(Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, and
immunodeficiency associated with Cushing's disease), and
developmental disorders (e.g., renal tubular acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker
muscular dystrophy, epilepsy, gonadal dyspolynucleotidesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and
mental retardation), Smith-Magenis syndrome, myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and neurofibromatosis, hypothyroidism, hydrocephalus,
seizure disorders such as Syndenham's chorea and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma,
cataract, and sensorineural hearing loss). BGS-19-related disorders
also include, but are not limited to, any disorders associated with
cell growth, disorders associated with cell differentiation,
disorders associated with embryopolynucleotidesis, and disorders
associated with morphopolynucleotidesis involving any tissue,
organ, and/or system (e.g., the brain, adrenal gland, kidney,
skeletal or reproductive system) of a subject.
[0077] As used herein, the phrase "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences at least 60% (65%,
70%, 75%, 80% or preferably 85% or more) identical to each other
typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989) pp. 6.3.1-6.3.6, which describes aqueous and
non-aqueous methods, either of which can be used. Another
preferred, non-limiting example of stringent hybridization
conditions are hybridization in 6.times. sodium chloride/sodium
citrate ("SSC") at about 45_C., followed by one or more washes in
2.0.times.SSC at 50.degree. C. (low stringency) or 0.2.times.SSC,
0.1% SDS at 50-65_C. (high stringency). Another preferred example
of stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50.degree. C. Another example of stringent hybridization conditions
are hybridization in 6.times. sodium chloride/sodium citrate (SSC)
at about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 55.degree. C. A further example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
60.degree. C. Preferably, stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 65.degree. C. Particularly preferred
stringency conditions (and the conditions that should be used if
the practitioner is uncertain about what conditions should be
applied to determine if a molecule is within a hybridization
limitation of the invention) are 0.5 M Sodium Phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. In one embodiment, an isolated nucleic acid
molecule of the invention that hybridizes under stringent
conditions to the sequence of the BGS-19 nucleic acid, or a
complement thereof, corresponds to a naturally-occurring nucleic
acid molecule.
[0078] As used herein, the phrase "BGS-19 polynucleotide
expression" refers to transcription of a BGS-19 polynucleotide that
produces BGS-19 pre-mRNA, BGS-19 mRNA, and/or translation of BGS-19
mRNA to produce BGS-19 protein.
[0079] As used herein, the phrase "therapeutic" or "therapeutic
agent" refers to a molecule or compound that assists in the
treatment of a disease. As such, a cancer therapeutic is a molecule
or compound that aids in the treatment of tumors or cancer. A
treatment protocol includes, but is not limited to, administration
of therapeutic agents, radiation therapy, dietary therapy, physical
therapy, and psychological therapy. Cancer therapeutics also
encompass a molecule or compound that aids in the prevention of
tumors or cancer, prevents the recurrence of tumors or cancer, or
prevents the spread or metastasis of tumors or cancer.
[0080] As used herein, the phrase "pharmaceutically acceptable"
refers to an agent that does not interfere with the effectiveness
of the biological activity of an active ingredient, and which may
be approved by a regulatory agency of the Federal government or a
state government, or is listed in the U.S. Pharmacopeia or other
polynucleotiderally recognized pharmacopeia for use in animals, and
more particularly for use in humans. Accordingly, suitable
pharmaceutically acceptable carriers include agents that do not
interfere with the effectiveness of a pharmaceutical
composition.
[0081] As used herein, the term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle. Such carriers can be sterile
liquids, such as saline solutions in water, or oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. A
saline solution is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions.
BRIEF DESCRIPTION OF THE FIGURES
[0082] FIGS. 1A-C show the full-length polynucleotide sequence (SEQ
ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the novel
human cell surface protein with immunoglobulin folds, BGS-19, of
the present invention. The standard one-letter abbreviation for
amino acids is used to illustrate the deduced amino acid sequence.
The polynucleotide sequence contains a sequence of 1985 nucleotides
(SEQ ID NO:1), encoding a polypeptide of 385 amino acids (SEQ ID
NO:2). An analysis of the BGS-19 polypeptide determined that it
comprised the following features: a putative signal sequence
located from about amino acid 1 to about amino acid 15 of SEQ ID
NO:2 represented by single underlining; a predicted transmembrane
domain located from about amino acid 250 to about amino acid 275
(SEQ ID NO:8) of SEQ ID NO:2 represented by double underlining; two
Ig-like domains located from about amino acid 16 to about amino
acid 113 (Ig-like domain 1; SEQ ID NO:9) of SEQ ID NO:2, and
located from about amino acid 140 to about amino acid 241 (Ig-like
domain 2; SEQ ID NO:10) of SEQ ID NO:2 represented by light
shading; and a predicted ITIM domain located from about amino acid
329 to about amino acid 334 of SEQ ID NO:2 represented by dark
shading.
[0083] FIGS. 2A-B show the partial polynucleotide sequence (SEQ ID
NO:3) and deduced amino acid sequence (SEQ ID NO:4) of the novel
human cell surface protein with immunoglobulin folds, BGS-19, of
the present invention. The standard one-letter abbreviation for
amino acids is used to illustrate the deduced amino acid sequence.
The polynucleotide sequence contains a sequence of 1632 nucleotides
(SEQ ID NO:3), encoding a polypeptide of 544 amino acids (SEQ ID
NO:4).
[0084] FIG. 3. Hydrophobicity plot of BGS-19 demonstrating a
putative signal sequence at the amino terminus and a transmembrane
domain (SEQ ID NO:8).
[0085] FIGS. 4A-E. Sequence alignment among BGS-19, Siglec-6 (SEQ
ID NO:5), Siglec-7 (SEQ ID NO:6), and Siglec-10 (SEQ ID NO:7) amino
acid sequences (Siglec-6 GenBank accession number: NP.sub.--001763,
Siglec-10 GenBank accession number: NP.sub.--149121). Sequence
homology between BGS-19 and Siglec-6 is 41.4% identity and 48.0%
similarity. Sequence homology between BGS-19 and Siglec-7 is 44.7%
identity and 50.8% similarity. Sequence homology between BGS-19 and
Siglec-10 is 46.6% identity and 51.7% similarity. Signal peptide
region, Ig-like domains, transmembrane domain, and ITIM motif are
indicated by underscoring.
[0086] FIG. 5. Schematic drawing of the domain structure of BGS-19,
indicating the amino acid boundaries of the signal peptide, Ig-like
domain 1, Ig-like domain 2, transmembrane domain and ITIM
motif.
[0087] FIG. 6. Bar graph showing significant expression of BGS-19
in bone marrow, lung, lymph node, small intestine, spinal cord, and
spleen.
[0088] FIG. 7 shows an expanded expression profile of the novel
human cell surface protein with immunoglobulin folds, BGS-19. The
figure illustrates the relative expression level of BGS-19 amongst
various mRNA tissue sources. As shown, the BGS-19 polypeptide was
expressed predominately in the ovary. Expression of BGS-19 was also
significantly expressed in the testis, adrenal gland, the
parenchyma of the spleen, throughout the stomach, and to a lesser
extent in the other tissues as shown. Expression data was obtained
by measuring the steady state BGS-19 mRNA levels by quantitative
PCR using the PCR primer pair provided as SEQ ID NO:85 and 86, and
Taqman probe (SEQ ID NO:87) as described herein.
[0089] FIG. 8 shows an expanded expression profile of the novel
human cell surface protein with immunoglobulin folds, BGS-19, of
the present invention. The figure illustrates the relative
expression level of BGS-19 amongst various mRNA tissue sources
isolated from normal and tumor tissues. As shown, the BGS-19
polypeptide was differentially expressed in ovarian cancer tissue
compared to its respective normal tissue. Expression data was
obtained by measuring the steady state BGS-19 mRNA levels by
quantitative PCR using the PCR primer pair provided as SEQ ID NO:85
and 86, and Taqman probe (SEQ ID NO:87) as described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0090] The present invention is based, at least in part, on the
discovery of a nucleic acid sequence encoding BGS-19, a novel human
cell surface receptor of the immunoglobulin superfamily
("IgSF").
[0091] The present invention provides isolated nucleic acid
molecules, that comprise, or alternatively consist of, a
polynucleotide encoding the BGS-19 protein having the amino acid
sequence shown in FIGS. 1A-C (SEQ ID NO:2) or the amino acid
sequence encoded by the cDNA clone, BGS-19 deposited as ATCC
Deposit Number PTA-3949 on Dec. 22, 2001. The ATCC is located at
10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC
deposit was made pursuant to the terms of the Budapest Treaty on
the international recognition of the deposit of microorganisms for
purposes of patent procedure. The deposited clone is inserted in
the pSport1 plasmid using the Not I and Sal I restriction
sites.
[0092] Accordingly, the present invention relates to isolated
BGS-19 polynucleotides derived from the BGS-19 polynucleotide and
variants thereof. The invention also relates to polypeptides
encoded by polynucleotides of the invention. The invention also
relates to antibodies directed to polypeptides encoded by the
polynucleotides of the invention. The invention also relates to
agonists and antagonists of a BGS-19 polynucleotide, BGS-19
polypeptide or complexes comprising a BGS-19 polynucleotide or
BGS-19 polypeptide.
[0093] The invention also relates to methods for detecting,
identifying and characterizing agonists and antagonists of a BGS-19
polynucleotide or polypeptide, such as the nature and strength of
binding to the BGS-19 polynucleotide or polypeptide.
[0094] The invention also relates to methods for synthesizing a
BGS-19 polynucleotide, BGS-19 polypeptide, BGS-19 agonist, or
BGS-19 antagonist, or variants thereof, including complexes among
them or with other compounds.
[0095] The invention also relates to diagnosing a BGS-19-related
disorder. The invention also relates to methods for monitoring the
progression of a BGS-19-related disorder, and methods for assessing
treatment efficacy.
[0096] The invention also relates to prevention or treatment of a
BGS-19-related disorder comprising administering a BGS-19
polynucleotide, BGS-19 polypeptide, BGS-19 agonist, and/or BGS-19
antagonist with or without additional therapeutic agents.
Isolated BGS-19 Polynucleotides
[0097] The present invention provides isolated polynucleotides
encoding a BGS-19 polypeptide and variants thereof. Also
encompassed by the present invention are specific portions of the
BGS-19 polynucleotide and the polypeptide encoded by such portions.
For example, individual subclones or subsequences used to assemble
the full-length (or nearly full-length) BGS-19 cDNA or
polynucleotide ("BGS-19 subsequences") are encompassed by the
BGS-19 polynucleotides of the invention. Accordingly, any
polypeptide encoded by such subclones or subsequences is
encompassed by the BGS-19 polypeptides of the invention, which
include, for example, the polynucleotides encoding the BGS-19
extracellular domain, BGS-19 intracellular domain, BGS-19 signal
peptide, BGS-19 Ig-like domain 1 (SEQ ID NO:9), BGS-19 Ig-like
domain 2 (SEQ ID NO:10), BGS-19 transmembrane domain, BGS-19 ITIM
motif, and any subcellular localization signals.
[0098] Furthermore, partially assembled subsequences or hybrid
molecules comprising BGS-19 subsequences or partially assembled
subsequences are encompassed by the BGS-19 polynucleotides of the
invention. Accordingly, any polypeptide encoded by such subclones
or subsequences is encompassed by the BGS-19 polypeptides of the
invention.
[0099] Similarly, the present invention encompasses specific
portions of a BGS-19 polynucleotide or BGS-19 polypeptide that can
be discerned as a domain or motif. Such domains and motifs include,
but are not limited to, Ig domains, Ig-like domains,
immunotyrosine-based inhibition motifs ("ITIMs"), signal sequences,
transmembrane domains, phosphorylation sites, exons, introns,
splice acceptor sites, splice donor sites, 5' and 3' regulatory
regions of the mRNA, mRNA capping regions, promoter regions,
transcriptional regulatory sites, enhancer sequences, extracellular
ligand-binding sites, and derivatives thereof. In a specific
embodiment, a BGS-19 polypeptide comprises an ITIM (Unkeless and
Jin, 1997, "Inhibitory receptors, ITIM sequences and phosphatases",
Curr Opin Immunol. 9:338-343).
[0100] Accordingly, a BGS-19 polynucleotide can comprise cDNA,
genomic DNA, introns, exons, promoter regions, 5' and 3' regulatory
regions of the polynucleotide, RNA, hnRNA, mRNA, regulatory regions
within RNAs, and depolynucleotiderate variants thereof. Promoter
sequences for BGS-19 can be determined by promoter-reporter
polynucleotide assays and in vitro binding assays.
[0101] In preferred embodiments, the present invention encompasses
a polynucleotide including the initiating start codon, in addition
to, the resulting encoded polypeptide of BGS-19. Specifically, the
present invention encompasses the polynucleotide corresponding to
nucleotides 140 thru 1294 of SEQ ID NO:1, and the polypeptide
corresponding to amino acids 1 thru 385 of SEQ ID NO:2. Also
encompassed are recombinant vectors comprising said encoding
sequence, and host cells comprising said vector.
[0102] In preferred embodiments, the present invention encompasses
a polynucleotide lacking the initiating start codon, in addition
to, the resulting encoded polypeptide of BGS-19. Specifically, the
present invention encompasses the polynucleotide corresponding to
nucleotides 143 thru 1294 of SEQ ID NO:1, and the polypeptide
corresponding to amino acids 2 thru 385 of SEQ ID NO:2. Also
encompassed are recombinant vectors comprising said encoding
sequence, and host cells comprising said vector.
[0103] Using all, or a portion, of the polynucleotide sequence of
BGS-19, as a hybridization probe, polynucleotides of the invention
can be isolated using standard hybridization and cloning techniques
(See, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0104] An isolated polynucleotide that encodes a variant
polypeptide can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of BGS-19. Such BGS-19 polynucleotide variants can result in one or
more amino acid substitutions, additions or deletions in the
encoded protein. Mutations can be introduced by standard
techniques, such as site-directed mutapolynucleotidesis and
PCR-mediated mutapolynucleotidesis. Preferably, conservative amino
acid substitutions are made at one or more predicted non-essential
amino acid residues. A "conservative amino acid substitution" is
one in which the amino acid residue is replaced with an amino acid
residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art.
These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic
acid, glutamic acid, asparagine, glutamine), uncharged polar side
chains (e.g., glycine, serine, threonine, tyrosine, cysteine),
non-polar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation
mutapolynucleotidesis, and the resultant mutants can be screened
for biological activity to identify mutants that retain or
antagonize activity. Following mutapolynucleotidesis, the encoded
protein can be expressed recombinantly and the activity of the
protein can be determined.
[0105] Nucleotide substitutions leading to amino acid substitutions
at "non-essential" amino acid residues can also be introduced. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequence without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologs of various species
can be non-essential for activity and thus would be likely targets
for alteration. Such polypeptides would retain biological
activity.
[0106] The present invention encompasses, in addition to the
polynucleotides disclosed herein, 1) any polynucleotide that
encodes a BGS-19 polypeptide of the invention; 2) any
polynucleotide that hybridizes to the complement of the sequence
depicted in FIGS. 2A-B or FIGS. 1A-C under highly stringent
conditions, e.g., washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel et al., 1989, Current Protocols in Molecular Biology,
Vol. 1, Green Publishing Associates, Inc., and John Wiley &
Sons, Inc., New York, at p. 2.10.3); and/or 3) any polynucleotide
sequence that hybridizes to the complement of the sequence depicted
in FIGS. 2A-B or FIGS. 1A-C under less stringent conditions, such
as moderately stringent conditions, e.g., washing in
0.2.times.SSC/0.1% SDS at 45.degree. C. (Ausubel et al., 1989,
supra).
[0107] In one embodiment, a variant BGS-19 polynucleotide sequence
hybridizes to a naturally-occurring BGS-19 polynucleotide under
stringent conditions. In another embodiment, a variant BGS-19
polynucleotide sequence hybridizes to a naturally-occurring BGS-19
polynucleotide under moderately stringent conditions. In another
embodiment, the BGS-19 polynucleotide hybridizes, under stringent
or moderately stringent conditions, to a fragment of a
naturally-occurring BGS-19 polynucleotide wherein the
naturally-occurring BGS-19 polynucleotide is not a polynucleotide
consisting of Genbank Accession Nos. gi|BI518708, gi|BF308356,
gi|BF205116, gi|BF969219, gi|BG826221, and/or gi|AA341128.
[0108] The term "hybridizes under highly stringent conditions" is
intended to describe conditions for hybridization and washing under
which nucleotide sequences at least 60% (65%, 70%, preferably 75%)
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989) pp. 6.3.1-6.3.6. A preferred, non-limiting
example of stringent hybridization conditions are hybridization in
6.times. sodium chloride/sodium citrate ("SSC") at about 45.degree.
C. followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Preferably, an isolated polynucleotide of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO: 1, 3 (or GenBank Accession No. AW225336), 5
(or GenBank Accession No. AW225339), 7, 9, 11, or 13, or a
complement thereof, corresponds to a naturally-occurring
polynucleotide.
[0109] Accordingly, the present invention contemplates
polynucleotide variants that are revealed from inter-species
comparisons of homologs of the BGS-19 polynucleotides. As such,
homologs of a BGS-19 polynucleotide of the invention that are found
in other species are encompassed by the present invention.
[0110] In addition to the polynucleotide sequence presented in
FIGS. 1 and 3, it will be appreciated by those skilled in the art
that DNA sequence polymorphisms that lead to changes in the amino
acid sequence may exist within a population (e.g., the human
population). Such polynucleotidetic polymorphisms may exist among
individuals within a population due to natural allelic variation.
An allele is one of a group of polynucleotides which occur
alternatively at a given polynucleotidetic locus. As used herein,
the phrase "allele" or "allelic variant" refers to a nucleotide
sequence which occurs at a given chromosomal locus, to a
polynucleotide molecule that encodes a polypeptide encoded by the
nucleotide sequence which occurs at the given chromosomal locus
(e.g., a cDNA molecule), or to a polypeptide encoded by the
nucleotide sequence. Allelic variations can typically result in
about 1-5% variance in the nucleotide sequence of a given
polynucleotide. Alternative alleles can be identified by sequencing
the polynucleotide of interest in a number of different
individuals. This can be readily carried out by using hybridization
probes to identify the same polynucleotidetic locus in a variety of
individuals, and to characterize the polymorphisms present at the
polynucleotidetic locus across the individuals. In one embodiment,
polymorphisms that are associated with a particular disease and/or
disorder are used as markers to diagnose said disease or
disorder.
[0111] The present invention also encompasses complexes formed by a
BGS-19 polynucleotide sequence and a binding partner and complexes
formed by a BGS-19 polypeptide and a binding partner. A binder
partner can be, but is not limited to, a polypeptide, carbohydrate
or lipid. In a specific embodiment, the present invention
encompasses the complex of BGS-19 and SH2 domain of phosphatases,
such as SHP1 and SHP2. In other specific embodiments, the present
invention encompasses the complexes of BGS-19 and polypeptides, and
fragments thereof, encoded by the nucleic acid sequences depicted
in FIGS. 1 and 3. The present invention also provides for methods
of identifying and isolating such binding partners.
[0112] The present invention also encompasses mature forms of the
polypeptide comprising, or alternatively consisting of, the
polypeptide sequence of SEQ ID NO:2, the polypeptide encoded by the
polynucleotide described as SEQ ID NO:1, and/or the polypeptide
sequence encoded by a cDNA in the deposited clone. The present
invention also encompasses polynucleotides encoding mature forms of
the present invention, such as, for example the polynucleotide
sequence of SEQ ID NO:1, and/or the polynucleotide sequence
provided in a cDNA of the deposited clone. Specifically, the
present invention encompasses the polynucleotide from about
nucleotide position 185 to about nucleotide position 1294 of SEQ ID
NO:1 (FIGS. 1A-C).
[0113] According to the signal hypothesis, proteins secreted by
eukaryotic cells have a signal or secretary leader sequence which
is cleaved from the mature protein once export of the growing
protein chain across the rough endoplasmic reticulum has been
initiated. Most eukaryotic cells cleave secreted proteins with the
same specificity. However, in some cases, cleavage of a secreted
protein is not entirely uniform, which results in two or more
mature species of the protein. Further, it has long been known that
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide.
[0114] Methods for predicting whether a protein has a signal
sequence, as well as the cleavage point for that sequence, are
available. For instance, the method of McGeoch, Virus Res.
3:271-286 (1985), uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje, Nucleic Acids Res.
14:4683-4690 (1986) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2, where
+1 indicates the amino terminus of the secreted protein. The
accuracy of predicting the cleavage points of known mammalian
secretory proteins for each of these methods is in the range of
75-80%. (von Heinje, supra.) However, the two methods do not always
produce the same predicted cleavage point(s) for a given
protein.
[0115] The established method for identifying the location of
signal sequences, in addition, to their cleavage sites has been the
SignalP program (v1.1) developed by Henrik Nielsen et al., Protein
Engineering 10: 1-6 (1997). The program relies upon the algorithm
developed by von Heinje, though provides additional parameters to
increase the prediction accuracy.
[0116] More recently, a hidden Markov model has been developed (H.
Neilson, et al., Ismb 1998;6:122-30), which has been incorporated
into the more recent SignalP (v2.0). This new method increases the
ability to identify the cleavage site by discriminating between
signal peptides and uncleaved signal anchors. The present invention
encompasses the application of the method disclosed therein to the
prediction of the signal peptide location, including the cleavage
site, to any of the polypeptide sequences of the present
invention.
[0117] As one of ordinary skill would appreciate, however, cleavage
sites sometimes vary from organism to organism and cannot be
predicted with absolute certainty. Accordingly, the polypeptide of
the present invention may contain a signal sequence. Polypeptides
of the invention which comprise a signal sequence have an
N-terminus beginning within 5 residues (i.e., + or -5 residues, or
Preferably at the -5, -4, -3, -2, -1, +1, +2, +3, +4, or +5
residue) of the predicted cleavage point. Similarly, it is also
recognized that in some cases, cleavage of the signal sequence from
a secreted protein is not entirely uniform, resulting in more than
one secreted species. These polypeptides, and the polynucleotides
encoding such polypeptides, are contemplated by the present
invention.
[0118] Moreover, the signal sequence identified by the above
analysis may not necessarily predict the naturally occurring signal
sequence. For example, the naturally occurring signal sequence may
be further upstream from the predicted signal sequence. However, it
is likely that the predicted signal sequence will be capable of
directing the secreted protein to the ER. Nonetheless, the present
invention provides the mature protein produced by expression of the
polynucleotide sequence of SEQ ID NO:1 and/or the polynucleotide
sequence contained in the cDNA of a deposited clone, in a mammalian
cell (e.g., COS cells, as described below). These polypeptides, and
the polynucleotides encoding such polypeptides, are contemplated by
the present invention.
[0119] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide
sequence of the present invention can be determined conventionally
using known computer programs. A preferred method for determining
the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the CLUSTALW
computer program (Thompson, J. D., et al., Nucleic Acids Research,
2(22):4673-4680, (1.994)), which is based on the algorithm of
Higgins, D. G., et al., Computer Applications in the Biosciences
(CABIOS), 8(2):189-191, (1992). In a sequence alignment the query
and subject sequences are both DNA sequences. An RNA sequence can
be compared by converting U's to T's. However, the CLUSTALW
algorithm automatically converts U's to T's when comparing RNA
sequences to DNA sequences. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
CLUSTALW alignment of DNA sequences to calculate percent identity
via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top
Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension
Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of
the subject nucleotide sequence, whichever is shorter. For multiple
alignments, the following CLUSTALW parameters are preferred: Gap
Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation
Penalty Range=8; End Gap Separation Penalty=Off; % Identity for
Alignment Delay=40%; Residue Specific Gaps:Off, Hydrophilic Residue
Gap=Off; and Transition Weighting=0. The pairwise and multiple
alignment parameters provided for CLUSTALW above represent the
default parameters as provided with the AlignX software program
(Vector NTI suite of programs, version 6.0).
[0120] The present invention encompasses the application of a
manual correction to the percent identity results, in the instance
where the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions.
If only the local pairwise percent identity is required, no manual
correction is needed. However, a manual correction may be applied
to determine the global percent identity from a global
polynucleotide alignment. Percent identity calculations based upon
global polynucleotide alignments are often preferred since they
reflect the percent identity between the polynucleotide molecules
as a whole (i.e., including any polynucleotide overhangs, not just
overlapping regions), as opposed to, only local matching
polynucleotides. Manual corrections for global percent identity
determinations are required since the CLUSTALW program does not
account for 5' and 3' truncations of the subject sequence when
calculating percent identity. For subject sequences truncated at
the 5' or 3' ends, relative to the query sequence, the percent
identity is corrected by calculating the number of bases of the
query sequence that are 5' and 3' of the subject sequence, which
are not matched/aligned, as a percent of the total bases of the
query sequence. Whether a nucleotide is matched/aligned is
determined by results of the CLUSTALW sequence alignment. This
percentage is then subtracted from the percent identity, calculated
by the above CLUSTALW program using the specified parameters, to
arrive at a final percent identity score. This corrected score may
be used for the purposes of the present invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed
by the CLUSTALW alignment, which are not matched/aligned with the
query sequence, are calculated for the purposes of manually
adjusting the percent identity score.
[0121] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
CLUSTALW alignment does not show a matched/alignment of the first
10 bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the CLUSTALW program. If
the remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by CLUSTALW
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are required for the purposes of the present invention.
BGS-19 Antisense Oligonucleotides
[0122] The present invention encompasses BGS-19 antisense
polynucleotides, i.e., molecules which are complementary to a sense
nucleic acid encoding a polypeptide of the invention, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. The antisense
polynucleotide can be complementary to an entire coding strand, or
to only a portion thereof, e.g., all or part of the protein coding
region (or open reading frame). An antisense polynucleotide can be
antisense to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a polypeptide of the
invention. The non-coding regions are the 5' and 3' sequences which
flank the coding region and are not translated into amino
acids.
[0123] The antisense oligonucleotides of the invention can be DNA
or RNA or chimeric mixtures, derivatives, or variants thereof. The
oligonucleotide can be modified at the base moiety, sugar moiety,
or phosphate backbone, which can, for example, improve the
oligonucleotide's pharmacokinetics and/or affect an
oligonucleotide's hybridization to the target mRNA. The
oligonucleotide can include other appended groups, such as for
example, peptides (e.g., for targeting host cell receptors in
vivo), agents facilitating transport across the cell membrane (See,
e.g., Letsinger et al., 1989, "Cholesteryl-conjugated
oligonucleotides: synthesis, properties, and activity as inhibitors
of replication of human immunodeficiency virus in cell culture",
Proc Natl Acad. Sci. 86:6553-6556; Lemaitre et al., 1987, "Specific
antiviral activity of a poly(L-lysine)-conjugated
oligodeoxyribonucleotide sequence complementary to vesicular
stomatitis virus N protein mRNA initiation site", Proc Natl Acad.
Sci. 84:648-652; PCT Publication No. WO 88/09810),
hybridization-triggered cleavage agents (See, e.g., van der Krol,
1988, "Modulation of eukaryotic polynucleotide expression by
complementary RNA or DNA sequences", Biotechniques 6:958-976), and
intercalating agents (See, e.g., Zon, 1988, "Oligonucleotide
analogues as potential chemotherapeutic agent", Pharm Res.
5:539-549). To this end, the oligonucleotide may be conjugated to
another molecule, which includes, but is not limited to, a peptide,
hybridization triggered cross-linking agent, transport agent, and
hybridization-triggered cleavage agent.
[0124] An antisense oligonucleotide can be, for example, about 8,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length.
An antisense polynucleotide of the invention can be constructed
using chemical synthesis and enzymatic ligation reactions using
procedures known in the art.
[0125] Antisense oligonucleotides may be single or double stranded.
Double stranded RNA's may be designed based upon the teachings of
Paddison et al., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); and
International Publication Nos. WO 01/29058, and WO 99/32619; which
are hereby incorporated herein by reference.
[0126] Various well-known modifications to the DNA molecules may be
introduced as a means of increasing intracellular stability and
half-life. For example, an antisense polynucleotide (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides.
Possible modifications include but are not limited to, the addition
of flanking sequences of ribo- or deoxynucleotides to the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the
oligodeoxyribonucleotide backbone. Examples of modified nucleotides
which can be used to polynucleotiderate a BGS-19 antisense
polynucleotide include, but are not limited to, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 2-methylthio-N6- isopentenyladenine,
2-thiocytosine, 2-thiouracil, 2,6-diaminopurine, 3-methylcytosine,
3-(3-amino-3-N-2-carboxypropyl) uracil, 4-acetylcytosine,
4-thiouracil, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 5-methylcytosine,
5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
5-methyl-2-thiouracil, 5-methyl-2-thiouracil, 5-methyluracil,
hypoxanthine, 7-methylguanine, beta-D-galactosylqueosine,
beta-D-mannosylqueosine, dihydrouracil, inosine,
N6-isopentenyladenine, N6-adenine, uracil-5-oxyacetic acid (v),
pseudouracil, queosine, wybutoxosine, xanthine, uracil-5-oxyacetic
acid methylester, (acp3)w and uracil-5-oxyacetic acid (v).
[0127] In another embodiment, the antisense oligonucleotide can
also comprise, one or more modified sugar moieties, which includes,
but is not limited to, 2-fluoroarabinose, arabinose, hexose, and
xylulose.
[0128] In another embodiment, the antisense oligonucleotide
comprises a modified phosphate backbone, which includes, but is not
limited to, phosphorothioate, phosphorodithioate,
phosphoramidothioate, phosphoramidate, phosphordiamidate,
methylphosphonate, alkyl phosphotriester, formacetal, and analogs
thereof.
[0129] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide can form double-stranded hybrids with complementary
RNA, but in contrast to the usual .beta.-oligonucleotides, the
nucleotide strands run parallel to each other (Gautier et al.,
1987, "Alpha-DNA. IV: Alpha-anomeric and beta-anomeric
tetrathymidylates covalently linked to intercalating
oxazolopyridocarbazole. Synthesis, physicochemical properties and
poly (rA) binding", Nucl Acids Res. 15:6625-6641). The
oligonucleotide can be a 2'-O-methylribonucleotide (Inoue et al.,
1987, "Synthesis and hybridization studies on two complementary
nona(2'-O-methyl) ribonucleotides", Nucl Acids Res. 15:6131-6148)
or a chimeric RNA-DNA analogue (Inoue et al., 1987,
"Sequence-dependent hydrolysis of RNA using modified
oligonucleotide splints and RNase H", FEBS Lett. 215:327-330).
[0130] The antisense oligonucleotides may be RNA or DNA, or
derivatives thereof. The particular form of antisense
oligonucleotide may affect the oligonucleotide's pharmacokinetic
parameters such as bioavailability, metabolism, and half-life. As
such, where appropriate, the invention contemplates antisense
oligonucleotide derivatives having properties that improve cellular
uptake, enhance nuclease resistance, improve binding to the target
sequence, or increase cleavage or degradation of the target
sequence. The antisense oligonucleotides may comprise bases
comprising, for example, phosphorothioates or methylphosphonates.
The antisense oligonucleotides, instead, can be mixed
oligonucleotides comprising combinations of phosphodiesters,
phosphorothioate, and/or methylphosphonate nucleotides, among
others. Such oligonucleotides may possess modifications which
comprise, but are not limited to, 2-0'-alkyl or 2-0'-halo sugar
modifications, backbone modifications (e.g., methylphosphonate,
phosphorodithioate, phosphordithioate, formacetal,
3'-thioformacetal, sulfone, sulfamate, nitroxide backbone,
morpholino derivatives and peptide nucleic acid ("PNA")
derivatives), or derivatives wherein the base moieties have been
modified. In another embodiment, antisense oligonucleotides
comprise conjugates of the oligonucleotides and derivatives thereof
(Goodchild, 1990, "Conjugates of oligonucleotides and modified
oligonucleotides: a review of their synthesis and properties",
Bioconjug Chem. 1: 165-87).
[0131] In one embodiment, the deoxyribose phosphate backbone of a
polynucleotide of the invention can be modified to incorporate
peptide nucleic acids ("PNAs") (See, e.g., Hyrup and Nielsen, 1996,
"Peptide nucleic acids (PNA): synthesis, properties and potential
applications", Bioorg Med. Chem. 4:5-23). As used herein, PNAs
refer to nucleic acid mimics, in which the deoxyribose phosphate
backbone is replaced by a pseudopeptide backbone. The neutral
backbone of PNAs allows for specific hybridization to DNA and RNA
under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using any suitable peptide synthesis
protocol known in the art (see, e.g., Hyrup and Nielsen, 1996
supra; Perry-O'Keefe et al., 1996, "Peptide nucleic acid pre-gel
hybridization: an alternative to southern hybridization", Proc Natl
Acad. Sci. 93:14670-14675).
[0132] PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antipolynucleotide
agents for sequence-specific modulation of polynucleotide
expression by, e.g., inducing transcription or translation arrest
or inhibiting replication. PNAs can also be used for analyzing
polynucleotide mutations by, for example, PNA-directed PCR
clamping, or as artificial restriction enzymes when used in
combination with other enzymes, such as for example, S1 nucleases
(Hyrup and Nielsen, 1996 supra), or as probes or primers for DNA
sequence and hybridization (Hyrup and Nielsen, 1996, supra;
Perry-O'Keefe et al., 1996, supra).
[0133] In another embodiment, PNAs can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic
or other helper groups to PNA, by the formation of PNA-DNA
chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras can be
polynucleotiderated which may combine the advantageous properties
of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g.,
RNase H and DNA polymerases, to interact with the DNA portion while
the PNA portion would provide high binding affinity and
specificity. PNA-DNA chimeras can be linked using linkers of
appropriate lengths selected in terms of base stacking, number of
bonds between the nucleobases, and orientation (Hyrup and Nielsen,
1996, supra).
[0134] The synthesis of PNA-DNA chimeras can be performed using
various methods known in the art (see, e.g., Hyrup and Nielsen,
1996, supra, and Finn et al., 1996, "Synthesis and properties of
DNA-PNA chimeric oligomers", Nucleic Acids Res. 24:3357-3363). For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs. Compounds such as 5'-(4-methoxytrityl)
amino-5'-deoxy-thymidine phosphoramidite can be used as a link
between the PNA and the 5' end of DNA (Mag and Engels, 1989,
"Synthesis and selective cleavage of oligodeoxyribonucleotides
containing non-chiral internucleotide phosphoramidate linkages",
Nucleic Acids Res. 17:5973-5988). PNA monomers are then coupled in
a stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al., 1996, supra).
Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment.
[0135] The target sequences may be RNA or DNA, and may be
single-stranded or double-stranded. Target molecules include, but
are not limited to, pre-mRNA, mRNA, and DNA. In a one embodiment,
the target molecule is a BGS-19 mRNA. In a preferred embodiment,
the target molecule is BGS-19 pre-mRNA or BGS-19 mRNA. In a
specific embodiment, the antisense oligonucleotides hybridize to a
portion anywhere along a BGS-19 pre-mRNA or mRNA. In another
embodiment, a BGS-19 antisense oligonucleotides are selected from
those oligonucleotides which hybridize to the translation
initiation site, donor splicing site, acceptor splicing site, sites
for transportation, or sites for degradation of a BGS-19 pre-mRNA
or mRNA.
[0136] The antisense polynucleotides of the invention can be
administered to a subject or polynucleotiderated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a selected polypeptide of the invention to thereby inhibit
expression, e.g., by inhibiting transcription and/or translation.
An example of a route of administration of antisense
polynucleotides of the invention includes direct injection at a
tissue site. Alternatively, antisense polynucleotides can be
modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense polynucleotides to peptides or antibodies
which bind to cell surface receptors or antigens. The antisense
polynucleotides can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense polynucleotide is placed under the control of a
strong pol II or pol III promoter are preferred.
[0137] For in vivo therapeutic use, a phosphorothioate derivative
of a BGS-19 antisense oligonucleotide can be useful, at least
partly because of greater resistance to degradation. In one
embodiment, a BGS-19 antisense oligonucleotide is a hybrid
oligonucleotide comprising phosphorothioate bases. In another
embodiment, a BGS-19 antisense oligonucleotide comprises at least
one phosphorothioate linkage. In yet another embodiment, a BGS-19
antisense oligonucleotide is comprised entirely of phosphorothioate
linkages. Methods for preparing oligonucleotide derivatives are
known in the art. See, e.g., Stein et al., 1988, "Physicochemical
properties of phosphorothioate oligodeoxynucleotides", Nucl. Acids
Res., 16:3209-3221; Blake et al., 1985, "Inhibition of rabbit
globin mRNA translation by sequence-specific
oligodeoxyribonucleotides", Biochemistry 24:6132-6138
(methylphosphonate); Morvan et al., 1986, "alpha-DNA. I. Synthesis,
characterization by high field .sup.1H-NMR, and base-pairing
properties of the unnatural hexadeoxyribonucleotide
alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)]",
Nucl Acids Res. 14:5019-5032 (alphadeoxynucleotides); Monia et al.,
1993, "Evaluation of 2'-modified oligonucleotides containing 2'
deoxy gaps as antisense inhibitors of polynucleotide expression",
J. Biol. Chem. 268:14514-22 (2'-O-methyl-ribonucleosides); Asseline
et al., 1984, "Nucleic acid-binding molecules with high affinity
and base sequence specificity: intercalating agents covalently
linked to oligodeoxynucleotides", Proc Natl Acad. Sci. 81:3297-3301
(acridine); Knorre et al., 1985, "Reactive oligonucleotide
derivatives and sequence-specific modification of nucleic acids",
Biochimie 67:785-789; Vlassov et al., 1986, "Complementary
addressed modification and cleavage of a single stranded DNA
fragment with alkylating oligonucleotide derivatives", Nucl Acids
Res. 14:4065-4076 (N-2-chlorocethylamine and phenazine); Webb and
Matteucci, 1986, "Hybridization triggered cross-linking of
deoxyoligonucleotides", Nucl Acids Res. 14:7661-7674
(5-methyl-N.sup.4--N.sup.4-ethanocytosine); Boutorin et-al. 1984,
FEBS Letters 172:43-46 (Fe-ethylenediamine tetraacetic acid
("EDTA") and analogues); Boutorin et al., 1989, "Synthesis of
alkylating oligonucleotide derivatives containing cholesterol or
phenazinium residues at their 3'-terminus and their interaction
with DNA within mammalian cells", FEBS Lett. 254:129-132; Chen and
Sigman, 1986, "Nuclease activity of 1,10-phenanthroline-copper:
sequence-specific targeting", Proc Natl Acad. Sci. 83:7147-7151
(5-glycylamido-1,10-o-phenanthroline); and Chu and Orgel, 1985,
"Nonenzymatic sequence-specific cleavage of single-stranded DNA",
Proc Natl Acad. Sci. 82:963-967 (diethylenetriaamine-pentaacetic
acid derivatives).
Isolated BGS-19 Polypeptides
[0138] One aspect of the invention pertains to an isolated BGS-19
protein, and biologically active portions thereof (e.g., ITIM
motifs, extracellular ligand binding domains), as well as
polypeptide fragments suitable for use as immunogens to raise
antibodies directed against a BGS-19 polypeptide of the invention.
In one embodiment, the native polypeptide can be isolated from
cells or tissues expressing a BGS-19 polypeptide using standard
protein purification techniques. In another embodiment,
polypeptides of the invention are produced from expression vectors
by recombinant DNA techniques. In yet another embodiment, a
polypeptide of the invention can be synthesized chemically using
standard peptide synthesis techniques.
[0139] An isolated or purified protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the protein is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free" indicates protein preparations in
which the protein is separated from cellular components of the
cells from which it is isolated or recombinantly produced. Thus,
protein that is substantially free of cellular material includes
protein preparations having less than 20%, 10%, or 5% (by dry
weight) of a contaminating protein. Similarly, when an isolated
BGS-19 polypeptide of the invention is recombinantly produced, it
is substantially free of culture medium. When the BGS-19 protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals.
[0140] Biologically active portions of a polypeptide of the
invention include polypeptides comprising amino acid sequences
identical to or derived from the amino acid sequence of the
protein, such that the variants sequences comprise conservative
substitutions or truncations. Typically, biologically active
portions comprise a domain or motif with at least one activity of
the corresponding protein. Domains or motifs include, but are not
limited to, a biologically active portion of a protein of the
invention. Polypeptides of the invention can comprise, for example,
a BGS-19 extracellular domain, BGS-19 intracellular domain, BGS-19
signal peptide, BGS-19 Ig-like domain 1 (SEQ ID NO:9), BGS-19
Ig-like domain 2 (SEQ ID NO:10), BGS-19 transmembrane domain,
BGS-19 ITIM motif, subcellular localization signals. In a specific
embodiment, a BGS-19 polypeptide comprises the signal domain at
amino acid residues 1-15, the Ig-like domain 1 at about amino acid
residues 16-113 (SEQ ID NO:9), the Ig-like domain 2 at about amino
acid residues 140-241 (SEQ ID NO:10), the transmembrane domain at
about amino acid residues 250-275 (SEQ ID NO:8), and the ITIM motif
at about amino acid residues 329-334. The BGS-19 polypeptide (SEQ
ID NO:2) has a predicted molecular weight of 42 kD.
[0141] In preferred embodiments, the following BGS-19 transmembrane
domain polypeptide is encompassed by the present invention:
AALGAGVAALLAFCSCL VVFRVKICR (SEQ ID NO:8). Polynucleotides encoding
this polypeptide are also provided. The present invention also
encompasses the use of this BGS-19 transmembrane domain polypeptide
as an immunogenic and/or antigenic epitope as described elsewhere
herein.
[0142] In preferred embodiments, the following BGS-19 Ig-like
domain polypeptides are encompassed by the present invention:
GSLNKDPSYSLQVQRQVPVPEGLC
VIVSCNLSYPRDGWDESTAAYGYWFKGRTSPKTGAPVATNNQSREVEMSTRD
RFQLTGDPGKGSCSLVIRDAQRED (SEQ ID NO:9), VTALTKKPDVYIP
ETLEPGQPVTVICVFNWAFKKCPAPSFSWTGAALSPRRTRPSTSQPSDPGVLELP
PIQMEHEGEFTCHAQHPLGSQHVSLSLSVHWKLE and/or QTVHFTVREAPQI (SEQ ID
NO:10). Polynucleotides encoding these polypeptides are also
provided. The present invention also encompasses the use of these
BGS-19 Ig-like domain polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0143] In one embodiment, a BGS-19 polypeptide has a transmembrane
domain, which is an amino acid sequence comprising at least about
20 to 40 amino acid residues in length and features hydrophobic
amino acid residues, such as alanine, leucine, isoleucine,
phenylalanine, proline, tyrosine, tryptophan, or valine. In a
preferred embodiment, a transmembrane domain comprises at least
about 20 to 40 amino acid residues, preferably 25-30 amino acid
residues, and has at least about 60% to 80% hydrophobic
residues.
[0144] In one embodiment, a BGS-19 polypeptide comprises a portion
of the amino sequence depicted in FIGS. 2A-B or FIGS. 1A-C. In one
embodiment, a BGS-19 polypeptide comprises at least 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the
amino sequence depicted in FIGS. 2A-B or FIGS. 1A-C. In further
embodiments, the BGS-19 polypeptide does not consist of the
sequence TABLE-US-00001 (SEQ ID NO: 11)
MLLLPLLLPVLGAGSLNKDPSYSLQVQRQVPVPEGLCVIVSCNLSYPRDG WDESTAAYGYWFKG;
(SEQ ID NO: 12) TSPKTGAPVATNNQSREVEMSTRDRFQLTGDPGKGSCSLVIRDAQREDEA
WYFFRVERGSRVRHSF; (SEQ ID NO: 13) LKVTALT; (SEQ ID NO: 14)
KPDVYIPETLEPG; (SEQ ID NO: 15)
RVKICRKEARKRAAAEQDVPSTLGPISQGHQHECSAGSSQDHPPPGAATY
TPGKGEEQELHYASLSFQ; (SEQ ID NO: 16)
GLRLWEPADQEAPSTTEYSEIKIHTGQPLRGPGFGLQLEREMSGMVP; (SEQ ID NO: 17)
RVKICRKEARKRAAAEQDVPSTLGPISQGHQHECSAGSSQDHPPPGAATY TPGKGEEQ; (SEQ
ID NO: 18) GPGFGLQLEREMSG; (SEQ ID NO: 19) LHYASLSFQGLRLW; (SEQ ID
NO: 20) RVKICRKEARKRAAAEQDVPSTLGPISQGHQHECSAGSSQDHP; (SEQ ID NO:
21) PGAATYT; or (SEQ ID NO: 22) GKGEEQELHYA.
[0145] In a specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence LLLPLLLPVL (SEQ ID NO:23). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
MLLLPLLLPVLG (SEQ ID NO:24). In another specific embodiment, a
BGS-19 polypeptide comprises the amino acid sequence VPEGLC (SEQ ID
NO:25). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence PVPEGLCV (SEQ ID NO:26). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence AYGYWFK (SEQ ID NO:27). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
AAYGYWFKG (SEQ ID NO:28). In another specific embodiment, a BGS-19
polypeptide comprises the amino acid sequence GAPVATN (SEQ ID
NO:29). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence TGAPVATNN (SEQ ID NO:30). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence GAPVATNNNQSREVEMSTR (SEQ ID NO:31). In another
specific embodiment, a BGS-19 polypeptide comprises the amino acid
sequence GAPVATNNNQSREVEMSTRDRFQLTGDP (SEQ ID NO:32). In another
specific embodiment, a BGS-19 polypeptide comprises the amino acid
sequence QSREVEMSTR (SEQ ID NO:33). In another specific embodiment,
a BGS-19 polypeptide comprises the amino acid sequence NQSREVEMSTRD
(SEQ ID NO:34). In another specific embodiment, a BGS-19
polypeptide comprises the amino acid sequence RFQLTGDP (SEQ ID
NO:35). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence DRFQLTGDPG (SEQ ID NO:36). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence KGSCSLVIRDAQ (SEQ ID NO:37). In another
specific embodiment, a BGS-19 polypeptide comprises the amino acid
sequence GKGSCSLVIRDAQR (SEQ ID NO:38). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
YFFRVERGS (SEQ ID NO:39). In another specific embodiment, a BGS-19
polypeptide comprises the amino acid sequence WYFFRVERGSR (SEQ ID
NO:40). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence FFLKVTALT (SEQ ID NO:41). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence AFFLKVTALTK (SEQ ID NO:42). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
KPDVYIPETLEPGQPVTVICVFNWAF (SEQ ID NO:43). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
KKPDVYIPETLEPGQPVTVICVFNWAFK (SEQ ID NO:44). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
CPAPSFSWTGAALS (SEQ ID NO:45). In another specific embodiment, a
BGS-19 polypeptide comprises the amino acid sequence
KCPAPSFSWTGAALSP (SEQ ID NO:46). In another specific embodiment, a
BGS-19 polypeptide comprises the amino acid sequence PSFSWTGAALS
(SEQ ID NO:47). In another specific embodiment, a BGS-19
polypeptide comprises the amino acid sequence APSFSWTGAALSP (SEQ ID
NO:48). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence PPIQMEH (SEQ ID NO:49). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence PPIQMEHE (SEQ ID NO:50). In another specific
embodiment, a BGS-19 polypeptide comprises the amino acid sequence
LPPIQMEHE (SEQ ID NO:51). In another specific embodiment, a BGS-19
polypeptide comprises the amino acid sequence GAALGAGVAALL (SEQ ID
NO:52). In another specific embodiment, a BGS-19 polypeptide
comprises the amino acid sequence LGAALGAGVAALL (SEQ ID NO:53). In
another specific embodiment, a BGS-19 polypeptide comprises the
amino acid sequence LGAALGAGVAALLA (SEQ ID NO:54). In another
specific embodiment, a BGS-19 polypeptide comprises the amino acid
sequence ELHYASLSF (SEQ ID NO:55). In another specific embodiment,
a BGS-19 polypeptide comprises the amino acid sequence QELHYASLSFQ
(SEQ ID NO:56).
[0146] In a particular embodiment, a BGS-19 polypeptide consists of
an amino acid sequence recited above. In another particular
embodiment, a BGS-19 polypeptide consists of an amino acid sequence
recited above with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive
amino acids of the native BGS-19 polypeptide flanking the recited
amino acid sequence. In a further embodiment, the 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 consecutive amino acids of the native BGS-19
polypeptide are the amino acids normally flanking the recited amino
acid sequence.
[0147] In another particular embodiment, a BGS-19 polypeptide
consists of an amino acid sequence recited above with 10, 20, 30,
40, 50, 60, 70, 80, 90 or 100 consecutive amino acids of the native
BGS-19 polypeptide flanking the recited amino acid sequence. In a
further embodiment, the 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100
consecutive amino acids of the native BGS-19 polypeptide are the
amino acids normally flanking the recited amino acid sequence.
[0148] The BGS-19 polypeptide has been shown to comprise two
glycosylation sites according to the Motif algorithm (Genetics
Computer Group, Inc.). As discussed more specifically herein,
protein glycosylation is thought to serve a variety of functions
including: augmentation of protein folding, inhibition of protein
aggregation, regulation of intracellular trafficking to organelles,
increasing resistance to proteolysis, modulation of protein
antigenicity, and mediation of intercellular adhesion.
[0149] Asparagine glycosylation sites have the following consensus
pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation
site. However, it is well known that that potential N-glycosylation
sites are specific to the consensus sequence Asn-Xaa-Ser/Thr.
However, the presence of the consensus tripeptide is not sufficient
to conclude that an asparagine residue is glycosylated, due to the
fact that the folding of the protein plays an important role in the
regulation of N-glycosylation. It has been shown that the presence
of proline between Asn and Ser/Thr will inhibit N-glycosylation;
this has been confirmed by a recent statistical analysis of
glycosylation sites, which also shows that about 50% of the sites
that have a proline C-terminal to Ser/Thr are not glycosylated.
Additional information relating to asparagine glycosylation may be
found in reference to the following publications, which are hereby
incorporated by reference herein: Marshall R. D., Annu. Rev.
Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.
Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.
209:331-336(1983); Gavel Y., von Heijne G., Protein Eng.
3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol.
Chem. 265:11397-11404(1990).
[0150] In preferred embodiments, the following asparagine
glycosylation site polypeptides are encompassed by the present
invention: VIVSCNLSYPRDGW (SEQ ID NO:62), and/or PVATNNQSREVEMS(SEQ
ID NO:63). Polynucleotides encoding these polypeptides are also
provided. The present invention also encompasses the use of these
BGS-19 asparagine glycosylation site polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0151] The BGS-19 polypeptides of the present invention were
determined to comprise several phosphorylation sites based upon the
Motif algorithm (Genetics Computer Group, Inc.). The
phosphorylation of such sites may regulate some biological activity
of the BGS-19 polypeptide. For example, phosphorylation at specific
sites may be involved in regulating the proteins ability to
associate or bind to other molecules (e.g., proteins, ligands,
substrates, DNA, etc.). In the present case, phosphorylation may
modulate the ability of the BGS-19 polypeptide to associate with
other immunoglobulin proteins, the ability of BGS-19 to serve as an
antigen receptors, cytokine receptors, as a receptor for
cell-surface molecules (e.g., other IgSF proteins, adhesion
molecules), and as a counter-receptor.
[0152] Specifically, the BGS-19 polypeptide was predicted to
comprise one tyrosine phosphorylation site using the Motif
algorithm (Genetics Computer Group, Inc.). Such sites are
phosphorylated at the tyrosine amino acid residue. The consensus
pattern for tyrosine phosphorylation sites are as follows:
[RK]-x(2)-[DE]-x(3)-Y, or or [RK]-x(3)-[DE]-x(2)-Y, where Y
represents the phosphorylation site and `x` represents an
intervening amino acid residue. Additional information specific to
tyrosine phosphorylation sites can be found in Patschinsky T.,
Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl.
Acad. Sci. U.S.A. 79:973-977(1982); Hunter T., J. Biol. Chem.
257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S.,
Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby
incorporated herein by reference.
[0153] In preferred embodiments, the following tyrosine
phosphorylation site polypeptides are encompassed by the present
invention: IRDAQREDEAWYFFRVE (SEQ ID NO:68). Polynucleotides
encoding these polypeptides are also provided. The present
invention also encompasses the use of these BGS-19 tyrosine
phosphorylation site polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0154] The BGS-19 polypeptide was predicted to comprise four PKC
phosphorylation sites using the Motif algorithm (Genetics Computer
Group, Inc.). In vivo, protein kinase C exhibits a preference for
the phosphorylation of serine or threonine residues. The PKC
phosphorylation sites have the following consensus pattern:
[ST]-x-[RK], where S or T represents the site of phosphorylation
and `x` an intervening amino acid residue. Additional information
regarding PKC phosphorylation sites can be found in Woodget J. R.,
Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and
Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H.,
Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985);
which are hereby incorporated by reference herein.
[0155] In preferred embodiments, the following PKC phosphorylation
site polypeptides are encompassed by the present invention:
FKGRTSPKTGAPV (SEQ ID NO:64), REVEMSTRDRFQL (SEQ ID NO:65),
KVTALTKKPDVYI (SEQ ID NO:66), and/or TGAALSPRRTRPS (SEQ ID NO:67).
Polynucleotides encoding these polypeptides are also provided. The
present invention also encompasses the use of these BGS-19 PKC
phosphorylation site polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0156] The BGS-19 polypeptide was predicted to comprise three
casein kinase II phosphorylation sites using the Motif algorithm
(Genetics Computer Group, Inc.). Casein kinase II (CK-2) is a
protein serine/threonine kinase whose activity is independent of
cyclic nucleotides and calcium. CK-2 phosphorylates many different
proteins. The substrate specificity [1] of this enzyme can be
summarized as follows: (1) Under comparable conditions Ser is
favored over Thr.; (2) An acidic residue (either Asp or Glu) must
be present three residues from the C-terminal of the phosphate
acceptor site; (3) Additional acidic residues in positions +1, +2,
+4, and +5 increase the phosphorylation rate. Most physiological
substrates have at least one acidic residue in these positions; (4)
Asp is preferred to Glu as the provider of acidic determinants; and
(5) A basic residue at the N-terminal of the acceptor site
decreases the phosphorylation rate, while an acidic one will
increase it.
[0157] A consensus pattern for casein kinase II phosphorylations
site is as follows: [ST]-x(2)-[DE], wherein `x` represents any
amino acid, and S or T is the phosphorylation site.
[0158] Additional information specific to casein kinase II
phosphorylation sites may be found in reference to the following
publication: Pinna L. A., Biochim. Biophys. Acta
1054:267-284(1990); which is hereby incorporated herein in its
entirety.
[0159] In preferred embodiments, the following casein kinase II
phosphorylation site polypeptide is encompassed by the present
invention: REVEMSTRDRFQLT (SEQ ID NO:69), ECSAGSSQDHPPPG (SEQ ID
NO:70), and/or DQEAPSTTEYSEIK (SEQ ID NO:71). Polynucleotides
encoding these polypeptides are also provided. The present
invention also encompasses the use of this casein kinase II
phosphorylation site polypeptide as an immunogenic and/or antigenic
epitope as described elsewhere herein.
[0160] The BGS-19 polypeptide was predicted to comprise seven
N-myristoylation sites using the Motif algorithm (Genetics Computer
Group, Inc.). An appreciable number of eukaryotic proteins are
acylated by the covalent addition of myristate (a C 14-saturated
fatty acid) to their N-terminal residue via an amide linkage. The
sequence specificity of the enzyme responsible for this
modification, myristoyl CoA:protein N-myristoyl transferase (NMT),
has been derived from the sequence of known N-myristoylated
proteins and from studies using synthetic peptides. The specificity
seems to be the following: i.) The N-terminal residue must be
glycine; ii.) In position 2, uncharged residues are allowed; iii.)
Charged residues, proline and large hydrophobic residues are not
allowed; iv.) In positions 3 and 4, most, if not all, residues are
allowed; v.) In position 5, small uncharged residues are allowed
(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) In
position 6, proline is not allowed.
[0161] A consensus pattern for N-myristoylation is as follows:
G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein `x` represents any amino
acid, and G is the N-myristoylation site.
[0162] Additional information specific to N-myristoylation sites
may be found in reference to the following publication: Towler D.
A., Gordon J. I., Adams S. P., Glaser L., Annu. Rev. Biochem.
57:69-99(1988); and Grand R. J. A., Biochem. J. 258:625-638(1989);
which is hereby incorporated herein in its entirety.
[0163] In preferred embodiments, the following N-myristoylation
site polypeptides are encompassed by the present invention:
TSPKTGAPVATNNQSR (SEQ ID NO:72), ETLEPGQPVTVICVFN (SEQ ID NO:73),
WKLEHGGGLGLGAALG (SEQ ID NO:74), LEHGGGLGLGAALGAG (SEQ ID NO:75),
HGGGLGLGAALGAGVA (SEQ ID NO:76), GGLGLGAALGAGVAAL (SEQ ID NO:77),
and/or LGAALGAGVAALLAFC (SEQ ID NO:78). Polynucleotides encoding
these polypeptides are also provided. The present invention also
encompasses the use of these N-myristoylation site polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0164] Moreover, in confirmation of the BGS-19 polypeptide
representing an immunoglobulin-like domain containing protein, it
has been shown to comprise one Ig_MHC site according to the Motif
algorithm (Genetics Computer Group, Inc.). The identification of
the BGS-19 polypeptide as containing an Ig_MHC site is consistent
with its potential involvement in immune and/or hematopoietic
modulation.
[0165] The basic structure of immunoglobulin (Ig) molecules is a
tetramer of two light chains and two heavy chains linked by
disulfide bonds. There are two types of light chains: kappa and
lambda, each composed of a constant domain (CL) and a variable
domain (VL). There are five types of heavy chains: alpha, delta,
epsilon, gamma and mu, all consisting of a variable domain (VH) and
three (in alpha, delta and gamma) or four (in epsilon and mu)
constant domains (CH1 to CH4).
[0166] The major histocompatibility complex (MHC) molecules are
made of two chains. In class I the alpha chain is composed of three
extracellular domains, a transmembrane region and a cytoplasmic
tail. The beta chain (beta-2-microglobulin) is composed of a single
extracellular domain. In class II, both the alpha and the beta
chains are composed of two extracellular domains, a transmembrane
region and a cytoplasmic tail.
[0167] It is known that the Ig constant chain domains and a single
extracellular domain in each type of MHC chains are related. These
homologous domains are approximately one hundred amino acids long
and include a conserved intradomain disulfide bond.
[0168] The consensus pattern for Ig_MHC domain signatures is the
following: [FY]-x-C-x-[VA]-x-H, wherein "x" represents any amino
acid.
[0169] Sequences known to belong to this class are the following:
Ig heavy chains type Alpha C region--All, in CH2 and CH3; Ig heavy
chains type Delta C region--All, in CH3; Ig heavy chains type
Epsilon C region--All, in CH1, CH3 and CH4; Ig heavy chains type
Gamma C region--All, in CH3 and also CH 1 in some cases; Ig heavy
chains type Mu C region--All, in CH2, CH3 and CH4; Ig light chains
type Kappa C region--In all CL except rabbit and Xenopus; Ig light
chains type Lambda C region--In all CL except rabbit; MHC class I
alpha chains--All, in alpha-3 domains; the cytomegalovirus MHC-1
homologous protein [6]; Beta-2-microglobulin--All; MHC class II
alpha chains--All, in alpha-2 domains; and MHC class II beta
chains--All, in beta-2 domains.
[0170] Additional information related to immunoglobulin domains and
MGC domains may be obtained through reference to the following
publications: Gough N., Trends Biochem. Sci. 6:203-205(1981); Klein
J., Figueroa F., Immunol. Today 7:41-44(1986); Figueroa F., Klein
J., Immunol. Today 7:78-81(1986); Orr H. T., Lancet D., Robb R. J.,
Lopez de Castro J. A., Strominger J. L., Nature 282:266-270(1979);
Cushley W., Owen M. J., Immunol. Today 4:88-92(1983); and Beck S.,
Barrel B. G., Nature 331:269-272(1988); which are hereby
incorporated by reference herein.
[0171] In preferred embodiments, the following Ig_MHC signature
domain polypeptide is encompassed by the present invention:
EHEGEFTCHAQHPLGSQ (SEQ ID NO:79). Polynucleotides encoding this
polypeptide is also provided. The present invention also
encompasses the use of the BGS-19 Ig_MHC signature domain
polypeptide as immunogenic and/or antigenic epitopes as described
elsewhere herein.
[0172] In preferred embodiments, the following N-terminal BGS-19
deletion polypeptides are encompassed by the present invention:
M1-K385, L2-K385, L3-K385, L4-K385, P5-K385, L6-K385, L7-K385,
L8-K385, P9-K385, V10-K385, L11-K385, G12-K385, A13-K385, G14-K385,
S15-K385, L16-K385, N17-K385, K18-K385, D19-K385, P20-K385,
S21-K385, Y22-K385, S23-K385, L24-K385, Q25-K385, V26-K385,
Q27-K385, R28-K385, Q29-K385, V30-K385, P31-K385, V32-K385,
P33-K385, E34-K385, G35-K385, L36-K385, C37-K385, V38-K385,
I39-K385, V40-K385, S41-K385, C42-K385, N43-K385, L44-K385,
S45-K385, Y46-K385, P47-K385, R48-K385, D49-K385, G50-K385,
W51-K385, D52-K385, E53-K385, S54-K385, T55-K385, A56-K385,
A57-K385, Y58-K385, G59-K385, Y60-K385, W61-K385, F62-K385,
K63-K385, G64-K385, R65-K385, T66-K385, S67-K385, P68-K385,
K69-K385, T70-K385, G71-K385, A72-K385, P73-K385, V74-K385,
A75-K385, T76-K385, N77-K385, N78-K385, Q79-K385, S80-K385,
R81-K385, E82-K385, V83-K385, E84-K385, M85-K385, S86-K385,
T87-K385, R88-K385, D89-K385, R90-K385, F91-K385, Q92-K385,
L93-K385, T94-K385, G95-K385, D96-K385, P97-K385, G98-K385,
K99-K385, G100-K385, S101-K385, C102-K385, S103-K385, L104-K385,
V105-K385, 1106-K385, R107-K385, D108-K385, A109-K385, Q110-K385,
R111-K385, E112-K385, D113-K385, E114-K385, A115-K385, W116-K385,
Y117-K385, F118-K385, F119-K385, R120-K385, V121-K385, E122-K385,
R123-K385, G124-K385, S125-K385, R126-K385, V127-K385, R128-K385,
H129-K385, S130-K385, F131-K385, L132-K385, S133-K385, N134-K385,
A135-K385, F136-K385, F137-K385, L138-K385, K139-K385, V140-K385,
T141-K385, A142-K385, L143-K385, T144-K385, K145-K385, K146-K385,
P147-K385, D148-K385, V149-K385, Y150-K385, I151-K385, P152-K385,
E153-K385, T154-K385, L155-K385, E156-K385, P157-K385, G158-K385,
Q159-K385, P160-K385, V161-K385, T162-K385, V163-K385, I164-K385,
C165-K385, V166-K385, F167-K385, N168-K385, W169-K385, A170-K385,
F171-K385, K172-K385, K173-K385, C174-K385, P175-K385, A176-K385,
P177-K385, S178-K385, F179-K385, S180-K385, W181-K385, T182-K385,
G183-K385, A184-K385, A185-K385, L186-K385, S187-K385, P188-K385,
R189-K385, R190-K385, T191-K385, R192-K385, P193-K385, S194-K385,
T195-K385, S196-K385, Q197-K385, P198-K385, S199-K385, D200-K385,
P201-K385, G202-K385, V203-K385, L204-K385, E205-K385, L206-K385,
P207-K385, P208-K385, I209-K385, Q210-K385, M211-K385, E212-K385,
H213-K385, E214-K385, G215-K385, E216-K385, F217-K385, T218-K385,
C219-K385, H220-K385, A221-K385, Q222-K385, H223-K385, P224-K385,
L225-K385, G226-K385, S227-K385, Q228-K385, H229-K385, V230-K385,
S231-K385, L232-K385, S233-K385, L234-K385, S235-K385, V236-K385,
H237-K385, W238-K385, K239-K385, L240-K385, E241-K385, H242-K385,
G243-K385, G244-K385, G245-K385, L246-K385, G247-K385, L248-K385,
G249-K385, A250-K385, A251-K385, L252-K385, G253-K385, A254-K385,
G255-K385, V256-K385, A257-K385, A258-K385, L259-K385, L260-K385,
A261-K385, F262-K385, C263-K385, S264-K385, C265-K385, L266-K385,
V267-K385, V268-K385, F269-K385, R270-K385, V271-K385, K272-K385,
1273-K385, C274-K385, R275-K385, K276-K385, E277-K385, A278-K385,
R279-K385, K280-K385, R281-K385, A282-K385, A283-K385, A284-K385,
E285-K385, Q286-K385, D287-K385, V288-K385, P289-K385, S290-K385,
T291-K385, L292-K385, G293-K385, P294-K385, 1295-K385, S296-K385,
Q297-K385, G298-K385, H299-K385, Q300-K385, H301-K385, E302-K385,
C303-K385, S304-K385, A305-K385, G306-K385, S307-K385, S308-K385,
Q309-K385, D310-K385, H311-K385, P312-K385, P313-K385, P314-K385,
G315-K385, A316-K385, A317-K385, T318-K385, Y319-K385, T320-K385,
P321-K385, G322-K385, K323-K385, G324-K385, E325-K385, E326-K385,
Q327-K385, E328-K385, L329-K385, H330-K385, Y331-K385, A332-K385,
S333-K385, L334-K385, S335-K385, F336-K385, Q337-K385, G338-K385,
L339-K385, R340-K385, L341-K385, W342-K385, E343-K385, P344-K385,
A345-K385, D346-K385, Q347-K385, E348-K385, A349-K385, P350-K385,
S351-K385, T352-K385, T353-K385, E354-K385, Y355-K385, S356-K385,
E357-K385, 1358-K385, K359-K385, I360-K385, H361-K385, T362-K385,
G363-K385, Q364-K385, P365-K385, L366-K385, R367-K385, G368-K385,
P369-K385, G370-K385, F371-K385, G372-K385, L373-K385, Q374-K385,
L375-K385, E376-K385, R377-K385, E378-K385, and/or M379-K385 of SEQ
ID NO:2. Polynucleotide sequences encoding these polypeptides are
also provided. The present invention also encompasses the use of
these N-terminal BGS-19 deletion polypeptides as immunogenic and/or
antigenic epitopes as described elsewhere herein.
[0173] In preferred embodiments, the following C-terminal BGS-19
deletion polypeptides are encompassed by the present invention:
M1-K385, M1-P384, M1-V383, M1-M382, M1-G381, M1-S380, M1-M379,
M1-E378, M1-R377, M1-E376, M1-L375, M1-Q374, M1-L373, M1-G372,
M1-F371, M1-G370, M1-P369, M1-G368, M1-R367, M1-L366, M1-P365,
M1-Q364, M1-G363, M1-T362, M1-H361, M1-1360, M1-K359, M1-1358,
M1-E357, M1-S356, M1-Y355, M1-E354, M1-T353, M1-T352, M1-S351,
M1-P350, M1-A349, M1-E348, M1-Q347, M1-D346, M1-A345, M1-P344,
M1-E343, M1-W342, M1-L341, M1-R340, M1-L339, M1-G338, M1-Q337,
M1-F336, M1-S335, M1-L334, M1-S333, M1-A332, M1-Y331, M1-H330,
M1-L329, M1-E328, M1-Q327, M1-E326, M1-E325, M1-G324, M1-K323,
M1-G322, M1-P321, M1-T320, M1-Y319, M1-T318, M1-A317, M1-A316,
M1-G315, M1-P314, M1-P313, M1-P312, M1-H311, M1-D310, M1-Q309,
M1-S308, M1-S307, M1-G306, M1-A305, M1-S304, M1-C303, M1-E302,
M1-H301, M1-Q300, M1-H299, M1-G298, M1-Q297, M1-S296, M1-1295,
M1-P294, M1-G293, M1-L292, M1-T291, M1-S290, M1-P289, M1-V288,
M1-D287, M1-Q286, M1-E285, M1-A284, M1-A283, M1-A282, M1-R281,
M1-K280, M1-R279, M1-A278, M1-E277, M1-K276, M1-R275, M1-C274,
M1-1273, M1-K272, M1-V271, M1-R270, M1-F269, M1-V268, M1-V267,
M1-L266, M1-C265, M1-S264, M1-C263, M1-F262, M1-A261, M1-L260,
M1-L259, M1-A258, M1-A257, M1-V256, M1-G255, M1-A254, M1-G253,
M1-L252, M1-A251, M1-A250, M1-G249, M1-L248, M1-G247, M1-L246,
M1-G245, M1-G244, M1-G243, M1-H242, M1-E241, M1-L240, M1-K239,
M1-W238, M1-H237, M1-V236, M1-S235, M1-L234, M1-S233, M1-L232,
M1-S231, M1-V230, M1-H229, M1-Q228, M1-S227, M1-G226, M1-L225,
M1-P224, M1-H223, M1-Q222, M1-A221, M1-H220, M1-C219, M1-T218,
M1-F217, M1-E216, M1-G215, M1-E214, M1-H213, M1-E212, M1-M211,
M1-Q210, M1-1209, M1-P208, M1-P207, M1-L206, M1-E205, M1-L204,
M1-V203, M1-G202, M1-P201, M1-D200, M1-S199, M1-P198, M1-Q197,
M1-S196, M1-T195, M1-S194, M1-P193, M1-R192, M1-T191, M1-R190,
M1-R189, M1-P188, M1-S187, M1-L186, M1-A185, M1-A184, M1-G183,
M1-T182, M1-W181, M1-S180, M1-F179, M1-S178, M1-P177, M1-A176,
M1-P175, M1-C174, M1-K173, M1-K172, M1-F171, M1-A170, M1-W169,
M1-N168, M1-F167, M1-V166, M1-C.sub.165, M1-164, M1-V163, M1-T162,
M1-V161, M1-P160, M1-Q159, M1-G158, M1-P157, M1-E156, M1-L155,
M1-T154, M1-E153, M1-P152, M1-I151, M1-Y150, M1-V149, M1-D148,
M1-P147, M1-K146, M1-K145, M1-T144, M1-L143, M1-A142, M1-T141,
M1-V140, M1-K139, M1-L138, M1-F137, M1-F136, M1-A135, M1-N134,
M1-S133, M1-L132, M1-F131, M1-S130, M1-H129, M1-R128, M1-V127,
M1-R126, M1-S125, M1-G124, M1-R123, M1-E122, M1-V121, M1-R120,
M1-F119, M1-F118, M1-Y117, M1-W116, M1-A115, M1-E114, M1-D113,
M1-E112, M1-R111, M1-Q110, M1-A109, M1-D108, M1-R107, M1-I106,
M1-V105, M1-L104, M1-S103, M1-C102, M1-S101, M1-G1000, M1-K99,
M1-G98, M1-P97, M1-D96, M1-G95, M1-T94, M1-L93, M1-Q92, M1-F91,
M1-R90, M1-D89, M1-R88, M1-T87, M1-S86, M1-M85, M1-E84, M1-V83,
M1-E82, M1-R81, M1-S80, M1-Q79, M1-N78, M1-N77, M1-T76, M1-A75,
M1-V74, M1-P73, M1-A72, M1-G71, M1-T70, M1-K69, M1-P68, M1-S67,
M1-T66, M1-R65, M1-G64, M1-K63, M1-F62, M1-W61, M1-Y60, M1-G59,
M1-Y58, M1-A57, M1-A56, M1-T55, M1-S54, M1-E53, M1-D52, M1-W51,
M1-G50, M1-D49, M1-R48, M1-P47, M1-Y46, M1-S45, M1-L44, M1-N43,
M1-C42, M1-S41, M1-V40, M1-139, M1-V38, M1-C37, M1-L36, M1-G35,
M1-E34, M1-P33, M1-V32, M1-P31, M1-V30, M1-Q29, M1-R28, M1-Q27,
M1-V26, M1-Q25, M1-L24, M1-S23, M1-Y22, M1-S21, M1-P20, M1-D19,
M1-K18, M1-N17, M1-L16, M1-S15, M1-G14, M1-A13, M1-G12, M1-L11,
M1-V10, M1-P9, M1-L8, and/or M1-L7 of SEQ ID NO:2. Polynucleotide
sequences encoding these polypeptides are also provided. The
present invention also encompasses the use of these C-terminal
BGS-19 deletion polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0174] The invention also provides chimeric or fusion proteins. As
used herein, a "chimeric protein" or "fusion protein" comprises all
or part (preferably biologically active) of a polypeptide of the
invention fused in-frame to a heterologous polypeptide. The
heterologous polypeptide can be fused to the N-terminus or
C-terminus of the polypeptide of the invention.
[0175] One useful fusion protein is a GST fusion protein in which
the polypeptide of the invention is fused to the C-terminus of GST
sequences. Such fusion proteins can facilitate the purification of
a recombinant polypeptide of the invention.
[0176] In another embodiment, the fusion protein comprises a
heterologous signal sequence at its N-terminus. For example, the
native signal sequence of a polypeptide of the invention can be
removed and replaced with a signal sequence from another protein.
For example, the gp67 secretory sequence of the baculovirus
envelope protein can be used as a heterologous signal sequence
(Ausubel et al., Current Protocols in Molecular Biolog, eds., John
Wiley & Sons, 1992). Other examples of eukaryotic heterologous
signal sequences include the secretory sequences of melittin and
human placental alkaline phosphatase (Stratapolynucleotide; La
Jolla, Calif.). In yet another example, useful prokaryotic
heterologous signal sequences include the phoA secretory signal
(Sambrook et al., Molecular Cloning: A Laboratorv Manual, 2nd ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989) and the protein A secretory signal
(Pharmacia Biotech; Piscataway, N.J.).
[0177] In yet another embodiment, the fusion protein is an
immunoglobulin fusion protein in which all or part of a polypeptide
of the invention is fused with sequences derived from a member of
the immunoglobulin protein family. The immunoglobulin fusion
proteins of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (soluble or membrane-bound) and a
protein on the surface of a cell (receptor), to thereby suppress
signal transduction in vivo. The immunoglobulin fusion protein can
be used to affect the bioavailability of a cognate ligand of a
polypeptide of the invention. Inhibition of ligand/receptor
interaction can be useful therapeutically, both for treating
proliferative disorders and for modulating (e.g., promoting or
inhibiting) cell survival. Moreover, the immunoglobulin fusion
proteins of the invention can be used as immunogens to produce
antibodies directed against a polypeptide of the invention in a
subject, to purify ligands and in screening assays to identify
molecules which inhibit the interaction of receptors with ligands.
The immunoglobulin fusion protein can, for example, comprise a
portion of a polypeptide of the invention fused with the
amino-terminus or the carboxyl-terminus of an immunoglobulin
constant region (see, e.g., U.S. Pat. Nos. 5,714,147; 5,116,964;
5,514,582; 5,455,165).
[0178] Chimeric and fusion proteins of the invention can be
produced by standard recombinant DNA techniques. In another
embodiment, the fusion polynucleotide can be synthesized by
conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of polynucleotide fragments can be
carried out using anchor primers which give rise to complementary
overhangs between two consecutive polynucleotide fragments which
can subsequently be annealed and reamplified to polynucleotiderate
a chimeric polynucleotide sequence (see, e.g., Ausubel et al.,
supra). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST
polypeptide). A polynucleotide encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
[0179] A signal sequence of a polypeptide of the invention can be
used to facilitate secretion and isolation of the secreted protein
or other proteins of interest. Signal sequences are typically
characterized by a core of hydrophobic amino acids which are
polynucleotiderally cleaved from the mature protein during
secretion in one or more cleavage events. Such signal peptides
comprise processing sites that allow cleavage of the signal
sequence from the mature proteins as they pass through the
secretory pathway. Thus, the invention pertains to the described
polypeptides having a signal sequence, as well as to the signal
sequence itself and to the polypeptide in the absence of the signal
sequence (i.e., the cleavage products). In one embodiment, a
polynucleotide encoding a signal sequence of the invention can be
operably linked in an expression vector to a protein of interest,
such as a protein which is ordinarily not secreted or is otherwise
difficult to isolate. The signal sequence directs secretion of the
protein, such as from a eukaryotic host into which the expression
vector is transformed, and the signal sequence is subsequently or
concurrently cleaved. The protein can then be readily purified from
the extracellular medium by art recognized methods. Alternatively,
the signal sequence can be linked to the protein of interest using
a sequence which facilitates purification, such as a GST
domain.
[0180] In another embodiment, the signal sequences of the present
invention can be used to identify regulatory sequences, e.g.,
promoters, enhancers, repressors. Since signal sequences are the
most amino-terminal sequences of a peptide, it is expected that the
nucleic acids which flank the signal sequence on its amino-terminal
side will be regulatory sequences which affect transcription. Thus,
a nucleotide sequence which encodes all or a portion of a signal
sequence can be used as a probe to identify and isolate signal
sequences and their flanking regions, and these flanking regions
can be studied to identify regulatory elements therein.
[0181] The present invention also pertains to variants of the
polypeptides of the invention. Such variants have an altered amino
acid sequence which can function as either agonists (mimetics) or
as antagonists. Variants can be polynucleotiderated by
mutapolynucleotidesis, e.g., discrete point mutation or truncation.
Moreover, variants of a polypeptide of the invention can be
produced by directed evolution techniques (see, e.g., U.S. Pat.
Nos. 6,309,883 and 6,238,884). For example, recursive ensemble
mutapolynucleotidesis, a technique which enhances the frequency of
functional mutants in the libraries, can be used in combination
with the screening assays to identify variants of a protein of the
invention (see, e.g., Arkin and Yourvan, 1992, "An algorithm for
protein engineering: simulations of recursive ensemble
mutapolynucleotidesis", Proc Natl Acad Sci 89:7811-7815; Delagrave
et al., 1993, "Recursive ensemble mutapolynucleotidesis", Protein
Eng. 6:327-331).
[0182] The polypeptides of the invention can exhibit
post-translational modifications, including, but not limited to,
glycosylations (e.g., N-linked or O-linked glycosylations),
myristylations, palmitylations, acetylations and phosphorylations
(e.g., serine/threonine or tyrosine). In one embodiment, the
polypeptides of the invention exhibit reduced levels of O-linked
glycosylation and/or N-linked glycosylation relative to
endogenously expressed. In another embodiment, the polypeptides of
the invention do not exhibit O-linked glycosylation or N-linked
glycosylation.
[0183] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, or substituted with another
amino acid. These alterations of the reference sequence may occur
at the amino- or carboxy-terminal positions of the reference amino
acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0184] As a practical matter, whether any particular polypeptide is
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
or 99.9% identical to, for instance, an amino acid sequence
referenced in Table 1 (SEQ ID NO:2) or to the amino acid sequence
encoded by cDNA contained in a deposited clone, can be determined
conventionally using known computer programs. A preferred method
for determining the best overall match between a query sequence (a
sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the CLUSTALW computer program (Thompson, J. D., et al., Nucleic
Acids Research, 2(22):4673-4680, (1994)), which is based on the
algorithm of Higgins, D. G., et al., Computer Applications in the
Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment
the query and subject sequences are both amino acid sequences. The
result of said global sequence alignment is in percent identity.
Preferred parameters used in a CLUSTALW alignment of DNA sequences
to calculate percent identity via pairwise alignments are:
Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3,
Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring
Method=Percent, Window Size=5 or the length of the subject
nucleotide sequence, whichever is shorter. For multiple alignments,
the following CLUSTALW parameters are preferred: Gap Opening
Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty
Range=8; End Gap Separation Penalty=Off; % Identity for Alignment
Delay-40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off;
and Transition Weighting=0. The pairwise and multiple alignment
parameters provided for CLUSTALW above represent the default
parameters as provided with the AlignX software program (Vector NTI
suite of programs, version 6.0).
[0185] The present invention encompasses the application of a
manual correction to the percent identity results, in the instance
where the subject sequence is shorter than the query sequence
because of N- or C-terminal deletions, not because of internal
deletions. If only the local pairwise percent identity is required,
no manual correction is needed. However, a manual correction may be
applied to determine the global percent identity from a global
polypeptide alignment. Percent identity calculations based upon
global polypeptide alignments are often preferred since they
reflect the percent identity between the polypeptide molecules as a
whole (i.e., including any polypeptide overhangs, not just
overlapping regions), as opposed to, only local matching
polypeptides. Manual corrections for global percent identity
determinations are required since the CLUSTALW program does not
account for N- and C-terminal truncations of the subject sequence
when calculating percent identity. For subject sequences truncated
at the N- and C-termini, relative to the query sequence, the
percent identity is corrected by calculating the number of residues
of the query sequence that are N- and C-terminal of the subject
sequence, which are not matched/aligned with a corresponding
subject residue, as a percent of the total bases of the query
sequence. Whether a residue is matched/aligned is determined by
results of the CLUSTALW sequence alignment. This percentage is then
subtracted from the percent identity, calculated by the above
CLUSTALW program using the specified parameters, to arrive at a
final percent identity score. This final percent identity score is
what may be used for the purposes of the present invention. Only
residues to the N- and C-termini of the subject sequence, which are
not matched/aligned with the query sequence, are considered for the
purposes of manually adjusting the percent identity score. That is,
only query residue positions outside the farthest N- and C-terminal
residues of the subject sequence.
[0186] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the CLUSTALW alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the CLUSTALW program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence, which are not
matched/aligned with the query. In this case the percent identity
calculated by CLUSTALW is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the CLUSTALW alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are required for the purposes of the
present invention.
[0187] In addition to the above method of aligning two or more
polynucleotide or polypeptide sequences to arrive at a percent
identity value for the aligned sequences, it may be desirable in
some circumstances to use a modified version of the CLUSTALW
algorithm which takes into account known structural features of the
sequences to be aligned, such as for example, the SWISS-PROT
designations for each sequence. The result of such a modified
CLUSTALW algorithm may provide a more accurate value of the percent
identity for two polynucleotide or polypeptide sequences. Support
for such a modified version of CLUSTALW is provided within the
CLUSTALW algorithm and would be readily appreciated to one of skill
in the art of bioinformatics.
Recombinant Vectors and Host Cells
[0188] Another aspect of the invention pertains to vectors,
preferably expression vectors, comprising a BGS-19 polynucleotide
or a polynucleotide encoding a BGS-19 polypeptide, BGS-19 agonist,
BGS-19 antagonist, inhibitor of a BGS-19 agonist, inhibitor of a
BGS-19 antagonist, or a variant thereof. In a particular
embodiment, an expression vector comprises a BGS-19 polynucleotide
encoding a BGS-19 polypeptide of the invention (or a portion
thereof).
[0189] Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. In polynucleotideral, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids
(vectors). However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses).
[0190] A recombinant expression vector of the invention comprises a
polynucleotide of the invention in a form suitable for expression
of the polynucleotide in a host cell. This means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operably linked to the polynucleotide to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology, Academic Press, San
Diego, Calif. (1990) p. 185.
[0191] Regulatory sequences include those which direct constitutive
expression of a nucleotide sequence in many types of host cell and
those which direct expression of the nucleotide sequence only in
certain host cells (e.g., tissue-specific regulatory sequences). It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by polynucleotides
as described herein.
[0192] The recombinant expression vectors of the invention can be
designed for expression of a polypeptide of the invention in
prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells
(using baculovirus expression vectors), yeast cells or mammalian
cells). Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology, Academic Press, San
Diego, Calif. (1990) p. 185. Alternatively, the recombinant
expression vector can be transcribed and translated in vitro, for
example using T7 promoter regulatory sequences and T7
polymerase.
[0193] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors comprising constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve at least three
purposes: 1) to increase expression of recombinant protein; 2) to
increase the solubility of the recombinant protein; and/or 3) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase ("GST"),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0194] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988, "Tightly regulated tac
promoter vectors useful for the expression of unfused and fused
proteins in Escherichiacoli", Gene 69:301-315) and pET 11d(Studier
et al., Gene Expression Technology: Methods in Enzymology, Academic
Press, San Diego, Calif. (1990) pp. 60-89). Target polynucleotide
expression from the pTrc vector relies on host RNA polymerase
transcription from a hybrid trp-lac fusion promoter. Target
polynucleotide expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lamda. prophage harboring a T7 gnl polynucleotide under the
transcriptional control of the lacUV 5 promoter.
[0195] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, Gene Expression Technology: Methods in Enzymology,
Academic Press, San Diego, Calif. (1990) pp. 119-128). Another
strategy is to alter the sequence of the polynucleotide to be
inserted into an expression vector so that the individual codons
for each amino acid are those preferentially utilized in E. coli
(Wada et al., 1992, "Codon usage tabulated from the GenBank
polynucleotidetic sequence data", Nucleic Acids Res. 20
Suppl:2111-2118). Such alteration of polynucleotides of the
invention can be carried out by standard DNA synthesis
techniques.
[0196] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al., 1987, "A novel leader
peptide which allows efficient secretion of a fragment of human
interleukin 1 beta in Saccharomyces cerevisiae", EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, 1982, "Structure of a
yeast pheromone polynucleotide (MF alpha): a putative alpha-factor
precursor contains four tandem copies of mature alpha-factor", Cell
30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2
(Invitrogen Corp., San Diego, Calif.), and pPicZ (Invitrogen Corp.,
San Diego, Calif.).
[0197] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf9 cells) include the pAc
series (Smith et al., 1983, "Production of human beta interferon in
insect cells infected with a baculovirus expression vector", Mol
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers,
1989, "High level expression of nonfused foreign polynucleotides
with Autographa californica nuclear polyhedrosis virus expression
vectors" Virology 170:31-39).
[0198] In yet another embodiment, a BGS-19 polynucleotide of the
invention is expressed in mammalian cells using a mammalian
expression vector. Examples of mammalian expression vectors include
pCDM8 (Seed, 1987, "An LFA-3 cDNA encodes a phospholipid-linked
membrane protein homologous to its receptor CD2", Nature 329:840)
and pMT2PC (Kaufman et al., 1987, "Translational efficiency of
polycistronic mRNAs and their utilization to express heterologous
polynucleotides in mammalian cells", EMBO J. 6:187-195). When used
in mammalian cells, the expression vector's control functions are
often provided by viral regulatory elements. For example, commonly
used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression
systems for both prokaryotic and eukaryotic cells see chapters 16
and 17 of Sambrook et al., Molecular Cloning: A Laboratorv Manual,
2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0199] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the BGS-19
polynucleotide preferentially in a particular cell type (e.g.,
using tissue-specific regulatory elements to express the
polynucleotide). Tissue-specific regulatory elements are known in
the art. For example, the regulatory elements of imprinted
polynucleotides can be used in vector constructs with BGS-19
polynucleotides. Non-limiting examples of suitable tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert et
al., 1987, "An albumin enhancer located 10 kb upstream functions
along with its promoter to direct efficient, liver-specific
expression in transgenic mice", Genes Dev. 1:268-277),
lymphoid-specific promoters (Calame and Eaton, 1988,
"Transcriptional controlling elements in the immunoglobulin and T
cell receptor loci", Adv. Immunol. 43:235-275), in particular
promoters of T cell receptors (Winoto and Baltimore, 1989, "A
novel, inducible and T cell-specific enhancer located at the 3' end
of the T cell receptor alpha locus", EMBO J. 8:729-733) and
immunoglobulins (Banerji et al., 1983, "A lymphocyte-specific
cellular enhancer is located downstream of the joining region in
immunoglobulin heavy chain polynucleotides", Cell 33:729-740; Queen
and Baltimore, 1983, "Immunoglobulin polynucleotide transcription
is activated by downstream sequence elements", Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle, 1989, "Multiplex polynucleotide regulation: a
two-tiered approach to transpolynucleotide regulation in transgenic
mice", Proc Natl Acad. Sci. 86:5473-5477), pancreas-specific
promoters (Edlund et al., 1985, "Cell-specific expression of the
rat insulin polynucleotide: evidence for role of two distinct 5'
flanking elements", Science 230:912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss, 1990, Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman, 1989, Genes Dev. 3:537-546).
[0200] The invention further provides a recombinant expression
vector comprising a BGS-19 polynucleotide of the invention cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operably linked to a regulatory sequence in a
manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to the mRNA
encoding a polypeptide of the invention. Regulatory sequences
operably linked to a polynucleotide cloned in the antisense
orientation can be chosen which direct the continuous expression of
the antisense RNA molecule in a variety of cell types, for instance
viral promoters and/or enhancers, or regulatory sequences can be
chosen which direct constitutive, tissue specific or cell type
specific expression of antisense RNA. The antisense expression
vector can be in the form of a recombinant plasmid, phagemid or
attenuated virus in which antisense polynucleotides are produced
under the control of a high efficiency regulatory region, the
activity of which can be determined by the cell type into which the
vector is introduced.
[0201] In another embodiment, the expression characteristics of an
endogenous BGS-19 polynucleotide within a cell, cell line or
microorganism may be modified by inserting a DNA regulatory element
heterologous to the endogenous polynucleotide of interest into the
genome of a cell, stable cell line or cloned microorganism such
that the inserted regulatory element is operatively linked with an
endogenous BGS-19 polynucleotide and controls, modulates or
activates the endogenous polynucleotide. For example, endogenous
polynucleotides of the invention which are normally
"transcriptionally silent", i.e., polynucleotides which are
normally not expressed, or are expressed only at very low levels in
a cell line or microorganism, may be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed polynucleotide product in that cell line or
microorganism. Alternatively, transcriptionally silent, endogenous
polynucleotides of the invention may be activated by insertion of a
promiscuous regulatory element that works across cell types.
[0202] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with and activates expression of an endogenous
BGS-19 polynucleotide, using techniques, such as targeted
homologous recombination, which are well known to those of skill in
the art (See, e.g., U.S. Pat. Nos. 5,272,071 and 5,968,502;
International Publication Nos. WO 91/06667 and WO 94/12650).
Alternatively, non-targeted techniques (e.g., non-homologous
recombination) well known in the art can be used (see, e.g.,
International Publication No. WO 99/15650).
[0203] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding polynucleotiderations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are nevertheless within the scope
of the term as used herein.
[0204] Accordingly, the present invention provides a host cell
having an expression vector comprising a BGS-19 polynucleotide or a
polynucleotide encoding a BGS-19 polypeptide, BGS-19 agonist,
BGS-19 antagonist, inhibitor of a BGS-19 agonist, inhibitor of a
BGS-19 antagonist, or a variant thereof. A host cell can be any
prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells,
yeast or mammalian cells).
[0205] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0206] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a polynucleotide that encodes a selectable marker
(e.g., for resistance to antibiotics) is polynucleotiderally
introduced into the host cells along with the polynucleotide of
interest. Preferred selectable markers include those which confer
resistance to drugs, such as G418, hygromycin and methotrexate.
Cells stably transfected with the introduced polynucleotide can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker polynucleotide will survive, while the other
cells die).
[0207] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce a BGS-19
polypeptide of the invention. Accordingly, the invention further
provides methods for producing a BGS-19 polypeptide of the
invention using the host cells of the invention. In one embodiment,
the method comprises culturing the host cell of invention (into
which a recombinant expression vector encoding a BGS-19 polypeptide
of the invention has been introduced) in a suitable medium such
that the BGS-19 polypeptide is produced. In another embodiment, the
method further comprises isolating the BGS-19 polypeptide from the
medium or the host cell.
[0208] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which a sequence encoding a BGS-19 polypeptide of the
invention has been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous sequences
encoding a polypeptide of the invention have been introduced into
their genome or homologous recombinant animals in which endogenous
sequences encoding a BGS-19 polypeptide of the invention sequences
have been altered. Such animals are useful for studying the
function and/or activity of the polypeptide and for identifying
and/or evaluating modulators of polypeptide activity. In addition
to particular polynucleotide expression and/or polypeptide
expression phenotypes, the transgenic animals of the invention can
exhibit any of the phenotypes (e.g., processes, disorder symptoms
and/or disorders), as are described in the sections above. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a
transpolynucleotide. Other examples of transgenic animals include
non-human primates, sheep, dogs, cows, goats, chickens, amphibians,
etc. A transpolynucleotide is exogenous DNA which is integrated
into the genome of a cell from which a transgenic animal develops
and which remains in the genome of the mature animal, thereby
directing the expression of an encoded polynucleotide product in
one or more cell types or tissues of the transgenic animal. As used
herein, an "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous polynucleotide has been altered by homologous
recombination between the endogenous polynucleotide and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0209] A transgenic animal of the invention can be created by
introducing a polynucleotide encoding a BGS-19 polypeptide of the
invention (or a homolog thereof) into the male pronuclei of a
fertilized oocyte, e.g., by microinjection, retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. Intronic sequences and polyadenylation signals can
also be included in the transpolynucleotide to increase the
efficiency of expression of the transpolynucleotide. A
tissue-specific regulatory sequence(s) can be operably linked to
the transpolynucleotide to direct expression of the polypeptide of
the invention to particular cells. Methods for polynucleotiderating
transgenic animals via embryo manipulation and microinjection,
particularly animals such as mice, have become conventional in the
art (see, e.g., U.S. Pat. Nos. 4,736,866; 4,870,009; 4,873,191;
Hogan, 1986, Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Wakayama et al., 1999,
"Mice cloned from embryonic stem cells", Proc Natl Acad. Sci.
96:14984-14989). Similar methods are used for production of other
transgenic animals. A transgenic founder animal can be identified
based upon the presence of the transpolynucleotide in its genome
and/or expression of mRNA encoding the transpolynucleotide in
tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the
transpolynucleotide. Moreover, transgenic animals carrying the
transpolynucleotide can further be bred to other transgenic animals
carrying other transpolynucleotides.
[0210] To create an homologous recombinant animal, a vector is
prepared which comprises at least a portion of a polynucleotide
encoding a polypeptide of the invention into which a deletion,
addition or substitution has been introduced to thereby alter,
e.g., functionally disrupt, the polynucleotide. In a preferred
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous polynucleotide is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector). Alternatively, the vector can
be designed such that, upon homologous recombination, the
endogenous polynucleotide is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to thereby alter the expression of the endogenous
protein). In the homologous recombination vector, the altered
portion of the polynucleotide is flanked at its 5' and 3' ends by
additional nucleic acid sequences of the polynucleotide to allow
for homologous recombination to occur between the exogenous
polynucleotide carried by the vector and an endogenous
polynucleotide in an embryonic stem cell. The additional flanking
nucleic acid sequences are of sufficient length for successful
homologous recombination with the endogenous polynucleotide.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the vector (See, e.g., Thomas and Capecchi,
1987, Cell. 51:503 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 polynucleotide
has been introduced by homologous recombination are selected (See,
e.g., L1 et al., 1992, Cell 69:915). The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, 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. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transpolynucleotide. Methods for constructing
homologous recombination vectors and homologous recombinant animals
are described further in Bradley, 1991, Current Opinion in
Biotechnology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO
91/01140, WO 92/0968 and WO 93/04169.
[0211] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transpolynucleotide. One example of such a system
is the cre/loxP recombinase system of bacteriophage P1 (see, e.g.,
Lakso et al., 1992, Proc Natl Acad. Sci. 89:6232-6236). Another
example of a recombinase system is the FLP recombinase system of
Saccharomyces cerevisiae (O'Gorman et al., 1991, Science
251:1351-1355). If a cre/loxP recombinase system is used to
regulate expression of the transpolynucleotide, animals comprising
transpolynucleotides encoding both the Cre recombinase and a
selected protein are required. Such animals can be provided through
the construction of "double" transgenic animals, e.g., by mating
two transgenic animals, one comprising a transpolynucleotide
encoding a selected protein and the other comprising a
transpolynucleotide encoding a recombinase.
[0212] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al., 1997, Nature 385:810-813 and PCT Publication Nos. WO
97/07668 and WO 97/07669.
[0213] Agonists and Antagonists of BGS-19 Polynucleotides and
Polypeptides
[0214] The present invention relates to agonists or antagonists of
a BGS-19 polynucleotide, BGS-19 polypeptide, or agonists or
antagonists of complexes comprising a BGS-19 polynucleotide and/or
BGS-19 polypeptide.
[0215] For example, variants of the polypeptides of the invention
have an altered amino acid sequence that can function as agonists
(e.g., mimetics) or as antagonists. An agonist can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of the protein. An antagonist of a
protein can inhibit one or more of the activities of the naturally
occurring form of the protein by, for example, competitively
binding to a downstream or upstream member of a cellular signaling
cascade which includes the protein of interest. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant having a
subset of the biological activities of the naturally occurring form
of the protein can have fewer side effects in a subject relative to
treatment with the naturally occurring form of the protein.
Variants of a protein of the invention which function as either
agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the protein of the invention for agonist or antagonist
activity. In one embodiment, a library of variants is
polynucleotiderated by combinatorial mutapolynucleotidesis at the
nucleic acid level. Such a library of variants can be produced by,
for example, enzymatically ligating a mixture of synthetic
oligonucleotides into polynucleotide sequences such that a
depolynucleotiderate set of potential protein sequences is
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g., for phage display). There are a
variety of methods which can be used to produce libraries of
potential variants of the polypeptides of the invention from a
depolynucleotiderate oligonucleotide sequence. Methods for
synthesizing depolynucleotiderate oligonucleotides are known in the
art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al.,
1984, Annu Rev Biochem. 53:323; Itakura et al., 1984, Science
198:1056; Ike et al., 1983, Nucleic Acid Res. 11:477).
[0216] In addition, libraries of fragments of the coding sequence
of a polypeptide of the invention can be used to polynucleotiderate
a variegated population of polypeptides for screening and
subsequent selection of variants. For example, a library of coding
sequence fragments can be polynucleotiderated by treating a double
stranded PCR fragment of the coding sequence of interest with a
nuclease under conditions wherein nicking occurs only about once
per molecule, denaturing the double stranded DNA, renaturing the
DNA to form double stranded DNA which can include sense/antisense
pairs from different nicked products, removing single stranded
portions from reformed duplexes by treatment with S1 nuclease, and
ligating the resulting fragment library into an expression vector.
By this method, an expression library can be derived which encodes
N-terminal and internal fragments of various sizes of the protein
of interest.
[0217] Several techniques are known in the art for screening
polynucleotide products of combinatorial libraries made by point
mutations or truncation, and for screening cDNA libraries for
polynucleotide products having a selected property. The most widely
used techniques, which are amenable to high through-put analysis,
for screening large polynucleotide libraries typically include
cloning the polynucleotide library into replicable expression
vectors, transforming appropriate cells with the resulting library
of vectors, and expressing the combinatorial polynucleotides under
conditions in which detection of a desired activity facilitates
isolation of the vector encoding the polynucleotide whose product
was detected.
[0218] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used to prepare individuals
for extraterrestrial travel, low gravity environments, prolonged
exposure to extraterrestrial radiation levels, low oxygen levels,
reduction of metabolic activity, exposure to extraterrestrial
pathogens, etc. Such a use may be administered either prior to an
extraterrestrial event, during an extraterrestrial event, or both.
Moreover, such a use may result in a number of beneficial changes
in the recipient, such as, for example, any one of the following,
non-limiting, effects: an increased level of hematopoietic cells,
particularly red blood cells which would aid the recipient in
coping with low oxygen levels; an increased level of B-cells,
T-cells, antigen presenting cells, and/or macrophages, which would
aid the recipient in coping with exposure to extraterrestrial
pathogens, for example; a temporary (i.e., reversible) inhibition
of hematopoietic cell production which would aid the recipient in
coping with exposure to extraterrestrial radiation levels; increase
and/or stability of bone mass which would aid the recipient in
coping with low gravity environments; and/or decreased metabolism
which would effectively facilitate the recipients ability to
prolong their extraterrestrial travel by any one of the following,
non-limiting means: (i) aid the recipient by decreasing their basal
daily energy requirements; (ii) effectively lower the level of
oxidative and/or metabolic stress in recipient (i.e., to enable
recipient to cope with increased extraterrestial radiation levels
by decreasing the level of internal oxidative/metabolic damage
acquired during normal basal energy requirements; and/or
[0219] (iii) enabling recipient to subsist at a lower metabolic
temperature (i.e., cryogenic, and/or sub-cryogenic
environment).
[0220] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used to increase the efficacy
of a pharmaceutical composition, either directly or indirectly.
Such a use may be administered in simultaneous conjunction with
said pharmaceutical, or separately through either the same or
different route of administration (e.g., intravenous for the
polynucleotide or polypeptide of the present invention, and orally
for the pharmaceutical, among others described herein.).
BGS-19 Antisense Oligonucleotides
[0221] The present invention relates to antagonists in the form of
antisense polynucleotides, i.e., molecules which are complementary
to a sense nucleic acid encoding a polypeptide of the invention,
e.g., complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. BGS-19 antisense
molecules are fully described in Section 5.2 supra.
BGS-19 Antibodies
[0222] The present invention also relates to agonists or
antagonists in the form of anti-BGS-19 antibodies. An isolated
polypeptide of the invention, or a fragment thereof, can be used as
an immunogen to polynucleotiderate antibodies using standard
techniques for polyclonal and monoclonal antibody preparation. The
full-length polypeptide or protein can be used or, alternatively,
the invention provides antigenic peptide fragments for use as
immunogens. The antigenic peptide of a protein of the invention
comprises at least 8 (preferably 10, 15, 20, or 30) consecutive
amino acid residues of the amino acid sequence depicted in FIGS.
2A-B or FIGS. 1A-C, and encompasses an epitope of the protein such
that an antibody raised against the peptide forms a specific immune
complex with the protein.
[0223] In one embodiment, the invention provides substantially
purified antibodies or fragments thereof, including human or
non-human antibodies or fragments thereof, which antibodies or
fragments specifically bind to a polypeptide of the invention
comprising an amino acid sequence selected from the group
consisting of: the amino acid sequence depicted in FIGS. 2A-B or
FIGS. 1A-C; a fragment of at least 8 contiguous amino acid residues
of the amino acid sequence depicted in FIGS. 2A-B or FIGS. 1A-C; an
amino acid sequence which is at least 95% identical to the amino
acid sequence presented in depicted in FIGS. 2A-B or FIGS. 1A-C,
wherein the percent identity is determined using the ALIGN program
of the GCG software package with a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4, and an amino acid
sequence which is encoded by a polynucleotide which hybridizes to
the polynucleotide consisting of the sequence depicted in FIGS.
2A-B or 3 under conditions of hybridization of 6.times.SSC at
45.degree. C. and washing in 0.2.times.SSC, 0.1% SDS at 65.degree.
C. In various embodiments, the substantially purified antibodies of
the invention, or fragments thereof, can be human, non-human,
chimeric and/or humanized antibodies.
[0224] Preferred epitopes encompassed by the antigenic peptide are
regions that are located on the surface of the protein, e.g.,
hydrophilic regions. Hydropathy plots of the polypeptides of the
invention, or similar analyses, can be used to identify hydrophilic
regions. In certain embodiments, BGS-19 polynucleotides are present
as part of larger polynucleotides comprising nucleotide sequences
that encode heterologous sequences (e.g., vector, expression
vector, or fusion protein). These nucleotides can then be used to
express proteins which can be used as immunogens to
polynucleotiderate an immune response, or more particularly, to
polynucleotiderate polyclonal or monoclonal antibodies specific to
the expressed protein.
[0225] An immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other
mammal). An appropriate immunogenic preparation can comprise, for
example, recombinantly expressed or chemically synthesized
polypeptide. The preparation can further include an adjuvant, such
as Freud's complete or incomplete adjuvant, or similar
immunostimulatory agent.
[0226] Accordingly, another aspect of the invention pertains to
antibodies directed against a polypeptide of the invention. The
term "antibody" as used herein refers to immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules,
i.e., molecules that comprise an antigen binding site which
specifically binds an antigen, such as a polypeptide of the
invention, e.g., an epitope of a polypeptide of the invention. A
molecule which specifically binds to a given polypeptide of the
invention is a molecule which binds the polypeptide, but does not
substantially bind other molecules in a sample, e.g., a biological
sample, which naturally comprises the polypeptide. Examples of
immunologically active portions of immunoglobulin molecules include
F(ab) and F(ab').sub.2 fragments which can be polynucleotiderated
by treating the antibody with an enzyme such as pepsin. The
invention provides polyclonal and monoclonal antibodies. The term
"monoclonal antibody" or "monoclonal antibody composition", as used
herein, refers to a population of antibody molecules that comprise
only one species of an antigen binding site capable of
immunoreacting with a particular epitope.
[0227] Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a BGS-19 polypeptide of the
invention as an immunogen. Preferred polyclonal antibody
compositions are ones that have been selected for antibodies
directed against a polypeptide or polypeptides of the invention.
Particularly preferred polyclonal antibody preparations are ones
that comprise only antibodies directed against a polypeptide or
polypeptides of the invention. Particularly preferred immunogen
compositions are those that comprise no other human proteins such
as, for example, immunogen compositions made using a non-human host
cell for recombinant expression of a polypeptide of the invention.
In such a manner, the only human epitope or epitopes recognized by
the resulting antibody compositions raised against this immunogen
will be present as part of a polypeptide or polypeptides of the
invention.
[0228] The antibody titer in the immunized subject can be monitored
over time by standard techniques, such as with an enzyme linked
immunosorbent assay ("ELISA") using immobilized polypeptide. If
desired, the antibody molecules can be isolated from the mammal
(e.g., from the blood) and further purified by well-known
techniques, such as protein A chromatography to obtain the IgG
fraction. Alternatively, antibodies specific for a BGS-19 protein
or polypeptide of the invention can be selected for (e.g.,
partially purified) or purified by, e.g., affinity chromatography.
For example, a recombinantly expressed and purified (or partially
purified) protein of the invention is produced as described herein,
and covalently or non-covalently coupled to a solid support such
as, for example, a chromatography column. The column can then be
used to affinity purify antibodies specific for the proteins of the
invention from a sample comprising antibodies directed against a
large number of different epitopes, thereby polynucleotiderating a
substantially purified antibody composition, i.e., one that is
substantially free of contaminating antibodies. By a substantially
purified antibody composition is meant, in this context, that the
antibody sample comprises at most only 30% (by dry weight) of
contaminating antibodies directed against epitopes other than those
on the desired protein or polypeptide of the invention, and
preferably at most 20%, yet more preferably at most 10%, and most
preferably at most 5% (by dry weight) of the sample is
contaminating antibodies. A purified antibody composition means
that at least 99% of the antibodies in the composition are directed
against the desired protein or polypeptide of the invention.
[0229] At an appropriate time after immunization, e.g., when the
specific antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein, 1975, Nature
256:495-497, the human B cell hybridoma technique (Kozbor et al.,
1983, Immunol Today 4:72), the EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
hybridomas is well known (See, e.g., Current Protocols in
Immunology, 1994, Coligan et al. (eds.) John Wiley & Sons,
Inc., New York, N.Y.). Hybridoma cells producing a monoclonal
antibody of the invention are detected by screening the hybridoma
culture supernatants for antibodies that bind the polypeptide of
interest, e.g., using a standard ELISA assay.
[0230] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
polynucleotiderating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the
Stratapolynucleotide SurfZAP Phage Display Kit, Catalog No.
240612). Additionally, examples of methods and reagents
particularly amenable for use in polynucleotiderating and screening
an antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al., 1991, Biotechnology 9:1370-1372; Hay et
al., 1992, Hum Antibod Hybridomas 3:81-85; Huse et al., 1989,
Science 246:1275-1281; Griffiths et al., 1993, EMBO J.
12:725-734.
[0231] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine mAb and a human immunoglobulin constant
region (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and
Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein
by reference in their entirety). Humanized antibodies are antibody
molecules from non-human species having one or more complementarity
determining regions ("CDRs") from the non-human species and a
framework region from a human immunoglobulin molecule. (See, e.g.,
Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by
reference in its entirety). Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, (see, e.g., PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al., 1988, Science 240:1041-1043; Liu et al.,
1987, Proc Natl Acad. Sci. 84:3439-3443; Liu et al., 1987, J.
Immunol. 139:3521-3526; Sun et al., 1987, Proc Natl Acad. Sci.
84:214-218; Nishimura et al., 1987, Cancer Res. 47:999-1005; Wood
et al., 1985, Nature 314:446-449; Shaw et al., 1988, J Natl Cancer
Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et
al., 1986, Biotechniques 4:214; U.S. Pat. No. 5,225,539; Jones et
al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science
239:1534; Beidler et al., 1988, J. Immunol. 141:4053-4060).
[0232] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced, for example, using transgenic mice which are incapable of
expressing endogenous immunoglobulin heavy and light chains
polynucleotides, but which can express human heavy and light chain
polynucleotides. The transgenic mice are immunized in the normal
fashion with a selected antigen, e.g., all or a portion of a
polypeptide of the invention. Monoclonal antibodies directed
against the antigen can be obtained using conventional hybridoma
technology. The human immunoglobulin transpolynucleotides harbored
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
IgG, IgA and IgE antibodies can be produced using techniques well
known in the art, (see, e.g., Lonberg and Huszar, 1995, Int. Rev.
Immunol. 13:65-93; U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825;
5,661,016; and 5,545,806. In addition, companies such as Abgenix,
Inc. (Fremont, Calif.), can be engaged to provide human antibodies
directed against a selected antigen using technology similar to
that described above.
[0233] Completely human antibodies which recognize a selected
epitope can be polynucleotiderated using a technique referred to as
"guided selection." In this approach a selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a completely human antibody recognizing the same
epitope (see, e.g., Jespers et al., 1994, Biotechnology
12:899-903).
[0234] An antibody directed against a BGS-19 polypeptide of the
invention (e.g., monoclonal antibody) can be used to isolate the
polypeptide by standard techniques, such as affinity chromatography
or immunoprecipitation. Moreover, such an antibody can be used to
detect the protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
polypeptide. The antibodies can also be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0235] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0236] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, a
thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or
endostatin; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-4 ("IL-4"), interleukin-6 ("IL-6"), interleukin-7
("IL-7"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
interleukin-10 ("IL-10"), interleukin-12 ("IL-12"), interleukin-15
("IL-15"), interferon-.gamma. ("IFN-.gamma."), interferon-a
("IFN-.alpha."), or other immune factors or growth factors.
[0237] Techniques for conjugating such therapeutic moiety to
antibodies are well known (see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
Alan R. Liss, Inc. (1985) pp. 243-256; Hellstrom et al.,
"Antibodies For Drug Delivery" in Controlled Drug Delivery(2nd
Ed.), Robinson et al. (eds.) Marcel Dekker, Inc. (1987) pp.
623-653; Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.) (1985) pp. 475-506;
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
Academic Press (1985) pp. 303-316; Thorpe et al., 1992, "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol Rev. 62:119-158).
[0238] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described in U.S.
Pat. No. 4,676,980.
[0239] An antibody with or without a therapeutic moiety conjugated
to it can be used as a therapeutic that is administered alone or in
combination with chemotherapeutic agents.
[0240] Alternatively, an antibody of the invention can be
conjugated to a second antibody to form an "antibody
heteroconjugate" as described in U.S. Pat. No. 4,676,980 or
alternatively, two antibodies can be conjugated to each other to
create a bispecific heteromers, or an "antibody heteropolymer" as
described in U.S. Pat. Nos. 5,470,570 and 5,487,890.
[0241] An antibody with or without a therapeutic moiety conjugated
to it can be used as a therapeutic that is administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s).
Functional Analysis of BGS-19
Human Expression Pattern of BGS-19
[0242] The expression profile of the BGS-19 polypeptide was
assessed by measuring the steady state levels of BGS-19 mRNA by
quantitative PCR. First strand cDNA was made from commercially
available mRNA (Clontech) and subjected to real time quantitative
PCR using a PE 5700 instrument (Applied Biosystems, Foster City,
Calif.) which detects the amount of DNA amplified during each cycle
by the fluorescent output of SYBR green, a DNA binding dye specific
for double strands. The BGS-19 polypeptide showed predominately
high expression levels in spleen; significantly in lung, spinal
cord, and to a lesser extent, in other tissues as shown in FIG.
6.
[0243] Based upon the observed homology, the polypeptide of the
present invention may share at least some biological activity with
immunoglobulin-like domain containing proteins, specifically with
human siglec proteins, more specifically with human siglec proteins
referenced elsewhere herein.
[0244] Expanded analysis of BGS-19 expression levels by TaqMan.TM.
quantitative PCR (See FIG. 7) extended the expression profile
achieved previously (FIG. 6). BGS-19 mRNA was expressed
predominately in the ovary (9000 fold over other tissues).
Significant expression was observed in the testis, adrenal gland,
the parenchyma of the spleen and throughout the stomach. BGS-19 was
expressed in non-ovarian tissues at levels that were approximately
nine fold less than that observed in the ovary.
[0245] Morever, an additional analysis of BGS-19 expression levels
by TaqMan.TM. quantitative PCR (see FIG. 8) in disease cells and
tissues indicated that the BGS-19 polypeptide is differentially
expressed in ovarian tumor tissues relative to normal ovarian
tissue. In the ovarian cancer results, an average of 5 samples
showed a 25-fold reduction in BGS-19 steady state RNA over that
observed in 5 normal samples (P=0.0145). These data suggest that
BGS-19 can be used as a diagnostic marker of various ovarian
cancers, and that replacing BGS-19 function may offer a novel
therapeutic approach to the treatment of ovarian cancer.
[0246] The strong homology to human immunoglobulin-like domain
containing proteins, combined with the predominate localized
expression in spleen tissue suggests the BGS-19 polynucleotides and
polypeptides, including modulators thereof, may be useful in
treating, diagnosing, prognosing, and/or preventing ovarian
diseases and/or disorders. Such disorders include the following,
non-limiting, diseases or disorders of the ovary, in addition to
disorders related to ovarian disorders: ovarian cancer;
dysfunctional uterine bleeding; amenorrhea; primary dysmenorrhea;
sexual dysfunction; infertility; pelvic inflammatory disease;
endometriosis; placental aromatase deficiency; premature menopause;
placental dysfunction; hormone deficiency; estrogen deficiency;
aberrant androgen metabolism; gaberrant onset of female puberty;
aberrant showing of female primary sexual characteristics; aberrant
showing of female secondary sexual characteristics; precocious
puberty; precocious pseudopuberty; incomplete isosexual precocity;
premature thelarche; premature adrenarche; premature pubarche;
polycystic ovarian disease; aberrant ovarian cycle; menorrhagia;
metrorrhagia; menometrorrhagia; dysmenorrhea; hypomenorrhea;
polymenorrhea; dysfunctional uterine bleeding; resistant-ovary
syndrome; and/or hermaphroditism.
[0247] The strong homology to human immunoglobulin-like domain
containing proteins, combined with the predominate localized
expression in spleen tissue suggests the BGS-19 polynucleotides and
polypeptides, including modulators thereof, may be useful in
treating, diagnosing, prognosing, and/or preventing immune diseases
and/or disorders. Representative uses are described elsewhere
herein. Briefly, the strong expression in immune tissue indicates a
role in regulating the proliferation; survival; differentiation;
and/or activation of hematopoietic cell lineages, including blood
stem cells.
[0248] The BGS-19 polypeptide may also be useful as a preventative
agent for immunological disorders including arthritis, asthma,
immunodeficiency diseases such as AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease,
sepsis, acne, neutropenia, neutrophilia, psoriasis,
hypersensitivities, such as T-cell mediated cytotoxicity; immune
reactions to transplanted organs and tissues, such as
host-versus-graft and graft-versus-host diseases, or autoimmunity
disorders, such as autoimmune infertility, lense tissue injury,
demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
The BGS-19 polypeptide may be useful for modulating cytokine
production, antigen presentation, or other processes, such as for
boosting immune responses, etc.
[0249] Moreover, the protein may represent a secreted factor that
influences the differentiation or behavior of other blood cells, or
that recruits hematopoietic cells to sites of injury. Thus, this
polynucleotide product is thought to be useful in the expansion of
stem cells and committed progenitors of various blood lineages, and
in the differentiation and/or proliferation of various cell types.
Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissuemarkers, to isolate cognate
ligands or receptors, to identify agents that modulate their
interactions, in addition to its use as a nutritional supplement.
Protein, as well as, antibodies directed against the protein may
show utility as a tumor marker and/or immunotherapy targets for the
above listed tissues.
[0250] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of the immune system, by activating or inhibiting the
proliferation, differentiation, or mobilization (chemotaxis) of
immune cells. Immune cells develop through a process called
hematopoiesis, producing myeloid (platelets, red blood cells,
neutrophils, and macrophages) and lymphoid (B and T lymphocytes)
cells from pluripotent stem cells. The etiology of these immune
diseases, disorders, and/or conditions may be polynucleotidetic,
somatic, such as cancer or some autoimmune diseases, disorders,
and/or conditions, acquired (e.g., by chemotherapy or toxins), or
infectious. Moreover, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention can be used as a
marker or detector of a particular immune system disease or
disorder.
[0251] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of hematopoietic cells. A polynucleotides or
polypeptides, or agonists or antagonists of the present invention
could be used to increase differentiation and proliferation of
hematopoietic cells, including the pluripotent stem cells, in an
effort to treat or prevent those diseases, disorders, and/or
conditions associated with a decrease in certain (or many) types
hematopoietic cells. Examples of immunologic deficiency syndromes
include, but are not limited to: blood protein diseases, disorders,
and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),
ataxia telangiectasia, common variable immunodeficiency, Digeorge
Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal
dysfunction, severe combined immunodeficiency (SCIDs),
Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
[0252] Moreover, a polynucleotides or polypeptides, or agonists or
antagonists of the present invention could also be used to modulate
hemostatic (the stopping of bleeding) or thrombolytic activity
(clot formation). For example, by increasing hemostatic or
thrombolytic activity, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention could be used to
treat or prevent blood coagulation diseases, disorders, and/or
conditions (e.g., afibrinopolynucleotidemia, factor deficiencies),
blood platelet diseases, disorders, and/or conditions (e.g.
thrombocytopenia), or wounds resulting from trauma, surgery, or
other causes. Alternatively, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention that can decrease
hemostatic or thrombolytic activity could be used to inhibit or
dissolve clotting. These molecules could be important in the
treatment or prevention of heart attacks (infarction), strokes, or
scarring.
[0253] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be useful in
treating, preventing, and/or diagnosing autoimmune diseases,
disorders, and/or conditions. Many autoimmune diseases, disorders,
and/or conditions result from inappropriate recognition of self as
foreign material by immune cells. This inappropriate recognition
results in an immune response leading to the destruction of the
host tissue. Therefore, the administration of a polynucleotides or
polypeptides, or agonists or antagonists of the present invention
that inhibits an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing autoimmune diseases, disorders, and/or
conditions.
[0254] Examples of autoimmune diseases, disorders, and/or
conditions that can be treated, prevented, and/or diagnosed or
detected by the present invention include, but are not limited to:
Addison's Disease, hemolytic anemia, antiphospholipid syndrome,
rheumatoid arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,
Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura,
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,
Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye disease.
[0255] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, and/or diagnosed by polynucleotides or
polypeptides, or agonists or antagonists of the present invention.
Moreover, these molecules can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0256] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to treat,
prevent, and/or diagnose organ rejection or graft-versus-host
disease (GVHD). Organ rejection occurs by host immune cell
destruction of the transplanted tissue through an immune response.
Similarly, an immune response is also involved in GVHD, but, in
this case, the foreign transplanted immune cells destroy the host
tissues. The administration of a polynucleotides or polypeptides,
or agonists or antagonists of the present invention that inhibits
an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD.
[0257] Similarly, a polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to modulate
inflammation. For example, the polypeptide or polynucleotide or
agonists or antagonist may inhibit the proliferation and
differentiation of cells involved in an inflammatory response.
These molecules can be used to treat, prevent, and/or diagnose
inflammatory conditions, both chronic and acute conditions,
including chronic prostatitis, granulomatous prostatitis and
malacoplakia, inflammation associated with infection (e.g., septic
shock, sepsis, or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine induced lung injury, inflammatory bowel disease, Crohn's
disease, or resulting from over production of cytokines (e.g., TNF
or IL-1.)
[0258] A polypeptide or polynucleotide and/or agonist or antagonist
of the present invention can be used to treat, prevent, and/or
diagnose infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be
treated, prevented, and/or diagnosed. The immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention may also directly inhibit the infectious agent, without
necessarily eliciting an immune response.
[0259] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated, prevented, and/or
diagnosed by a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention. Examples of viruses, include,
but are not limited to Examples of viruses, include, but are not
limited to the following DNA and RNA viruses and viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue,
EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B,
and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia polynucleotides or polypeptides, or agonists or
antagonists of the invention, can be used to treat, prevent, and/or
diagnose any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis B). In an additional specific embodiment polynucleotides,
polypeptides, or agonists or antagonists of the invention are used
to treat patients nonresponsive to one or more other commercially
available hepatitis vaccines. In a further specific embodiment
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose AIDS.
[0260] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated, prevented, and/or diagnosed by
a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention include, but not limited to, include, but not
limited to, the following Gram-Negative and Gram-positive bacteria
and bacterial families and fungi: Actinomycetales (e.g.,
Corynebacterium, Mycobacterium, Norcardia), Cryptococcus
neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,
Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella
typhi, and Salmonella paratyphi), Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Meisseria meningitidis, Pasteurellacea Infections (e.g.,
Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B),
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,
Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and
Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the
following diseases or symptoms, including, but not limited to:
bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g.,
AIDS related infections), paronychia, prosthesis-related
infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B),
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. Polynucleotides or polypeptides,
agonists or antagonists of the invention, can be used to treat,
prevent, and/or diagnose any of these symptoms or diseases. In
specific embodiments, polynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: tetanus, Diptheria, botulism, and/or meningitis type
B.
[0261] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
include, but not limited to, the following families or class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium
falciparium, Plasmodium malariae and Plasmodium ovale). These
parasites can cause a variety of diseases or symptoms, including,
but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal disease (e.g., dysentery, giardiasis), liver disease,
lung disease, opportunistic infections (e.g., AIDS related),
malaria, pregnancy complications, and toxoplasmosis.
polynucleotides or polypeptides, or agonists or antagonists of the
invention, can be used totreat, prevent, and/or diagnose any of
these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
[0262] Preferably, treatment or prevention using a polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention could either be by administering an effective amount of a
polypeptide to the patient, or by removing cells from the patient,
supplying the cells with a polynucleotide of the present invention,
and returning the engineered cells to the patient (ex vivo
therapy). Moreover, the polypeptide or polynucleotide of the
present invention can be used as an antigen in a vaccine to raise
an immune response against infectious disease.
[0263] A polypeptide of the present invention may be used to screen
for molecules that bind to the polypeptide or for molecules to
which the polypeptide binds. The binding of the polypeptide and the
molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the polypeptide or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides,
proteins (e.g., receptors), or small molecules.
[0264] Preferably, the molecule is closely related to the natural
ligand of the polypeptide, e.g., a fragment of the ligand, or a
natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2):Chapter
5 (1991).) Similarly, the molecule can be closely related to the
natural receptor to which the polypeptide binds, or at least, a
fragment of the receptor capable of being bound by the polypeptide
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[0265] Preferably, the screening for these molecules involves
producing appropriate cells which express the polypeptide, either
as a secreted protein or on the cell membrane. Preferred cells
include cells from mammals, yeast, Drosophila, or E. coli. Cells
expressing the polypeptide (or cell membrane containing the
expressed polypeptide) are then preferably contacted with a test
compound potentially containing the molecule to observe binding,
stimulation, or inhibition of activity of either the polypeptide or
the molecule.
[0266] The assay may simply test binding of a candidate compound to
the polypeptide, wherein binding is detected by a label, or in an
assay involving competition with a labeled competitor. Further, the
assay may test whether the candidate compound results in a signal
polynucleotiderated by binding to the polypeptide.
[0267] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing a polypeptide, measuring polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule
activity or binding to a standard.
[0268] Preferably, an ELISA assay can measure polypeptide level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure polypeptide level
or activity by either binding, directly or indirectly, to the
polypeptide or by competing with the polypeptide for a
substrate.
[0269] Additionally, the receptor to which a polypeptide of the
invention binds can be identified by numerous methods known to
those of skill in the art, for example, ligand panning and FACS
sorting (Coligan, et al., Current Protocols in Immun., 1(2),
Chapter 5, (1991)). For example, expression cloning is employed
wherein polyadenylated RNA is prepared from a cell responsive to
the polypeptides, for example, NIH3T3 cells which are known to
contain multiple receptors for the FGF family proteins, and SC-3
cells, and a cDNA library created from this RNA is divided into
pools and used to transfect COS cells or other cells that are not
responsive to the polypeptides. Transfected cells which are grown
on glass slides are exposed to the polypeptide of the present
invention, after they have been labeled. The polypeptides can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase.
[0270] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[0271] As an alternative approach for receptor identification, the
labeled polypeptides can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE analysis and exposed to
X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and
subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of
depolynucleotiderate oligonucleotide probes to screen a cDNA
library to identify the polynucleotides encoding the putative
receptors.
[0272] Moreover, the techniques of polynucleotide-shuffling,
motif-shuffling, exon-shuffling, and/or codon-shuffling
(collectively referred to as "DNA shuffling") may be employed to
modulate the activities of polypeptides of the invention thereby
effectively polynucleotiderating agonists and antagonists of
polypeptides of the invention. See polynucleotiderally, U.S. Pat.
Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and
Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O.,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and
Blasco, R. Biotechniques 24(2):308-13 (1998) (each of these patents
and publications are hereby incorporated by reference). In one
embodiment, alteration of polynucleotides and corresponding
polypeptides of the invention may be achieved by DNA shuffling. DNA
shuffling involves the assembly of two or more DNA segments into a
desired polynucleotide sequence of the invention molecule by
homologous, or site-specific, recombination. In another embodiment,
polynucleotides and corresponding polypeptides of the invention may
be altered by being subjected to random mutapolynucleotidesis by
error-prone PCR, random nucleotide insertion or other methods prior
to recombination. In another embodiment, one or more components,
motifs, sections, parts, domains, fragments, etc., of the
polypeptides of the invention may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules. In preferred embodiments, the
heterologous molecules are family members. In further preferred
embodiments, the heterologous molecule is a growth factor such as,
for example, platelet-derived growth factor (PDGF), insulin-like
growth factor (IGF-I), transforming growth factor (TGF)-alpha,
epidermal growth factor (EGF), fibroblast growth factor (FGF),
TGF-beta, bone morphopolynucleotidetic protein (BMP)-2, BMP-4,
BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,
OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,
inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and
glial-derived neurotrophic factor (GDNF).
[0273] Other preferred fragments are biologically active fragments
of the polypeptides of the invention. Biologically active fragments
are those exhibiting activity similar, but not necessarily
identical, to an activity of the polypeptide. The biological
activity of the fragments may include an improved desired activity,
or a decreased undesirable activity.
[0274] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of the
polypeptide of the present invention. An example of such an assay
comprises combining a mammalian fibroblast cell, a the polypeptide
of the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell
would normally proliferate. A control assay may be performed in the
absence of the compound to be screened and compared to the amount
of fibroblast proliferation in the presence of the compound to
determine if the compound stimulates proliferation by determining
the uptake of 3[H] thymidine in each case. The amount of fibroblast
cell proliferation is measured by liquid scintillation
chromatography which measures the incorporation of 3[H] thymidine.
Both agonist and antagonist compounds may be identified by this
procedure.
[0275] In another method, a mammalian cell or membrane preparation
expressing a receptor for a polypeptide of the present invention is
incubated with a labeled polypeptide of the present invention in
the presence of the compound. The ability of the compound to
enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system
following interaction of a compound to be screened and the receptor
is measured and the ability of the compound to bind to the receptor
and elicit a second messenger response is measured to determine if
the compound is a potential agonist or antagonist. Such second
messenger systems include but are not limited to, cAMP guanylate
cyclase, ion channels or phosphoinositide hydrolysis.
[0276] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat, prevent, and/or diagnose disease or to bring
about a particular result in a patient (e.g., blood vessel growth)
by activating or inhibiting the polypeptide/molecule. Moreover, the
assays can discover agents which may inhibit or enhance the
production of the polypeptides of the invention from suitably
manipulated cells or tissues. Therefore, the invention includes a
method of identifying compounds which bind to the polypeptides of
the invention comprising the steps of: (a) incubating a candidate
binding compound with the polypeptide; and (b) determining if
binding has occurred. Moreover, the invention includes a method of
identifying agonists/antagonists comprising the steps of: (a)
incubating a candidate compound with the polypeptide, (b) assaying
a biological activity, and (b) determining if a biological activity
of the polypeptide has been altered.
[0277] Also, one could identify molecules bind a polypeptide of the
invention experimentally by using the beta-pleated sheet regions
contained in the polypeptide sequence of the protein. Accordingly,
specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, the amino acid sequence of each beta
pleated sheet regions in a disclosed polypeptide sequence.
Additional embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, any combination or all of contained in
the polypeptide sequences of the invention. Additional preferred
embodiments of the invention are directed to polypeptides which
comprise, or alternatively consist of, the amino acid sequence of
each of the beta pleated sheet regions in one of the polypeptide
sequences of the invention. Additional embodiments of the invention
are directed to polypeptides which comprise, or alternatively
consist of, any combination or all of the beta pleated sheet
regions in one of the polypeptide sequences of the invention.
[0278] The strong homology to human inmunoglobulin-like domain
containing proteins, combined with the localized expression in lung
tissue also emphasizes the potential utility for BGS-19
polynucleotides and polypeptides in treating, diagnosing,
prognosing, and/or preventing pulmonary diseases and disorders
which include the following, not limiting examples: ARDS,
emphysema, cystic fibrosis, interstitial lung disease, chronic
obstructive pulmonary disease, bronchitis,
lymphangioleiomyomatosis, pneumonitis, eosinophilic pneumonias,
granulomatosis, pulmonary infarction, pulmonary fibrosis,
pneumoconiosis, alveolar hemorrhage, neoplasms, lung abscesses,
empyema, and increased susceptibility to lung infections (e.g.,
immumocompromised, HIV, etc.), for example.
[0279] Moreover, polynucleotides and polypeptides, including
fragments and/or antagonists thereof, have uses which include,
directly or indirectly, treating, preventing, diagnosing, and/or
prognosing the following, non-limiting, pulmonary infections:
pnemonia, bacterial pnemonia, viral pnemonia (for example, as
caused by Influenza virus, Respiratory syncytial virus,
Parainfluenza virus, Adenovirus, Coxsackievirus, Cytomegalovirus,
Herpes simplex virus, Hantavirus, etc.), mycobacteria pnemonia (for
example, as caused by Mycobacterium tuberculosis, etc.) mycoplasma
pnemonia, fungal pnemonia (for example, as caused by Pneumocystis
carinii, Histoplasma capsulatum, Coccidioides immitis, Blastomyces
dermatitidis, Candida sp., Cryptococcus neoformans, Aspergillus
sp., Zygomycetes, etc.), Legionnaires' Disease, Chlamydia pnemonia,
aspiration pnemonia, Nocordia sp. Infections, parasitic pnemonia
(for example, as caused by Strongyloides, Toxoplasma gondii, etc.)
necrotizing pnemonia, in addition to any other pulmonary disease
and/or disorder (e.g., non-pneumonia) implicated by the causative
agents listed above or elsewhere herein.
[0280] The strong homology to human immunoglobulin-like domain
containing proteins, combined with the localized expression in
spinal cord suggests the BGS-19 polynucleotides and polypeptides
may be useful in treating, diagnosing, prognosing, and/or
preventing neurodepolynucleotiderative disease states, behavioral
disorders, or inflammatory conditions. Briefly, the uses include,
but are not limited to the detection, treatment, and/or prevention
of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette Syndrome, meningitis, encephalitis, demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal cord injuries, ischemia and infarction,
aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors,
including disorders in feeding, sleep patterns, balance, and
perception. In addition, elevated expression of this polynucleotide
product in regions of the brain indicates it plays a role in normal
neural function. Potentially, this polynucleotide product is
involved in synapse formation, neurotransmission, learning,
cognition, homeostasis, or neuronal differentiation or survival.
Furthermore, the protein may also be used to determine biological
activity, to raise antibodies, as tissue markers, to isolate
cognate ligands or receptors, to identify agents that modulate
their interactions, in addition to its use as a nutritional
supplement. Protein, as well as, antibodies directed against the
protein may show utility as a tumor marker and/or immunotherapy
targets for the above listed tissues.
[0281] Nervous system diseases, disorders, and/or conditions, which
can be treated, prevented, and/or diagnosed with the compositions
of the invention (e.g., polypeptides, polynucleotides, and/or
agonists or antagonists), include, but are not limited to, nervous
system injuries, and diseases, disorders, and/or conditions which
result in either a disconnection of axons, a diminution or
depolynucleotideration of neurons, or demyelination. Nervous system
lesions which may be treated, prevented, and/or diagnosed in a
patient (including human and non-human mammalian patients)
according to the invention, include but are not limited to, the
following lesions of either the central (including spinal cord,
brain) or peripheral nervous systems: (1) ischemic lesions, in
which a lack of oxygen in a portion of the nervous system results
in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord infarction or ischemia; (2) traumatic
lesions, including lesions caused by physical injury or associated
with surgery, for example, lesions which sever a portion of the
nervous system, or compression injuries; (3) malignant lesions, in
which a portion of the nervous system is destroyed or injured by
malignant tissue which is either a nervous system associated
malignancy or a malignancy derived from non-nervous system tissue;
(4) infectious lesions, in which a portion of the nervous system is
destroyed or injured as a result of infection, for example, by an
abscess or associated with infection by human immunodeficiency
virus, herpes zoster, or herpes simplex virus or with Lyme disease,
tuberculosis, syphilis; (5) depolynucleotiderative lesions, in
which a portion of the nervous system is destroyed or injured as a
result of a depolynucleotiderative process including but not
limited to depolynucleotideration associated with Parkinson's
disease, Alzheimer's disease, Huntington's chorea, or amyotrophic
lateral sclerosis (ALS); (6) lesions associated with nutritional
diseases, disorders, and/or conditions, in which a portion of the
nervous system is destroyed or injured by a nutritional disorder or
disorder of metabolism including but not limited to, vitamin B12
deficiency, folic acid deficiency, Wernicke disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary
depolynucleotideration of the corpus callosum), and alcoholic
cerebellar depolynucleotideration; (7) neurological lesions
associated with systemic diseases including, but not limited to,
diabetes (diabetic neuropathy, Bell's palsy), systemic lupus
erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by
toxic substances including alcohol, lead, or particular
neurotoxins; and (9) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including, but not limited to, multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[0282] In a preferred embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to protect neural cells from the damaging effects of cerebral
hypoxia. According to this embodiment, the compositions of the
invention are used to treat, prevent, and/or diagnose neural cell
injury associated with cerebral hypoxia. In one aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose neural cell injury associated with cerebral ischemia. In
another aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with cerebral infarction. In another aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose or prevent neural cell injury associated with a stroke. In
a further aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with a heart attack.
[0283] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515
(1990)); increased sprouting of neurons may be detected by methods
known in the art, such as, for example, the methods set forth in
Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al.
(Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0284] In specific embodiments, motor neuron diseases, disorders,
and/or conditions that may be treated, prevented, and/or diagnosed
according to the invention include, but are not limited to,
diseases, disorders, and/or conditions such as infarction,
infection, exposure to toxin, trauma, surgical damage,
depolynucleotiderative disease or malignancy that may affect motor
neurons as well as other components of the nervous system, as well
as diseases, disorders, and/or conditions that selectively affect
neurons such as amyotrophic lateral sclerosis, and including, but
not limited to, progressive spinal muscular atrophy, progressive
bulbar palsy, primary lateral sclerosis, infantile and juvenile
muscular atrophy, progressive bulbar paralysis of childhood
(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome,
and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth
Disease).
Screening Assays
[0285] The present invention also relates to screening assays
particularly useful in drug discovery efforts. Thus, the invention
provides methods for screening for compounds that bind and/or
modulate a BGS-19 polynucleotide or polypeptide. Accordingly, in
one embodiment, the invention provides a method for detecting an
analyte that binds a BGS-19 polypeptide comprising the steps of
contacting the BGS-19 polypeptide, or a variant thereof, with an
analyte under conditions that allow the BGS-19 polypeptide to be
bound by the analyte, and detecting binding of the BGS-19
polypeptide to the analyte, wherein detection of binding indicates
presence of an analyte that binds the BGS-19 polypeptide. In
particular embodiments, such methods can be used to detect and
identify compounds that bind or affect the pharmacokinetics (e.g.,
catalytic activity) of a polypeptide of the invention.
[0286] In particular embodiments, the analyte is a protein.
Accordingly, in one embodiments, the present invention provides a
method for identifying a BGS-19-binding protein comprising the
steps of contacting a BGS-19 polypeptide, or a variant thereof,
with an array comprising a plurality of proteins, and detecting
binding of the BGS-19 polypeptide to a protein on the array,
wherein detection of binding indicates presence of a BGS-19-binding
protein.
[0287] The present invention also relates to methods for detecting
an analyte that binds a BGS-19 polynucleotide comprising the steps
of contacting the BGS-19 polynucleotide, or a variant thereof, with
an analyte under conditions that allow the BGS-19 polynucleotide to
be bound by the analyte, and detecting binding of the BGS-19
polynucleotide to the analyte, wherein detection of binding
indicates presence of an analyte that binds the BGS-19
polynucleotide. In particular embodiments, such methods can be used
to detect and identify compounds that modulate transcription or
translation of a BGS-19 polynucleotide product.
Methods for Detecting Modulators of a BGS-19 Polynucleotide or
Polypeptide
[0288] The present invention relates to methods for detecting and
identifying proteins that bind BGS-19 DNA sequences, such proteins
including, but not limited to, proteins that affect DNA
conformation and proteins that modulate transcriptional activity
(e.g., transcription factors, proteins that bind enhancers). In
particular embodiments, the present invention provides methods for
detecting and identifying factors that bind BGS-19 RNA sequences,
such factors including, but not limited to, proteins, steroid
hormones, or other small molecules. In further embodiments, the
BGS-19 RNA-binding factors modulate translational efficacy and/or
affect RNA stability.
[0289] The present invention also relates to methods for detecting
and identifying BGS-19 agonists, antagonists, as well as activators
and inhibitors thereof. In specific non-limiting embodiments,
BGS-19 agonists, BGS-19 antagonists, inhibitors of BGS-19 agonists,
activators of BGS-19 agonists, inhibitors of BGS-19 antagonists and
activators of BGS-19 antagonists are small molecules (i.e., less
than 500 daltons) that bind a BGS-19 polynucleotide or BGS-19
polypeptide of the invention.
[0290] The present invention also relates to methods for screening
for proteins that bind specific domains of a BGS-19 polypeptide,
wherein the domains exhibit a biological activity. In one
embodiment, the invention provides a method for identifying a
protein having a SH2 domain comprising the steps of contacting a
SH2-binding domain of a BGS-19 polypeptide, or a variant thereof,
with an analyte under conditions that allow the SH2-binding domain
to be bound by the analyte, and detecting binding of the
SH2-binding domain to the analyte, wherein detection of binding
indicates the presence of a protein having a SH2 domain. In a
further embodiment, the SH2-binding domain of the BGS-19 protein
comprises an immunotyrosine-based inhibition motif ("ITIM").
[0291] Accordingly, the present invention provides isolated BGS-19
polynucleotides, or derivatives thereof, as probes that can be used
to screen for DNA-binding proteins including, but not limited to,
proteins that affect DNA conformation or modulate transcriptional
activity (e.g., enhancers, transcription factors). In another
embodiment, such probes can be used to screen for RNA-binding
factors, including but not limited to proteins, steroid hormones,
or other small molecules. In yet another embodiment, such probes
can be used to detect and identify molecules that bind or affect
the pharmacokinetics or activity (e.g., enzymatic activity) of a
polypeptide of the invention.
[0292] In one embodiment, a screening assay of the invention can
identify a test compound that is useful for increasing or
decreasing the translation of a BGS-19 mRNA, for example, by
binding to one or more regulatory elements in the 5' untranslated
region, the 3' untranslated region, or the coding region of the
mRNA. Compounds that bind to mRNA can, inter alia, increase or
decrease the rate of mRNA processing, alter its transport through
the cell, prevent or enhance binding of the mRNA to ribosomes,
suppressor proteins or enhancer proteins, or alter mRNA stability.
Accordingly, compounds that increase or decrease mRNA translation
can be used to treat or prevent disease. For example, diseases such
as cancer, associated with overproduction of proteins, such as Ras,
can be treated or prevented by decreasing translation of the mRNA
that codes for the overproduced protein, thus inhibiting production
of the protein. Conversely, the symptoms of diseases associated
with decreased protein function, such as hemophilia, may be treated
by increasing translation of mRNA coding for the protein whose
function is decreased, e.g., factor IX in some forms of
hemophilia.
[0293] Accordingly, in one embodiment, a compound identified by a
screening assay of the invention inhibits the production of a
BGS-19 protein. In a further embodiment, the compound inhibits the
translation of a BGS-19 mRNA.
[0294] The invention provides a method for identifying modulators,
ie., candidate or test compounds or agents (e.g., peptides,
peptidomimetics, small molecules or other drugs) which bind to
polypeptide of the invention or have a stimulatory or inhibitory
effect on, for example, expression or activity of a polypeptide of
the invention.
[0295] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of the membrane-bound form of a polypeptide of the
invention or biologically active portion thereof. The test
compounds of the present invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, 1997, Anticancer Drug Des.
12:145).
[0296] Examples of methods for the synthesis of molecular libraries
can be found in the art (see, e.g., DeWitt et al., 1993, Proc Natl
Acad. Sci. 90:6909; Erb et al., 1994, Proc Natl Acad. Sci.
91:11422; Zuckermann et al., 1994, J Med. Chem. 37:2678; Cho et
al., 1993, Science 261:1303; Carrell et al., 1994, Angew Chem Int
Ed Encl. 33:2059; Carell et al., 1994, Angew Chem Int Ed Encl.
33:2061; Gallop et al., 1994, J Med. Chem. 37:1233).
[0297] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (Patent Nos. 5,571,698;
5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc Natl
Acad. Sci. 89:1865-1869) or phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc Natl Acad. Sci. 87:6378-6382; Felici, 1991, J Mol. Biol.
222:301-310).
[0298] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a membrane-bound form of a polypeptide of the
invention, or a biologically active portion thereof, on the cell
surface is contacted with a test compound and the ability of the
test compound to bind to the polypeptide determined. The cell, for
example, can be a yeast cell or a cell of mammalian origin.
Determining the ability of the test compound to bind to the
polypeptide can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the polypeptide or biologically active
portion thereof can be determined by detecting the labeled compound
in a complex. For example, test compounds can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radio-emission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a preferred embodiment, the
assay comprises contacting a cell which expresses a membrane-bound
form of a polypeptide of the invention, or a biologically active
portion thereof, on the cell surface with a known compound which
binds the polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with the polypeptide, wherein
determining the ability of the test compound to interact with the
polypeptide comprises determining the ability of the test compound
to preferentially bind to the polypeptide or a biologically active
portion thereof as compared to the known compound.
[0299] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a membrane-bound form of a
polypeptide of the invention, or a biologically active portion
thereof, on the cell surface with a test compound and determining
the ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the polypeptide or biologically active
portion thereof. Determining the ability of the test compound to
modulate the activity of the polypeptide or a biologically active
portion thereof can be accomplished, for example, by determining
the ability of the polypeptide protein to bind to or interact with
a target molecule.
[0300] Determining the ability of a polypeptide of the invention to
bind to or interact with a target molecule can be accomplished by
one of the methods described above for determining direct binding.
As used herein, a "target molecule" is a molecule with which a
selected polypeptide (e.g., a BGS-19 polypeptide of the invention)
binds or interacts with in nature, for example, a molecule on the
surface of a cell which expresses the selected protein, a molecule
on the surface of a second cell, a molecule in the extracellular
milieu, a molecule associated with the internal surface of a cell
membrane or a cytoplasmic molecule. A target molecule can be a
polypeptide of the invention or some other polypeptide or protein.
For example, a target molecule can be a component of a signal
transduction pathway which facilitates transduction of an
extracellular signal (e.g., a signal polynucleotiderated by binding
of a compound to a polypeptide of the invention) through the cell
membrane and into the cell or a second intercellular protein which
has catalytic activity or a protein which facilitates the
association of downstream signaling molecules with a polypeptide of
the invention. Determining the ability of a polypeptide of the
invention to bind to or interact with a target molecule can be
accomplished by determining the activity of the target molecule.
For example, the activity of the target molecule can be determined
by detecting induction of a cellular second messenger of the target
(e.g., intracellular Ca.sup.+2, diacylglycerol, IP3, etc.),
detecting catalytic/enzymatic activity of the target on an
appropriate substrate, detecting the induction of a reporter
polynucleotide (e.g., a regulatory element that is responsive to a
polypeptide of the invention operably linked to a polynucleotide
encoding a detectable marker, e.g., luciferase), or detecting a
cellular response, for example, cellular differentiation, or cell
proliferation.
[0301] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a BGS-19 polypeptide of
the invention or biologically active portion thereof with a test
compound and determining the ability of the test compound to bind
to the polypeptide or biologically active portion thereof. Binding
of the test compound to the polypeptide can be determined either
directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the polypeptide of the
invention or biologically active portion thereof with a known
compound which binds the polypeptide to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with the polypeptide,
wherein determining the ability of the test compound to interact
with the polypeptide comprises determining the ability of the test
compound to preferentially bind to the polypeptide or biologically
active portion thereof as compared to the known compound.
[0302] In another embodiment, an assay is a cell-free assay
comprising contacting a BGS-19 polypeptide of the invention or
biologically active portion thereof with a test compound and
determining the ability of the test compound to modulate (e.g.,
stimulate or inhibit) the activity of the polypeptide or
biologically active portion thereof. Determining the ability of the
test compound to modulate the activity of the polypeptide can be
accomplished, for example, by determining the ability of the
polypeptide to bind to a target molecule by one of the methods
described above for determining direct binding. In an alternative
embodiment, determining the ability of the test compound to
modulate the activity of the polypeptide can be accomplished by
determining the ability of the polypeptide of the invention to
further modulate the target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
[0303] In yet another embodiment, the cell-free assay comprises
contacting a BGS-19 polypeptide of the invention or biologically
active portion thereof with a known compound which binds the
polypeptide to form an assay mixture, contacting the assay mixture
with a test compound, and determining the ability of the test
compound to interact with the polypeptide, wherein determining the
ability of the test compound to interact with the polypeptide
comprises determining the ability of the polypeptide to
preferentially bind to or modulate the activity of a target
molecule.
[0304] The cell-free assays of the present invention are amenable
to use of both a soluble form or the membrane-bound form of a
polypeptide of the invention. In the case of cell-free assays
comprising the membrane-bound form of the polypeptide, it may be
desirable to utilize a solubilizing agent such that the
membrane-bound form of the polypeptide is maintained in solution.
Examples of such solubilizing agents include non-ionic detergents
such as n-octylglucoside, n-dodecylglucoside, n-octylmaltoside,
octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton
X-100, Triton X-114, Thesit, isotridecypoly(ethylene glycol
ether).sub.n, 3-[(3-cholamidopropyl) dimethylamminio]-1-propane
sulfonate (CHAPS), 3-[(3-cholamidopropyl)
dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or
N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.
[0305] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either the
BGS-19 polypeptide of the invention or its target molecule to
facilitate separation of complexed from uncomplexed forms of one or
both of the proteins, as well as to accommodate automation of the
assay. Binding of a test compound to the polypeptide, or
interaction of the polypeptide with a target molecule in the
presence and absence of a candidate compound, can be accomplished
in any vessel suitable for comprising the reactants. Examples of
such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the
proteins to be bound to a matrix. For example,
glutathione-5-transferase fusion proteins or
glutathione-5-transferase fusion proteins can be adsorbed onto
glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or A polypeptide of the invention, and
the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads or microtiter plate wells are
washed to remove any unbound components and complex formation is
measured either directly or indirectly, for example, as described
above. Alternatively, the complexes can be dissociated from the
matrix, and the level of binding or activity of the polypeptide of
the invention can be determined using standard techniques.
[0306] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the polypeptide of the invention or its target molecule can
be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated polypeptide of the invention or target molecules can
be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques
well known in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptide of the
invention or target molecules but which do not interfere with
binding of the polypeptide of the invention to its target molecule
can be derivatized to the wells of the plate, and unbound target or
polypeptide of the invention trapped in the wells by antibody
conjugation. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
polypeptide of the invention or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the polypeptide of the invention or target
molecule.
[0307] In another embodiment, modulators of expression of a BGS-19
polypeptide of the invention are identified in a method in which a
cell is contacted with a candidate compound and the expression of
the selected mRNA or protein (i.e., the mRNA or protein
corresponding to a polypeptide or polynucleotide of the invention)
in the cell is determined. The level of expression of the selected
mRNA or protein in the presence of the candidate compound is
compared to the level of expression of the selected mRNA or protein
in the absence of the candidate compound. The candidate compound
can then be identified as a modulator of expression of the
polypeptide of the invention based on this comparison. For example,
when expression of the selected mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of the selected mRNA or protein
expression. Alternatively, when expression of the selected mRNA or
protein is less (statistically significantly less) in the presence
of the candidate compound than in its absence, the candidate
compound is identified as an inhibitor of the selected mRNA or
protein expression. The level of the selected mRNA or protein
expression in the cells can be determined by methods described
herein.
[0308] In yet another aspect of the invention, a BGS-19 polypeptide
of the inventions can be used as "bait proteins" in a two-hybrid
assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317;
Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J Biol.
Chem. 268:12046-12054; Bartel et al., 1993, Biotechniques
14:920-924; Iwabuchi et al., 1993, Oncopolynucleotide 8:1693-1696;
and PCT Publication No. WO 94/10300), to identify other proteins,
which bind to or interact with the polypeptide of the invention and
modulate activity of the polypeptide of the invention. Such binding
proteins are also likely to be involved in the propagation of
signals by the polypeptide of the inventions as, for example,
upstream or downstream elements of a signaling pathway involving
the polypeptide of the invention.
[0309] The invention also provides a method for screening for
compounds (e.g., potentially useful drugs) that bind a BGS-19
polynucleotide or polypeptide. In one embodiment, test compounds
are assayed for binding to a BGS-19 polynucleotide or polypeptide.
In another embodiment, test compounds are assayed for binding to a
complex comprising a BGS-19 polynucleotide (e.g., transcriptional
complex) or a BGS-19 polypeptide (hetero- or homo-dimer or
multimer). In a further embodiment, test compounds are assayed for
binding to a BGS-19 polypeptide when bound to a second, different
polypeptide.
[0310] The invention also provides a method for screening for
compounds (e.g., potentially useful drugs) that inhibit the binding
of a BGS-19 polynucleotide or polypeptide to an analyte, target
molecule or binding partner. In one embodiment, test compounds are
assayed to prevent formation of complexes comprising a BGS-19
polynucleotide (e.g., transcriptional complex) or a BGS-19
polypeptide (hetero- or homo-dimer or multimer). In a further
embodiment, test compounds are assayed for ability to inhibit
binding of a BGS-19 polypeptide to a second, different
polypeptide.
[0311] In particular embodiments, the test compounds are assayed
for the ability to interfere with existing complexes or existing
interactions of a BGS-19 polynucleotide or polypeptide with another
compound. In other embodiments, the test compound is incubated
first with the BGS-19 polynucleotide or polypeptide, prior to
addition of the analyte, target molecule or binding partner, after
which the ability to inhibit binding is assayed. In yet other
embodiments, the test compound is incubated first with the analyte,
target molecule or binding partner, prior to addition of the BGS-19
polynucleotide or polypeptide, after which the ability to inhibit
binding is assayed.
[0312] For example, and not by way of limitation, polynucleotides,
including those of the invention, that are modulated in cells by
treatment with an agent (e.g. compound, drug or small molecule)
which modulates activity or expression of a polypeptide of the
invention (e.g., as identified in a screening assay described
herein) can be identified. Thus, to study the effect of agents on
cellular proliferation disorders, for example, in a clinical trial,
cells can be isolated and RNA prepared and analyzed for the levels
of expression of a polynucleotide of the invention and other
polynucleotides implicated in the disorder. The levels of
polynucleotide expression (i.e., a polynucleotide expression
pattern) can be quantified by Northern blot analysis or RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods as described herein, or by
measuring the levels of activity of a polynucleotide of the
invention or other polynucleotides. In this way, the polynucleotide
expression pattern can serve as a marker, indicative of the
physiological response of the cells to the agent. Accordingly, this
response state may be determined before, and at various points
during, treatment of the individual with the agent.
[0313] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent;
[0314] (ii) detecting the level of the polypeptide or
polynucleotide of the invention in the pre-administration sample;
(iii) obtaining one or more post-administration samples from the
subject; (iv) detecting the level the of the polypeptide or
polynucleotide of the invention in the post-administration samples;
(v) comparing the level of the polypeptide or polynucleotide of the
invention in the pre-administration sample with the level of the
polypeptide or polynucleotide of the invention in the
post-administration sample or samples; and (vi) altering the
administration of the agent to the subject accordingly. For
example, increased administration of the agent may be desirable to
increase the expression or activity of the polypeptide to higher
levels than detected, i.e., to increase the effectiveness of the
agent. Alternatively, decreased administration of the agent may be
desirable to decrease expression or activity of the polypeptide to
lower levels than detected, i.e., to decrease the effectiveness of
the agent.
[0315] This invention further pertains to uses of agents identified
by the above-described screening assays.
Drug Screening
[0316] The present invention also relates to screening assays
particularly useful in drug discovery efforts. Thus, the invention
provides methods for screening for compounds that bind and/or
modulate a BGS-19 polynucleotide or polypeptide. Accordingly, in
one embodiment, the invention provides a method for detecting an
analyte that binds a BGS-19 polypeptide comprising the steps of
contacting the BGS-19 polypeptide, or a variant thereof, with an
analyte under conditions that allow the BGS-19 polypeptide to be
bound by the analyte, and detecting binding of the BGS-19
polypeptide to the analyte, wherein detection of binding indicates
presence of an analyte that binds the BGS-19 polypeptide. In
particular embodiments, such methods can be used to detect and
identify compounds that bind or affect the pharmacokinetics (e.g.,
catalytic activity) of a polypeptide of the invention.
[0317] In particular embodiments, the analyte is a protein.
Accordingly, in one embodiments, the present invention provides a
method for identifying a BGS-19-binding protein comprising the
steps of contacting a BGS-19 polypeptide, or a variant thereof,
with an array comprising a plurality of proteins, and detecting
binding of the BGS-19 polypeptide to a protein on the array,
wherein detection of binding indicates presence of a BGS-19-binding
protein.
[0318] Using a BGS-19 polynucleotide, BGS-19 polypeptide, and/or a
BGS-19-binding protein, a screening assay against, for example, a
defined collection of molecules or a biological sample, can
polynucleotiderate a signature of the metabolic state or biological
response. Many protein arrays, antigen arrays, DNA arrays etc.,
known in the art, can be screened using a BGS-19 polynucleotide,
polypeptide, antagonist, agonist and/or a BGS-19-binding protein to
determine binding patterns. Many similar screening assays are known
in the art and can be adapted for the present invention to, for
example, determine a diagnosis or prognosis or monitor treatment or
progression of a disorder.
[0319] Further contemplated is the use of the polypeptides of the
present invention, or the polynucleotides encoding these
polypeptides, to screen for molecules which modify the activities
of the polypeptides of the present invention. Such a method would
include contacting the polypeptide of the present invention with a
selected compound(s) suspected of having antagonist or agonist
activity, and assaying the activity of these polypeptides following
binding.
[0320] This invention is particularly useful for screening
therapeutic compounds by using the polypeptides of the present
invention, or binding fragments thereof, in any of a variety of
drug screening techniques. The polypeptide or fragment employed in
such a test may be affixed to a solid support, expressed on a cell
surface, free in solution, or located intracellularly. One method
of drug screening utilizes eukaryotic or prokaryotic host cells
which are stably transformed with recombinant nucleic acids
expressing the polypeptide or fragment. Drugs are screened against
such transformed cells in competitive binding assays. One may
measure, for example, the formulation of complexes between the
agent being tested and a polypeptide of the present invention.
[0321] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the polypeptides of the present invention. These methods comprise
contacting such an agent with a polypeptide of the present
invention or a fragment thereof and assaying for the presence of a
complex between the agent and the polypeptide or a fragment
thereof, by methods well known in the art. In such a competitive
binding assay, the agents to screen are typically labeled.
Following incubation, free agent is separated from that present in
bound form, and the amount of free or uncomplexed label is a
measure of the ability of a particular agent to bind to the
polypeptides of the present invention.
[0322] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the polypeptides of the present invention, and is described in
great detail in European Patent Application 84/03564, published on
Sep. 13, 1984, which is incorporated herein by reference herein.
Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds are reacted
with polypeptides of the present invention and washed. Bound
polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in
the aforementioned drug screening techniques. In addition,
non-neutralizing antibodies may be used to capture the peptide and
immobilize it on the solid support.
[0323] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding polypeptides of the present invention specifically compete
with a test compound for binding to the polypeptides or fragments
thereof. In this manner, the antibodies are used to detect the
presence of any peptide which shares one or more antigenic epitopes
with a polypeptide of the invention.
[0324] The human BGS-19 polypeptides and/or peptides of the present
invention, or immunogenic fragments or oligopeptides thereof, can
be used for screening therapeutic drugs or compounds in a variety
of drug screening techniques. The fragment employed in such a
screening assay may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. The
reduction or abolition of activity of the formation of binding
complexes between the ion channel protein and the agent being
tested can be measured. Thus, the present invention provides a
method for screening or assessing a plurality of compounds for
their specific binding affinity with a BGS-19 polypeptide, or a
bindable peptide fragment, of this invention, comprising providing
a plurality of compounds, combining the BGS-19 polypeptide, or a
bindable peptide fragment, with each of a plurality of compounds
for a time sufficient to allow binding under suitable conditions
and detecting binding of the BGS-19 polypeptide or peptide to each
of the plurality of test compounds, thereby identifying the
compounds that specifically bind to the BGS-19 polypeptide or
peptide.
[0325] Methods of identifying compounds that modulate the activity
of the novel human BGS-19 polypeptides and/or peptides are provided
by the present invention and comprise combining a potential or
candidate compound or drug modulator of potassium channel beta
subunit biological activity with an BGS-19 polypeptide or peptide,
for example, the BGS-19 amino acid sequence as set forth in SEQ ID
NOS:2, and measuring an effect of the candidate compound or drug
modulator on the biological activity of the BGS-19 polypeptide or
peptide. Such measurable effects include, for example, physical
binding interaction; the ability to cleave a suitable potassium
channel beta subunit substrate; effects on native and cloned
BGS-19-expressing cell line; and effects of modulators or other
potassium channel beta subunit-mediated physiological measures.
[0326] Another method of identifying compounds that modulate the
biological activity of the novel BGS-19 polypeptides of the present
invention comprises combining a potential or candidate compound or
drug modulator of a potassium channel beta subunit biological
activity with a host cell that expresses the BGS-19 polypeptide and
measuring an effect of the candidate compound or drug modulator on
the biological activity of the BGS-19 polypeptide. The host cell
can also be capable of being induced to express the BGS-19
polypeptide, e.g., via inducible expression. Physiological effects
of a given modulator candidate on the BGS-19 polypeptide can also
be measured. Thus, cellular assays for particular potassium channel
beta subunit modulators may be either direct measurement or
quantification of the physical biological activity of the BGS-19
polypeptide, or they may be measurement or quantification of a
physiological effect. Such methods preferably employ a BGS-19
polypeptide as described herein, or an overexpressed recombinant
BGS-19 polypeptide in suitable host cells containing an expression
vector as described herein, wherein the BGS-19 polypeptide is
expressed, overexpressed, or undergoes upregulated expression.
[0327] Another aspect of the present invention embraces a method of
screening for a compound that is capable of modulating the
biological activity of a BGS-19 polypeptide, comprising providing a
host cell containing an expression vector harboring a nucleic acid
sequence encoding a BGS-19 polypeptide, or a functional peptide or
portion thereof (e.g., SEQ ID NOS:2); determining the biological
activity of the expressed BGS-19 polypeptide in the absence of a
modulator compound; contacting the cell with the modulator compound
and determining the biological activity of the expressed BGS-19
polypeptide in the presence of the modulator compound. In such a
method, a difference between the activity of the BGS-19 polypeptide
in the presence of the modulator compound and in the absence of the
modulator compound indicates a modulating effect of the
compound.
[0328] Essentially any chemical compound can be employed as a
potential modulator or ligand in the assays according to the
present invention. Compounds tested as potassium channel beta
subunit modulators can be any small chemical compound, or
biological entity (e.g., protein, sugar, nucleic acid, lipid). Test
compounds will typically be small chemical molecules and peptides.
Generally, the compounds used as potential modulators can be
dissolved in aqueous or organic (e.g., DMSO-based) solutions. The
assays are designed to screen large chemical libraries by
automating the assay steps and providing compounds from any
convenient source. Assays are typically run in parallel, for
example, in microtiter formats on microtiter plates in robotic
assays. There are many suppliers of chemical compounds, including
Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich
(St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland), for example. Also, compounds may be synthesized by
methods known in the art.
[0329] High throughput screening methodologies are particularly
envisioned for the detection of modulators of the novel BGS-19
polynucleotides and polypeptides described herein. Such high
throughput screening methods typically involve providing a
combinatorial chemical or peptide library containing a large number
of potential therapeutic compounds (e.g., ligand or modulator
compounds). Such combinatorial chemical libraries or ligand
libraries are then screened in one or more assays to identify those
library members (e.g., particular chemical species or subclasses)
that display a desired characteristic activity. The compounds so
identified can serve as conventional lead compounds, or can
themselves be used as potential or actual therapeutics.
[0330] A combinatorial chemical library is a collection of diverse
chemical compounds polynucleotiderated either by chemical synthesis
or biological synthesis, by combining a number of chemical building
blocks (i.e., reagents such as amino acids). As an example, a
linear combinatorial library, e.g., a polypeptide or peptide
library, is formed by combining a set of chemical building blocks
in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide or peptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks.
[0331] The preparation and screening of combinatorial chemical
libraries is well known to those having skill in the pertinent art.
Combinatorial libraries include, without limitation, peptide
libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept.
Prot. Res., 37:487-493; and Houghton et al., 1991, Nature,
354:84-88). Other chemistries for polynucleotiderating chemical
diversity libraries can also be used. Nonlimiting examples of
chemical diversity library chemistries include, peptides (PCT
Publication No. WO 91/019735), encoded peptides (PCT Publication
No. WO 93/20242), random bio-oligomers (PCT Publication No. WO
92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers
such as hydantoins, benzodiazepines and dipeptides (Hobbs et al.,
1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous
polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc.,
114:6568), nonpeptidal peptidomimetics with glucose scaffolding
(Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217-9218),
analogous organic synthesis of small compound libraries (Chen et
al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Cho et
al., 1993, Science, 261:1303), and/or peptidyl phosphonates
(Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody
libraries (e.g., Vaughn et al., 1996, Nature Biotechnology,
14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g.,
Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No.
5,593,853), small organic molecule libraries (e.g.,
benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S.
Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; and the like).
[0332] Devices for the preparation of combinatorial libraries are
commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,
Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, a large number of combinatorial libraries are
commercially available (e.g., ComGenex, Princeton, N.J.; Asinex,
Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd.,
Moscow, Russia; 3 D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md., and the like).
[0333] In one embodiment, the invention provides solid phase based
in vitro assays in a high throughput format, where the cell or
tissue expressing an ion channel is attached to a solid phase
substrate. In such high throughput assays, it is possible to screen
up to several thousand different modulators or ligands in a single
day. In particular, each well of a microtiter plate can be used to
perform a separate assay against a selected potential modulator,
or, if concentration or incubation time effects are to be observed,
every 5-10 wells can test a single modulator. Thus, a single
standard microtiter plate can assay about 96 modulators. If 1536
well plates are used, then a single plate can easily assay from
about 100 to about 1500 different compounds. It is possible to
assay several different plates per day; thus, for example, assay
screens for up to about 6,000-20,000 different compounds are
possible using the described integrated systems.
[0334] In another of its aspects, the present invention encompasses
screening and small molecule (e.g., drug) detection assays which
involve the detection or identification of small molecules that can
bind to a given protein, i.e., a BGS-19 polypeptide or peptide.
Particularly preferred are assays suitable for high throughput
screening methodologies.
[0335] In such binding-based detection, identification, or
screening assays, a functional assay is not typically required. All
that is needed is a target protein, preferably substantially
purified, and a library or panel of compounds (e.g., ligands,
drugs, small molecules) or biological entities to be screened or
assayed for binding to the protein target. Preferably, most small
molecules that bind to the target protein will modulate activity in
some manner, due to preferential, higher affinity binding to
functional areas or sites on the protein.
[0336] An example of such an assay is the fluorescence based
thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP,
Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920
to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News,
20(8)). The assay allows the detection of small molecules (e.g.,
drugs, ligands) that bind to expressed, and preferably purified,
ion channel polypeptide based on affinity of binding determinations
by analyzing thermal unfolding curves of protein-drug or ligand
complexes. The drugs or binding molecules determined by this
technique can be further assayed, if desired, by methods, such as
those described herein, to determine if the molecules affect or
modulate function or activity of the target protein.
[0337] To purify a BGS-19 polypeptide or peptide to measure a
biological binding or ligand binding activity, the source may be a
whole cell lysate that can be prepared by successive freeze-thaw
cycles (e.g., one to three) in the presence of standard protease
inhibitors. The BGS-19 polypeptide may be partially or completely
purified by standard protein purification methods, e.g., affinity
chromatography using specific antibody described infra, or by
ligands specific for an epitope tag engineered into the recombinant
BGS-19 polypeptide molecule, also as described herein. Binding
activity can then be measured as described.
[0338] Compounds which are identified according to the methods
provided herein, and which modulate or regulate the biological
activity or physiology of the BGS-19 polypeptides according to the
present invention are a preferred embodiment of this invention. It
is contemplated that such modulatory compounds may be employed in
treatment and therapeutic methods for treating a condition that is
mediated by the novel BGS-19 polypeptides by administering to an
individual in need of such treatment a therapeutically effective
amount of the compound identified by the methods described
herein.
[0339] In addition, the present invention provides methods for
treating an individual in need of such treatment for a disease,
disorder, or condition that is mediated by the BGS-19 polypeptides
of the invention, comprising administering to the individual a
therapeutically effective amount of the BGS-19-modulating compound
identified by a method provided herein.
[0340] The human BGS-19 polypeptides and/or peptides of the present
invention, or immunogenic fragments or oligopeptides thereof, can
be used for screening therapeutic drugs or compounds in a variety
of drug screening techniques. The fragment employed in such a
screening assay may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. The
reduction or abolition of activity of the formation of binding
complexes between the ion channel protein and the agent being
tested can be measured. Thus, the present invention provides a
method for screening or assessing a plurality of compounds for
their specific binding affinity with a BGS-19 polypeptide, or a
bindable peptide fragment, of this invention, comprising providing
a plurality of compounds, combining the BGS-19 polypeptide, or a
bindable peptide fragment, with each of a plurality of compounds
for a time sufficient to allow binding under suitable conditions
and detecting binding of the BGS-19 polypeptide or peptide to each
of the plurality of test compounds, thereby identifying the
compounds that specifically bind to the BGS-19 polypeptide or
peptide.
[0341] Methods of identifying compounds that modulate the activity
of the novel human BGS-19 polypeptides and/or peptides are provided
by the present invention and comprise combining a potential or
candidate compound or drug modulator of potassium channel beta
subunit biological activity with an BGS-19 polypeptide or peptide,
for example, the BGS-19 amino acid sequence as set forth in SEQ ID
NO:2, and measuring an effect of the candidate compound or drug
modulator on the biological activity of the BGS-19 polypeptide or
peptide. Such measurable effects include, for example, physical
binding interaction; the ability to cleave a suitable potassium
channel beta subunit substrate; effects on native and cloned
BGS-19-expressing cell line; and effects of modulators or other
potassium channel beta subunit-mediated physiological measures.
[0342] Another method of identifying compounds that modulate the
biological activity of the novel BGS-19 polypeptides of the present
invention comprises combining a potential or candidate compound or
drug modulator of a potassium channel beta subunit biological
activity with a host cell that expresses the BGS-19 polypeptide and
measuring an effect of the candidate compound or drug modulator on
the biological activity of the BGS-19 polypeptide. The host cell
can also be capable of being induced to express the BGS-19
polypeptide, e.g., via inducible expression. Physiological effects
of a given modulator candidate on the BGS-19 polypeptide can also
be measured. Thus, cellular assays for particular potassium channel
beta subunit modulators may be either direct measurement or
quantification of the physical biological activity of the BGS-19
polypeptide, or they may be measurement or quantification of a
physiological effect. Such methods preferably employ a BGS-19
polypeptide as described herein, or an overexpressed recombinant
BGS-19 polypeptide in suitable host cells containing an expression
vector as described herein, wherein the BGS-19 polypeptide is
expressed, overexpressed, or undergoes upregulated expression.
[0343] Another aspect of the present invention embraces a method of
screening for a compound that is capable of modulating the
biological activity of a BGS-19 polypeptide, comprising providing a
host cell containing an expression vector harboring a nucleic acid
sequence encoding a BGS-19 polypeptide, or a functional peptide or
portion thereof (e.g., SEQ ID NOS:2); determining the biological
activity of the expressed BGS-19 polypeptide in the absence of a
modulator compound; contacting the cell with the modulator compound
and determining the biological activity of the expressed BGS-19
polypeptide in the presence of the modulator compound. In such a
method, a difference between the activity of the BGS-19 polypeptide
in the presence of the modulator compound and in the absence of the
modulator compound indicates a modulating effect of the
compound.
[0344] Essentially any chemical compound can be employed as a
potential modulator or ligand in the assays according to the
present invention. Compounds tested as potassium channel beta
subunit modulators can be any small chemical compound, or
biological entity (e.g., protein, sugar, nucleic acid, lipid). Test
compounds will typically be small chemical molecules and peptides.
Generally, the compounds used as potential modulators can be
dissolved in aqueous or organic (e.g., DMSO-based) solutions. The
assays are designed to screen large chemical libraries by
automating the assay steps and providing compounds from any
convenient source. Assays are typically run in parallel, for
example, in microtiter formats on microtiter plates in robotic
assays. There are many suppliers of chemical compounds, including
Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich
(St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland), for example. Also, compounds may be synthesized by
methods known in the art.
[0345] High throughput screening methodologies are particularly
envisioned for the detection of modulators of the novel BGS-19
polynucleotides and polypeptides described herein. Such high
throughput screening methods typically involve providing a
combinatorial chemical or peptide library containing a large number
of potential therapeutic compounds (e.g., ligand or modulator
compounds). Such combinatorial chemical libraries or ligand
libraries are then screened in one or more assays to identify those
library members (e.g., particular chemical species or subclasses)
that display a desired characteristic activity. The compounds so
identified can serve as conventional lead compounds, or can
themselves be used as potential or actual therapeutics.
[0346] A combinatorial chemical library is a collection of diverse
chemical compounds polynucleotiderated either by chemical synthesis
or biological synthesis, by combining a number of chemical building
blocks (i.e., reagents such as amino acids). As an example, a
linear combinatorial library, e.g., a polypeptide or peptide
library, is formed by combining a set of chemical building blocks
in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide or peptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks.
[0347] The preparation and screening of combinatorial chemical
libraries is well known to those having skill in the pertinent art.
Combinatorial libraries include, without limitation, peptide
libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept.
Prot. Res., 37:487-493; and Houghton et al., 1991, Nature,
354:84-88). Other chemistries for polynucleotiderating chemical
diversity libraries can also be used. Nonlimiting examples of
chemical diversity library chemistries include, peptides (PCT
Publication No. WO 91/019735), encoded peptides (PCT Publication
No. WO 93/20242), random bio-oligomers (PCT Publication No. WO
92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers
such as hydantoins, benzodiazepines and dipeptides (Hobbs et al.,
1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous
polypeptides (Hagihara et al., 1992,J. Amer. Chem. Soc., 114:6568),
nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et
al., 1992, J. Amer. Chem. Soc., 114:9217-9218), analogous organic
synthesis of small compound libraries (Chen et al., 1994, J. Amer.
Chem. Soc., 116:2661), oligocarbamates (Cho et al., 1993, Science,
261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J.
Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat.
No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996,
Nature Biotechnology, 14(3):309-314) and PCT/US96/10287),
carbohydrate libraries (e.g., Liang et al., 1996, Science,
274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule
libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,
page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; and the like).
[0348] Devices for the preparation of combinatorial libraries are
commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,
Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, a large number of combinatorial libraries are
commercially available (e.g., ComGenex, Princeton, N.J.; Asinex,
Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd.,
Moscow, Russia; 3 D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md., and the like).
[0349] In one embodiment, the invention provides solid phase based
in vitro assays in a high throughput format, where the cell or
tissue expressing an ion channel is attached to a solid phase
substrate. In such high throughput assays, it is possible to screen
up to several thousand different modulators or ligands in a single
day. In particular, each well of a microtiter plate can be used to
perform a separate assay against a selected potential modulator,
or, if concentration or incubation time effects are to be observed,
every 5-10 wells can test a single modulator. Thus, a single
standard microtiter plate can assay about 96 modulators. If 1536
well plates are used, then a single plate can easily assay from
about 100 to about 1500 different compounds. It is possible to
assay several different plates per day; thus, for example, assay
screens for up to about 6,000-20,000 different compounds are
possible using the described integrated systems.
[0350] In another of its aspects, the present invention encompasses
screening and small molecule (e.g., drug) detection assays which
involve the detection or identification of small molecules that can
bind to a given protein, i.e., a BGS-19 polypeptide or peptide.
Particularly preferred are assays suitable for high throughput
screening methodologies.
[0351] In such binding-based detection, identification, or
screening assays, a functional assay is not typically required. All
that is needed is a target protein, preferably substantially
purified, and a library or panel of compounds (e.g., ligands,
drugs, small molecules) or biological entities to be screened or
assayed for binding to the protein target. Preferably, most small
molecules that bind to the target protein will modulate activity in
some manner, due to preferential, higher affinity binding to
functional areas or sites on the protein.
[0352] An example of such an assay is the fluorescence based
thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP,
Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920
to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News,
20(8)). The assay allows the detection of small molecules (e.g.,
drugs, ligands) that bind to expressed, and preferably purified,
ion channel polypeptide based on affinity of binding determinations
by analyzing thermal unfolding curves of protein-drug or ligand
complexes. The drugs or binding molecules determined by this
technique can be further assayed, if desired, by methods, such as
those described herein, to determine if the molecules affect or
modulate function or activity of the target protein.
[0353] To purify a BGS-19 polypeptide or peptide to measure a
biological binding or ligand binding activity, the source may be a
whole cell lysate that can be prepared by successive freeze-thaw
cycles (e.g., one to three) in the presence of standard protease
inhibitors. The BGS-19 polypeptide may be partially or completely
purified by standard protein purification methods, e.g., affinity
chromatography using specific antibody described infra, or by
ligands specific for an epitope tag engineered into the recombinant
BGS-19 polypeptide molecule, also as described herein. Binding
activity can then be measured as described.
[0354] Compounds which are identified according to the methods
provided herein, and which modulate or regulate the biological
activity or physiology of the BGS-19 polypeptides according to the
present invention are a preferred embodiment of this invention. It
is contemplated that such modulatory compounds may be employed in
treatment and therapeutic methods for treating a condition that is
mediated by the novel BGS-19 polypeptides by administering to an
individual in need of such treatment a therapeutically effective
amount of the compound identified by the methods described
herein.
[0355] In addition, the present invention provides methods for
treating an individual in need of such treatment for a disease,
disorder, or condition that is mediated by the BGS-19 polypeptides
of the invention, comprising administering to the individual a
therapeutically effective amount of the BGS-19-modulating compound
identified by a method provided herein.
Detection Assays
[0356] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete polynucleotide sequences)
can be used in numerous ways as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
polynucleotides on a chromosome and, thus, locate polynucleotide
regions associated with polynucleotidetic disease; (ii) identify an
individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
Chromosome Mapping
[0357] Once the sequence (or a portion of the sequence) of a
polynucleotide has been isolated, this sequence can be used to map
the location of the polynucleotide on a chromosome. Accordingly,
BGS-19 polynucleotides described herein or fragments thereof, can
be used to map the location of the corresponding polynucleotides on
a chromosome. The mapping of the sequences to chromosomes is an
important first step in correlating these sequences with
chromosomal aberrations associated with BGS-19-related disease.
[0358] Briefly, polynucleotides can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
sequence of a polynucleotide of the invention. Computer analysis of
the sequence of a polynucleotide of the invention can be used to
rapidly select primers that do not span more than one exon in the
genomic DNA, thus complicating the amplification process. These
primers can then be used for PCR screening of somatic cell hybrids
containing individual human chromosomes. Only those hybrids
comprising the human polynucleotide corresponding to the
polynucleotide sequences will yield an amplified fragment (see,
e.g., Eustachio et al., 1983, Science 220:919-924).
[0359] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the polynucleotides of the invention to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a polynucleotide to
its chromosome include in situ hybridization (see, e.g., Fan et
al., 1990, Proc Natl Acad. Sci. 87:6223-6227), pre-screening with
labeled flow-sorted chromosomes ("CITE"), and pre-selection by
hybridization to chromosome specific cDNA libraries. Fluorescence
in situ hybridization ("FISH") of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise
chromosomal location in one step (see, e.g., Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New
York, 1988).
[0360] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the polynucleotides actually are preferred for mapping purposes.
Coding sequences are more likely to be conserved within
polynucleotide families, thus increasing the chance of cross
hybridizations during chromosomal mapping.
[0361] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with polynucleotidetic map data (see, e.g., V.
McKusick, Mendelian Inheritance in Man,
http://www.ncbi.nlm.nih.gov/Omimi/). The relationship between
polynucleotides and disease, mapped to the same chromosomal region,
can then be identified through linkage analysis or co-inheritance
of physically adjacent polynucleotides (see, e.g., Egeland et al.,
1987, Nature 325:783-787).
[0362] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a polynucleotide of the invention can be determined. If a mutation
is observed in some or all of the affected individuals but not in
any unaffected individuals, then the mutation is likely to be the
causative agent of the particular disease. Comparison of affected
and unaffected individuals polynucleotiderally involves first
looking for structural alterations in the chromosomes such as
deletions or translocations that are visible from chromosome
spreads or detectable using PCR based on that DNA sequence.
Ultimately, complete sequencing of polynucleotides from several
individuals can be performed to confirm the presence of a mutation
and to distinguish mutations from polymorphisms.
[0363] Furthermore, the polynucleotides disclosed herein can be
used to perform searches against "mapping databases", e.g.,
BLAST-type search, such that the chromosome position of the
polynucleotide is identified by sequence homology or identity with
known sequence fragments which have been mapped to chromosomes.
[0364] In addition, a polypeptide and fragments and sequences
thereof and antibodies specific thereto can be used to map the
location of the polynucleotide encoding the polypeptide on a
chromosome. This mapping can be carried out by specifically
detecting the presence of the polypeptide in members of a panel of
somatic cell hybrids between cells of a first species of animal
from which the protein originates and cells from a second species
of animal and then determining which somatic cell hybrid(s)
expresses the polypeptide and noting the chromosome(s) from the
first species of animal that it contains (see, e.g., Pajunen et
al., 1988, Cytopolynucleotidet Cell Genet. 47:37-41; Van Keuren et
al., 1986, Hum Genet. 74:34-40). Alternatively, the presence of the
polypeptide in the somatic cell hybrids can be determined by
assaying an activity or property of the polypeptide, for example,
enzymatic activity, (see, e.g., Bordelon-Riser et al., 1979,
Somatic Cell Genetics 5:597-613; Owerbach et al., 1978, Proc Natl
Acad. Sci. 75:5640-5644).
Tissue Typing
[0365] The BGS-19 polynucleotides can also be used to identify
individuals from minute biological samples. The United States
military, for example, is considering the use of restriction
fragment length polymorphism ("RFLP") for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0366] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the polynucleotides described herein can
be used to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0367] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The polynucleotides of
the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of
these sequences, and to a greater degree in the noncoding regions.
It is estimated that allelic variation between individual humans
occurs with a frequency at about once per each 500 bases. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes. Because greater numbers of polymorphisms
occur in the noncoding regions, fewer sequences are necessary to
differentiate individuals.
[0368] If a panel of reagents from the polynucleotides described
herein is used to polynucleotiderate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
Uses For Forensic Biology
[0369] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
polynucleotidetic typing of biological evidence found at a crime
scene as a means for positively identifying, for example, a
perpetrator of a crime. To make such an identification, PCR
technology can be used to amplify DNA sequences taken from very
small biological samples such as tissues, e.g., hair or skin, or
body fluids, e.g., blood, saliva, or semen found at a crime scene.
The amplified sequence can then be compared to a standard, thereby
allowing identification of the origin of the biological sample.
This can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such probes
can be used to identify tissue by species and/or by organ type.
[0370] The BGS-19 polynucleotides of the invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme polynucleotiderated fragments. Sequences
targeted to non-coding regions are particularly appropriate for
this use as greater numbers of polymorphisms occur in the
non-coding regions, making it easier to differentiate individuals
using this technique. Examples of polynucleotide reagents include
the polynucleotides of the invention or portions thereof, e.g.,
fragments derived from non-coding regions having a length of at
least 20 or 30 bases.
[0371] Accordingly, the BGS-19 polynucleotides of the invention can
be used to provide polynucleotide reagents, e.g., labeled probes
that can be used in, for example, to identify a specific cell type
or tissue type by in situ hybridization technique.
Predictive Medicine
[0372] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. As such, the present invention contemplates use
of the BGS-19 polynucleotides, BGS-19 polypeptides, BGS-19 agonists
and/or BGS-19 antagonists of the invention to screen, diagnose,
stage, prevent and/or treat disorders characterized by aberrant
expression or activity of the BGS-19 polynucleotide and/or
polypeptides of the invention. Such disorders include, but are not
limited to, cancers, immune related disorders, developmental
disorders.
[0373] One aspect of the present invention relates to diagnostic
assays for determining expression of a polypeptide or
polynucleotide of the invention and/or activity of a polypeptide of
the invention, in the context of a biological sample (e.g., blood,
serum, cells, tissue) to thereby determine whether an individual is
afflicted with a disease or disorder, or is at risk of developing a
disorder, associated with aberrant expression or activity of a
polypeptide of the invention, such as a proliferative disorder,
e.g., cancer. Accordingly, the present invention provides a method
for diagnosing a BGS-19-related disorder, comprising comparing an
amount of BGS-19 polynucleotide or BGS-19 polypeptide expressed in
a normal tissue to an amount expressed in a diseased tissue.
[0374] The invention also provides for prognostic (or predictive)
assays for determining whether an individual is at risk of
developing a disorder associated with aberrant expression or
activity of a polypeptide of the invention. For example, mutations
in a polynucleotide of the invention can be assayed in a biological
sample. Such assays can be used for prognostic or predictive
purpose to thereby prophylactically treat an individual prior to
the onset of a disorder characterized by or associated with
aberrant expression or activity of a polypeptide of the invention.
Accordingly, the present invention provides a method for
determining a prognosis of a BGS-19-related disorder, comprising
the step of comparing an amount of BGS-19 polynucleotide or BGS-19
polypeptide expressed in a biological sample at a first stage of a
disease to an amount of BGS-19 polynucleotide or BGS-19 polypeptide
expressed in the sample at a second stage of the disease.
[0375] Another aspect of the invention provides methods for
expression of a BGS-19 polynucleotide or BGS-19 polypeptide of the
invention or activity of a BGS-19 polypeptide of the invention in
an individual to thereby select appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics"). Pharmacogenomics allows for the selection of
agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual based on the genotype of the individual (e.g., the
genotype of the individual examined to determine the ability of the
individual to respond to a particular agent).
[0376] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds) on the in
vivo expression or activity of a BGS-19 polypeptide of the
invention. These and other agents are described in further detail
in the following sections.
Diagnostic Assays
[0377] The present invention provides a method for diagnosing a
BGS-19-related disorder, comprising comparing an amount of BGS-19
polynucleotide or BGS-19 polypeptide expressed in a normal tissue
to an amount expressed in a diseased tissue. An exemplary method
for detecting the presence or absence of a BGS-19 polypeptide or
BGS-19 polynucleotide of the invention in a biological sample
involves obtaining a biological sample from a test subject and
contacting the biological sample with a compound or an agent
capable of detecting a BGS-19 polypeptide or polynucleotide (e.g.,
mRNA, genomic DNA) of the invention such that the presence of a
polypeptide or polynucleotide of the invention is detected in the
biological sample.
[0378] In a specific embodiment, the invention provides a method
for diagnosing a
[0379] BGS-19-related disorder in a subject comprising the steps of
contacting a BGS-19 antibody with a sample, suspected of containing
a BGS-19 polypeptide, from said subject under conditions that allow
the BGS-19 polypeptide to be bound by the BGS-19 antibody and
detecting or measuring binding of the BGS-19 antibody to the BGS-19
polypeptide, wherein detection or measurement of binding indicates
presence or amount, respectively, of the BGS-19 polypeptide, and
wherein the BGS-19-related disorder is determined to be present
when the presence or amount of detected BGS-19 polypeptide differs
from a control value representing the amount of BGS-19 polypeptide
present in an analogous sample from a subject not having the
BGS-19-related disorder.
[0380] A preferred agent for detecting mRNA or genomic DNA encoding
a polypeptide of the invention is a labeled nucleic acid probe
capable of hybridizing to mRNA or genomic DNA encoding a
polypeptide of the invention. The nucleic acid probe can be, for
example, a full-length cDNA, such as that presented in FIGS. 1A-C
or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 100, 250 or 500 contiguous nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
a mRNA or genomic DNA encoding a polypeptide of the invention,
excluding a polynucleotide consisting of Genbank Accession Nos.
gi|BI518708, gi|BF308356, gi|BF205116, gi|BF969219, gi|BG826221,
and/or gi|AA341128. Other suitable probes for use in the diagnostic
assays of the invention are described herein.
[0381] A preferred agent for detecting a BGS-19 polypeptide of the
invention is an antibody capable of binding to a polypeptide of the
invention, preferably an antibody with a detectable label.
Antibodies can be polyclonal, or more preferably, monoclonal. An
intact antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2)
can be used. The term "labeled", with regard to the probe or
antibody, is intended to encompass direct labeling of the probe or
antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin.
[0382] The detection methods of the invention can be used to detect
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
mRNA include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of a polypeptide of the invention
include enzyme linked immunosorbent assay (ELISA), Western
blotting, immunoprecipitation and immunofluorescence. In vitro
techniques for detection of genomic DNA include Southern
hybridizations. Furthermore, in vivo techniques for detection of a
polypeptide of the invention include introducing into a subject a
labeled antibody directed against the polypeptide. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques.
[0383] In one embodiment, the biological sample comprises protein
molecules from the test subject. Alternatively, the biological
sample can comprise mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0384] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting a
BGS-19 polypeptide of the invention or mRNA or genomic DNA encoding
a polypeptide of the invention, such that the presence of the
polypeptide or mRNA or genomic DNA encoding the polypeptide is
detected in the biological sample, and comparing the presence of
the polypeptide or mRNA or genomic DNA encoding the polypeptide in
the control sample with the presence of the polypeptide or mRNA or
genomic DNA encoding the polypeptide in the test sample.
[0385] Probes based on the sequence of a BGS-19 polynucleotide of
the invention can be used to detect transcripts or genomic
sequences encoding the same protein molecule encoded by a selected
polynucleotide. The probe comprises a label group attached thereto,
e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor. Such probes can be used as part of a diagnostic
test kit for examining protein expression in cells or tissues, such
as by measuring levels of a polynucleotide encoding the protein in
a sample of cells from a subject, e.g., detecting mRNA levels or
determining whether a polynucleotide encoding the protein has been
mutated or deleted.
[0386] Antibodies directed against wild-type or mutant BGS-19
polynucleotides or polypeptides, or conserved variants or peptide
fragments thereof, may also be used as diagnostics and prognostics,
as described herein. Such diagnostic methods, may be used to detect
abnormalities in the level of BGS-19 polynucleotide expression, or
abnormalities in the structure and/or temporal, tissue, cellular,
or subcellular location of BGS-19 polynucleotide product.
Antibodies, or fragments of antibodies, such as those described
below, may be used to screen potentially therapeutic compounds in
vitro to determine their effects on BGS-19 polynucleotide
expression and BGS-19 peptide production. The compounds which have
beneficial effects on cancer can be identified and a
therapeutically effective dose determined.
[0387] The tissue or cell type to be analyzed will
polynucleotiderally include those which are known, or suspected, to
express the BGS-19 polynucleotide such as, for example, bone
marrow, brain, heart, kidney, liver, lung, lymph node, pancreas,
prostate, small intestine, spinal cord, spleen, stomach, thymus and
uterus. Many protein isolation methods are well known in the art
(see, e.g., Harlow and Lane, 1988, Antibodies: A Laboratorv Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
which is incorporated herein by reference in its entirety). The
isolated cells can be derived from cell culture or from a patient.
The analysis of cells taken from culture may be a necessary step to
test the effect of compounds on the expression of the BGS-19
polynucleotide.
[0388] Preferred diagnostic methods for the detection of BGS-19
polynucleotide products or conserved variants or peptide fragments
thereof, may involve, for example, immunoassays wherein the BGS-19
polynucleotide products or conserved variants, including
polynucleotide products which are the result of alternatively
spliced transcripts, or peptide fragments are detected by their
interaction with an anti-BGS-19 polynucleotide product-specific
antibody.
[0389] For example, antibodies, or fragments of antibodies, useful
in the present invention may be used to quantitatively or
qualitatively detect the presence of BGS-19 polynucleotide products
or conserved variants or peptide fragments thereof. The antibodies
(or fragments thereof) useful in the present invention may,
additionally, be employed histologically, as in immunofluorescence
or immunoelectron microscopy, for in situ detection of BGS-19
polynucleotide products or conserved variants or peptide fragments
thereof. In situ detection may be accomplished by removing a
histological specimen from a patient, such as paraffin embedded
sections of lymphoid tissues and applying thereto a labeled
antibody of the present invention. The antibody (or fragment) is
preferably applied by overlaying the labeled antibody (or fragment)
onto a biological sample. When a BGS-19 polynucleotide product is
present in the cytoplasm, the antibody of the invention can be
introduced inside the cell, for example, by making the cell
membrane permeable. Through the use of such a procedure, it is
possible to determine not only the presence of a BGS-19
polynucleotide product, or conserved variants or peptide fragments,
but also the distribution of a BGS-19 in a cell, tissue, or organ
of interest. Using the present invention, those of ordinary skill
will readily perceive that any of a wide variety of histological
methods (such as staining procedures) can be modified in order to
achieve such in situ detection.
[0390] Immunoassays for BGS-19 polynucleotide products or conserved
variants or peptide fragments thereof will typically comprise
incubating a sample, such as a biological fluid, a tissue extract,
freshly harvested cells, or lysates of cells which have been
incubated in cell culture, in the presence of a detectably labeled
antibody capable of identifying BGS-19 polynucleotide products or
conserved variants or peptide fragments thereof, and detecting the
bound antibody by any of a number of techniques well-known in the
art.
[0391] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled BGS-19 polynucleotide specific antibody. The
solid phase support may then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on solid
support may then be detected by conventional means.
[0392] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0393] The binding activity of a given lot of anti-BGS-19
polynucleotide product antibody may be determined according to
standard methods. Those skilled in the art will be able to
determine operative and optimal assay conditions for each
determination by using standard techniques.
[0394] One of the ways in which the BGS-19 polynucleotide
peptide-specific antibody can be detectably labeled is by linking
the same to an enzyme and use in an enzyme immunoassay (Voller,
1978, "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978,
Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly
Publication, Walkersville, Md.); Voller et al., 1978, J Clin
Pathol. 31:507-520; Butler, 1981, Meth Enzymol. 73:482-523; Maggio,
Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa et
al., Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme
which is bound to the antibody will react with an appropriate
substrate, preferably a chromogenic substrate, in such a manner as
to produce a chemical moiety which can be detected, for example, by
spectrophotometric, fluorimetric or by visual means. Enzymes which
can be used to detectably label the antibody include, but are not
limited to, malate dehydrogenase, staphylococcal nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0395] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect BGS-19
peptides through the use of a radioimmunoassay ("RIA") (See, e.g.,
B. Weintraub, Principles of Radioimmunoassays. Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
gamma counter or a scintillation counter or by autoradiography.
[0396] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine. The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid ("DTPA") or ethylenediaminetetraacetic acid ("EDTA"). The
antibody also can be detectably labeled by coupling it to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0397] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0398] Any of numerous immunoassays can be used in the practice of
the instant invention. Antibodies, or antibody fragments comprising
the binding domain, are known in the art or can be obtained by
procedures standard in the art such as those described in Harlow
and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988.
Detecting and Staging Cancer in a Patient
[0399] Cancer can be detected and staged in a patient using a
BGS-19 polynucleotide and polypeptide of the invention. In one
embodiment of the present invention, measurement of at least one
BGS-19 polynucleotide products or fragments thereof, or soluble
BGS-19 polynucleotide products can be used to detect cancer in a
subject or to stage the cancer in a subject.
[0400] Staging refers to the grouping of patients according to the
extent of their disease. Staging is useful in choosing treatment
for individual patients, estimating prognosis, and comparing the
results of different treatment programs. Staging of cancer is
performed initially on a clinical basis, according to the physical
examination and laboratory radiologic evaluation. The most widely
used clinical staging system is the one adopted by the
International Union against Cancer (UICC) and the American Joint
Committee on Cancer (AJCC) Staging and End Results Reporting. It is
based on the tumor-nodes-metastases (TNM) system as detailed in the
1988 Manual for Staging of Cancer.
[0401] Accordingly, in an exemplary embodiment, the invention
provides a method for staging a disease in a subject comprising the
steps of contacting a BGS-19 binding protein with a sample,
suspected of containing a BGS-19 polypeptide, from the subject
under conditions that allow the BGS-19 polypeptide to be bound by
the BGS-19 binding protein and detecting or measuring binding of
the BGS-19 binding protein to the BGS-19 polypeptide, wherein
detection or measurement of binding indicates presence or amount,
respectively, of the BGS-19 polypeptide, and wherein the stage of
the disease is determined when the presence or amount of detected
BGS-19 polypeptide is compared with the amount of BGS-19
polypeptide present in an analogous sample from a subject having a
particular stage of the disease. In a further embodiment, the
disease is a BGS-19-related disorder.
[0402] In another embodiment, the invention provides a method for
staging a BGS-19-related disorder in a subject comprising the steps
of contacting a BGS-19 antibody with a sample, suspected of
containing a BGS-19 polypeptide, from the subject under conditions
that allow the BGS-19 polypeptide to be bound by the BGS-19
antibody and detecting or measuring binding of the BGS-19 antibody
to the BGS-19 polypeptide, wherein detection or measurement of
binding indicates presence or amount, respectively, of the BGS-19
polypeptide, and wherein the stage of the BGS-19-related disorder
in the subject is determined when the presence or amount of
detected BGS-19 polypeptide is compared with the amount of BGS-19
polypeptide present in an analogous sample from a subject having a
particular stage of the BGS-19-related disorder.
Prognostic Assays
[0403] The methods described herein can furthermore be utilized as
prognostic assays to identify subjects having or at risk of
developing a disease or disorder associated with aberrant
expression or activity of a BGS-19 polypeptide of the invention.
For example, the assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with aberrant expression or activity of a BGS-19
polypeptide of the invention, e.g., an immunologic disorder, or
proliferative disorders.
[0404] Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing such a disease
or disorder. Thus, the present invention provides a method in which
a test sample is obtained from a subject and a BGS-19 polypeptide
or polynucleotide (e.g., mRNA, genomic DNA) of the invention is
detected, wherein the presence of the polypeptide or polynucleotide
is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the polypeptide. As used herein, a "test sample" refers to a
biological sample obtained from a subject of interest. For example,
a test sample can be a biological fluid (e.g., serum), cell sample,
or tissue.
[0405] The prognostic assays described herein, for example, can be
used to identify a subject having or at risk of developing
disorders such as cancers. In another example, prognostic assays
described herein can be used to identify a subject having or at
risk of developing related disorders associated with expression of
polypeptides of the invention.
[0406] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant expression or activity
of a polypeptide of the invention. For example, such methods can be
used to determine whether a subject can be effectively treated with
a specific agent or class of agents (e.g., agents of a type which
decrease activity of the polypeptide). Thus, the present invention
provides methods for determining whether a subject can be
effectively treated with an agent for a disorder associated with
aberrant expression or activity of a polypeptide of the invention
in which a test sample is obtained and the polypeptide or
polynucleotide encoding the polypeptide is detected (e.g., wherein
the presence of the polypeptide or polynucleotide is diagnostic for
a subject that can be administered the agent to treat a disorder
associated with aberrant expression or activity of the
polypeptide).
[0407] The methods of the invention can also be used to detect
polynucleotidetic lesions or mutations in a BGsS19 polynucleotide
of the invention, thereby determining if a subject with the
lesioned polynucleotide is at risk for a disorder characterized by
aberrant expression or activity of a polypeptide of the invention.
In preferred embodiments, the methods include detecting, in a
sample of cells from the subject, the presence or absence of a
polynucleotidetic lesion or mutation characterized by at least one
of an alteration affecting the integrity of a polynucleotide
encoding the polypeptide of the invention, or the mis-expression of
the polynucleotide encoding the polypeptide of the invention. For
example, such polynucleotidetic lesions or mutations can be
detected by ascertaining the existence of at least one of: 1) a
deletion of one or more nucleotides from the BGS-19 polynucleotide;
2) an addition of one or more nucleotides to the BGS-19
polynucleotide;
[0408] 3) a substitution of one or more nucleotides of the BGS-19
polynucleotide; 4) a chromosomal rearrangement of the BGS-19
polynucleotide; 5) an alteration in the level of a messenger RNA
transcript of the BGS-19 polynucleotide; 6) an aberrant
modification of the BGS-19 polynucleotide, such as of the
methylation pattern of the genomic DNA; 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of the
BGS-19 polynucleotide; 8) a non-wild type level of the protein
encoded by the BGS-19 polynucleotide; 9) an allelic loss of a the
BGS-19 polynucleotide; and 10) an inappropriate post-translational
modification of the protein encoded by the BGS-19 polynucleotide.
As described herein, there are a large number of assay techniques
known in the art which can be used for detecting lesions in a
polynucleotide.
[0409] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction ("PCR") (See,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (see,
e.g., Landegran et al., 1988, Science 241:1077-1080; Nakazawa et
al., 1994, Proc Natl Acad. Sci. 91:360-364), the latter of which
can be particularly useful for detecting point mutations in a
polynucleotide (See, e.g., Abravaya et al., 1995, Nucleic Acids
Res. 23:675-682). This method can include the steps of collecting a
sample of cells from a patient, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the cells of the sample, contacting the
nucleic acid sample with one or more primers which specifically
hybridize to the selected polynucleotide under conditions such that
hybridization and amplification of the polynucleotide (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or ligation chain reaction can be useful as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0410] Alternative amplification methods include self sustained
sequence replication (Guatelli et al., 1990, Proc Natl Acad. Sci.
87:1874-1878), transcriptional amplification system (Kwoh et al.,
1989, Proc Natl Acad. Sci. 86:1173-1177), Q-Beta Replicase (Lizardi
et al., 1988, Biotechnology 6:1197), or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques well known to those of skill in the art.
These detection schemes are especially useful for the detection of
polynucleotides if such molecules are present in very low
numbers.
[0411] In an alternative embodiment, mutations in a selected
polynucleotide from a sample cell can be identified by alterations
in restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined by gel electrophoresis and compared. Differences in
fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can be used to score
for the presence of specific mutations by development or loss of a
ribozyme cleavage site.
[0412] In other embodiments, polynucleotidetic mutations can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high-density arrays comprising hundreds or thousands
of oligonucleotides probes (Cronin et al., 1996, Human Mutation
7:244-255; Kozal et al., 1996, Nature Medicine 2:753-759). For
example, polynucleotidetic mutations can be identified in
two-dimensional arrays containing light-polynucleotiderated DNA
probes as described in Cronin et al., supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type polynucleotide and
the other complementary to the mutant polynucleotide.
[0413] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
selected polynucleotide and detect mutations by comparing the
sequence of the sample nucleic acids with the corresponding
wild-type (control) sequence (see, e.g., Maxim and Gilbert, 1977,
Proc Natl Acad. Sci. 74:560; Sanger, 1977, Proc Natl Acad. Sci.
74:5463). It is also contemplated that any of a variety of
automated sequencing procedures can be utilized when performing the
diagnostic assays, including sequencing by mass spectrometry (see,
e.g., PCT Publication No. WO 94/16101; Cohen et al., 1996 Adv
Chromatogr. 36:127-162; Naeve et al., 1995, "Accuracy of automated
DNA sequencing: a multi-laboratory comparison of sequencing
results", Biotechniques. 19:448-453; Griffin et al., 1993, Appl
Biochem Biotechnol. 38:147-159).
[0414] Other methods for detecting mutations in a selected
polynucleotide include methods in which protection from cleavage
agents is used to detect mismatched bases in RNA/RNA or RNA/DNA
heteroduplexes (Myers et al., 1985, Science 230:1242). In
polynucleotideral, the technique of "mismatch cleavage" entails
providing heteroduplexes formed by hybridizing (labeled) RNA or DNA
comprising the wild-type sequence with potentially mutant RNA or
DNA obtained from a tissue sample. The double-stranded duplexes are
treated with an agent which cleaves single-stranded regions of the
duplex such as which will exist due to base pair mismatches between
the control and sample strands. RNA/DNA duplexes can be treated
with RNase to digest mismatched regions, and DNA/DNA hybrids can be
treated with S1 nuclease to digest mismatched regions.
[0415] In other embodiments, either DNA/DNA or RNA/DNA duplexes can
be treated with hydroxylamine or osmium tetroxide and with
piperidine in order to digest mismatched regions. After digestion
of the mismatched regions, the resulting material is then separated
by size on denaturing polyacrylamide gels to determine the site of
mutation (see, e.g., Cotton et al., 1988, Proc Natl Acad. Sci.
85:4397; Saleeba et al., 1992, Methods Enzymol. 217:286-295). In a
preferred embodiment, the control DNA or RNA can be labeled for
detection.
[0416] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in cDNAs
obtained from samples of cells. For example, the mutY enzyme of E.
coli cleaves A at G/A mismatches and the thymidine DNA glycosylase
from HeLa cells cleaves T at G/T mismatches (Hsu et al., 1994,
Carcinopolynucleotidesis 15:1657-1662). Accordingly, in one
embodiment, a probe based on a selected sequence, e.g., a wild-type
sequence, is hybridized to a cDNA or other DNA product from a test
cell(s). The duplex is treated with a DNA mismatch repair enzyme,
and the cleavage products, if any, can be detected from
electrophoresis protocols or the like (see, e.g., U.S. Pat. No.
5,459,039).
[0417] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in polynucleotides. For
example, single strand conformation polymorphism may be used to
detect differences in electrophoretic mobility between mutant and
wild type nucleic acids (see, e.g., Orita et al., 1989, Proc Natl
Acad. Sci. 86:2766; Cotton, 1993, Mutat Res. 285:125-144; Hayashi,
1992, Genet Anal Tech Appl. 9:73-79). Single-stranded DNA fragments
of sample and control nucleic acids will be denatured and allowed
to renature. The secondary structure of single-stranded nucleic
acids varies according to sequence, and the resulting alteration in
electrophoretic mobility enables the detection of even a single
base change. The DNA fragments may be labeled or detected with
labeled probes. The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In a preferred embodiment,
the subject method utilizes heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al., 1991, Trends Genet.
7:5).
[0418] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(Myers et al., 1985, Nature 313:495). When denaturing gradient gel
electrophoresis is used as the method of analysis, DNA will be
modified to insure that it does not completely denature, for
example by adding a GC clamp of approximately 40 bp of high-melting
GC-rich DNA by PCR. In a further embodiment, a temperature gradient
is used in place of a denaturing gradient to identify differences
in the mobility of control and sample DNA (Rosenbaum and Reissner,
1987, Biophys Chem. 265:12753).
[0419] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al., 1986, Nature 324:163); Saiki
et al., 1989, Proc Natl Acad. Sci. 86:6230). Such allele specific
oligonucleotides are hybridized to PCR amplified target DNA or a
number of different mutations when the oligonucleotides are
attached to the hybridizing membrane and hybridized with labeled
target DNA.
[0420] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al., 1989, Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent or reduce
polymerase extension (Prossner, 1993, Tibtech 11:238). In addition,
it maybe desirable to introduce a novel restriction site in the
region of the mutation to create cleavage-based detection
(Gasparini et al., 1992, Mol. Cell. Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany, 1991, Proc Natl Acad.
Sci. 88:189). In such cases, ligation will occur only if there is a
perfect match at the 3' end of the 5' sequence making it possible
to detect the presence of a known mutation at a specific site by
looking for the presence or absence of amplification.
[0421] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
polynucleotide probe or antibody reagent described herein, which
may be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a polynucleotide encoding a polypeptide of the
invention. Furthermore, any cell type or tissue, e.g., preferably
peripheral blood leukocytes, in which the polypeptide of the
invention is expressed may be utilized in the prognostic assays
described herein.
Pharmacogenomics
[0422] Pharmacogenomics relates to clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons (see, e.g.,
Linder, 1997, Clin Chem. 43:254-266. In polynucleotideral, two
types of pharmacopolynucleotidetic conditions can be
differentiated. Genetic conditions transmitted as a single factor
altering the way drugs act on the body are referred to as "altered
drug action." Genetic conditions transmitted as single factors
altering the way the body acts on drugs are referred to as "altered
drug metabolism". These pharmacopolynucleotidetic conditions can
occur either as rare defects or as polymorphisms.
[0423] Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, the pharmacogenomics of the individual permits the selection
of effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate
dosages and therapeutic regimens. Accordingly, the activity of a
BGS-19 polypeptide of the invention, expression of a BGS-19
polynucleotide of the invention, or mutation content of the BGS-19
polynucleotide of the invention in an individual can be determined
to thereby select appropriate agent(s) for therapeutic or
prophylactic treatment of the individual.
[0424] Accordingly, in addition to the nucleotide sequence
presented, it will be appreciated by those skilled in the art that
DNA sequence polymorphisms that lead to changes in the amino acid
sequence may exist within a population (e.g., the human
population). Such polynucleotidetic polymorphisms may exist among
individuals within a population due to natural allelic variation.
An allele is one of a group of polynucleotides which occur
alternatively at a given polynucleotidetic locus. As used herein,
the phrase "allelic variant" refers to a nucleotide sequence which
occurs at a given locus or to a polypeptide encoded by the
nucleotide sequence. Such natural allelic variations can typically
result in 1-5% variance in the nucleotide sequence of a given
polynucleotide. Alternative alleles can be identified by sequencing
the polynucleotide of interest in a number of different individuals
or by using hybridization probes to identify the same
polynucleotidetic locus in a variety of individuals. Any and all
such nucleotide variations and resulting amino acid polymorphisms
or variations that are the result of natural allelic variation and
that do not alter the functional activity are intended to be within
the scope of the invention.
Prophylactic Methods
[0425] The present invention provides for prophylactic and
therapeutic methods of treating a subject at risk of or having the
BGS-19-related disorder. Such the BGS-19-related disorder includes,
but is not limited to, immune related disorders, developmental
disorders, autoimmune disease, and cancer.
[0426] Accordingly, the present invention provides for prophylactic
and therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant expression or activity of the BGS-19 polypeptide of the
invention. For example, disorders characterized by aberrant
expression or activity of the polypeptides.
[0427] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant expression or activity of the BGS-19 polypeptide of the
invention, by administering to the subject an agent which modulates
expression of at least one activity of the BGS-19 polypeptide.
Subjects at risk for a disease which is caused or contributed to by
aberrant expression or activity of the BGS-19 polypeptide of the
invention can be identified by, for example, any or a combination
of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the aberrancy, such
that a disease or disorder is prevented or, alternatively, delayed
in its progression. Depending on the type of aberrancy, for
example, an agonist or antagonist agent can be used for treating
the subject.
[0428] The prophylactic agents described herein can be used to
treat a subject at risk of developing a BGS-19-related disorder.
The appropriate prophylactic agent can be determined based on
screening assays described herein.
Methods of Treatment
[0429] The present invention provides for methods for the
prevention and/or treatment of a BGS-19-related disorder comprising
administering to a patient in need thereof a BGS-19 polynucleotide,
BGS-19 polypeptide, BGS-19 agonist, BGS-19 antagonist, or an
inhibitor of a BGS-19 agonist or antagonist.
[0430] More particularly, the present invention relates to uses of
BGS-19 polynucleotides, polypeptides, and BGS-19 antagonists for
the prevention, diagnosis, prognosis and management of immune
related disorders and cancer. The invention contemplates uses of
BGS-19 polynucleotides, polypeptides, and BGS-19 antagonists (e.g.,
antibodies directed against BGS-19 polypeptides of the invention)
to treat such diseases.
[0431] The invention pertains to methods of modulating BGS-19
expression or activity of a BGS-19 polypeptide of the invention for
therapeutic purposes. The modulatory method of the invention
involves contacting a cell with an agent that modulates one or more
of the activities of the polypeptide. An agent that modulates
activity can be an agent as described herein, such as a nucleic
acid or a protein, a naturally-occurring cognate ligand of the
polypeptide, a peptide, a peptidomimetic, or other small molecules.
In one embodiment, the agent stimulates one or more of the
biological activities of the polypeptide. Examples of such
stimulatory agents include the active polypeptide of the invention
and a polynucleotide encoding the polypeptide of the invention that
has been introduced into the cell.
[0432] In another embodiment, the agent inhibits one or more of the
biological activities of the polypeptide of the invention. Examples
of such inhibitory agents include antisense polynucleotides and
antibodies. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject or in the
vicinity of the cells). As such, the present invention provides
methods of treating an individual afflicted with a disease or
disorder characterized by aberrant expression or activity of a
BGS-19 polypeptide of the invention. In one embodiment, the method
involves administering an agent (e.g., an agent identified by a
screening assay described herein), or combination of agents that
modulates (e.g., upregulates or downregulates) expression or
activity. In another embodiment, the method involves administering
a BGS-9 polypeptide of the invention or a polynucleotide of the
invention as therapy to compensate for reduced or aberrant
expression or activity of the polypeptide.
[0433] Stimulation of activity is desirable in situations in which
activity or expression is abnormally low or downregulated and/or in
which increased activity is likely to have a beneficial effect.
Conversely, inhibition of activity is desirable in situations in
which activity or expression is abnormally high or upregulated
and/or in which decreased activity is likely to have a beneficial
effect.
Cancers and Therapeutics
[0434] BGS-19 polynucleotides, BGS-19 polypeptides, BGS-19
agonists, BGS-19 antagonists, inhibitors of such agonists or
antagonists, and variants thereof, can be used to modulate the
development and progression of non-cancerous cell-proliferative
disorders such as, but not limited to, deregulated proliferation
(e.g., hyperdysplasia, hyper-IgM syndrome, or lymphoproliferative
disorders), cirrhosis of the liver (a condition in which scarring
has overtaken normal liver repolynucleotideration processes),
treatment of keloid (hypertrophic scar) formation (disfiguring of
the skin in which the scarring process interferes with normal
renewal), psoriasis (a common skin condition characterized by
excessive proliferation of the skin and delay in proper cell fate
determination), benign tumors, fibrocystic conditions, tissue
hypertrophy (e.g., prostatic hyperplasia), ovarian cancer or
proliferative ovarian disorder.
[0435] BGS-19 polynucleotides, BGS-19 polypeptides, BGS-19
agonists, BGS-19 antagonists, inhibitors of such agonists or
antagonists, and variants thereof, can also be used to modulate the
development and progression of cancers such as, but not limited to,
neoplasms, tumors, carcinomas, sarcomas, adenomas or myeloid
lymphoma tumors, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leimyosarcoma,
rhabdotheliosarcoma, colon sarcoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hematoma, bile duct
carcinoma, melanoma, choriocarcinoma, semicoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependynoma, pinealoma, hemangioblastoma, retinoblastoma),
leukemias, (e.g. acute lymphocytic leukemia), acute myelocytic
leukemia (myelolastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia), or
polycythemia vera, or lymphomas (Hodgkin's disease and
non-Hodgkin's diseases), multiple myelomas and Waldenstrom's
macroglobulinemia. In particular, the BGS-19 polynucleotides,
polypeptides, and modulators thereof, can be used to modulate the
development and progression of hormone-sensitive cancers, such as
but not limited to cancer of the breast, ovary, uterus, prostate,
testis, skin and brain.
Antisense Therapy
[0436] The present invention provides compositions and methods for
the use of a BGS-19 antisense oligonucleotide to prevent or treat a
BGS-19-related disorder, such as cancer. The invention also
provides pharmaceutical compositions comprising a BGS-19 antisense
oligonucleotide, as well as methods for their prophylactic and
therapeutic use. An antisense oligonucleotide, or an analogue or
derivative thereof, refers to a range of chemical species that
recognize polynucleotide target sequences through Watson-and-Crick
hydrogen bonding interactions with the nucleotide bases of the
target sequences. The target sequences may be RNA or DNA, and may
be single-stranded or double-stranded. Target molecules include,
but are not limited to, pre-mRNA, mRNA, and DNA. Also encompassed
by the invention are drug delivery means and therapeutic regimens
for the pharmaceutical compositions of the invention.
[0437] In one embodiment, a BGS-19 antisense oligonucleotide is
administered to a human to prevent or treat a BGS-19 related
disorder, wherein BGS-19 mRNA or protein is expressed at
above-normal levels.
[0438] In another embodiment, a BGS-19 antisense oligonucleotide is
administered to a human at a high dose to prevent or treat a BGS-19
related disorder.
[0439] In another embodiment, a BGS-19 antisense oligonucleotide is
administered to a human at a low or reduced dose to prevent or
treat a BGS-19 related disorder.
[0440] Aside from affecting diseased tissue, a BGS-19 antisense
oligonucleotide can affect normal tissues, which include tissues
containing cells that normally express a BGS-19 polynucleotide.
Additionally, a BGS-19 antisense oligonucleotide can affect normal
tissues that, although not expressing a BGS-19 polynucleotide, are
compromised by diseased tissues. In a particular embodiment, a
BGS-19 antisense oligonucleotide can protect normal tissues that do
or do not normally express a BGS-19 polynucleotide.
[0441] In a specific embodiment, the invention provides for a
BGS-19 antisense oligonucleotide that is administered to a human,
in combination with one of more additional therapeutic agents. In a
further embodiment, the additional therapeutic agent is a second,
different antisense oligonucleotide. In another further embodiment,
the additional therapeutic agent is a chemotherapeutic agent.
Antibody Therapy
[0442] The present invention provides for methods for preventing or
treating a BGS-19 related disorder comprising administering to a
patient in need thereof, an antibody that can bind a BGS-19
polypeptide.
[0443] Accordingly, by following procedures well known in the art
for developing antibodies useful in clinical settings, an antibody
directed to a BGS-19 polypeptide can be used for antibody therapy.
Depending on the disease to be treated, a dose of approximately 1
.mu.g/kg to 20 mg/kg of a composition comprising an antibody of the
present invention is administered to the patient. In one
embodiment, the dose of antibody is 1 .mu.g/kg to 100 .mu.g/kg. In
another embodiment, the dose of antibody is 101 .mu.g/kg to 999
.mu.g/kg. In another embodiment, the dose of antibody is 1 mg/kg to
5 mg/kg. In yet another embodiment, the dose of antibody is 6 mg/kg
to 10 mg/kg. In yet another embodiment, the dose of antibody is 11
mg/kg to 20 mg/kg. The progress of an antibody therapy can be
monitored using standard techniques and assays (See, e.g.,
International Patent Publication No. WO 94/04188).
Small Molecule Therapy
[0444] The present invention also encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. The factors to consider in choosing an appropriate dose
of a small molecule agent will be understood by the ordinarily
skilled physician, veterinarian, or scientist. The dose(s) of the
small molecule will vary, for example, depending upon the identity,
size, and condition of the subject or sample being treated, further
depending upon the route by which the composition is to be
administered, if applicable, and the effect which the practitioner
desires the small molecule to have upon the polynucleotide or
polypeptide of the invention. Exemplary doses include milligram or
microgram amounts of the small molecule per kilogram of subject or
sample weight (e.g., about 1 microgram per kilogram to about 500
milligrams per kilogram, about 100 micrograms per kilogram to about
5 milligrams per kilogram, or about 1 microgram per kilogram to
about 50 micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or polynucleotide
of the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, polynucleotideral health, gender, and diet of the
subject, the time of administration, the route of administration,
the rate of excretion, any drug combination, and the degree of
expression or activity to be modulated.
[0445] The human BGS-19 polypeptides and/or peptides of the present
invention, or immunogenic fragments or oligopeptides thereof, can
be used for screening therapeutic drugs or compounds in a variety
of drug screening techniques. The fragment employed in such a
screening assay may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. The
reduction or abolition of activity of the formation of binding
complexes between the ion channel protein and the agent being
tested can be measured. Thus, the present invention provides a
method for screening or assessing a plurality of compounds for
their specific binding affinity with a BGS-19 polypeptide, or a
bindable peptide fragment, of this invention, comprising providing
a plurality of compounds, combining the BGS-19 polypeptide, or a
bindable peptide fragment, with each of a plurality of compounds
for a time sufficient to allow binding under suitable conditions
and detecting binding of the BGS-19 polypeptide or peptide to each
of the plurality of test compounds, thereby identifying the
compounds that specifically bind to the BGS-19 polypeptide or
peptide.
[0446] Methods of identifying compounds that modulate the activity
of the novel human BGS-19 polypeptides and/or peptides are provided
by the present invention and comprise combining a potential or
candidate compound or drug modulator of immunoglobulin domain
containing polypeptide biological activity with an BGS-19
polypeptide or peptide, for example, the BGS-19 amino acid sequence
as set forth in SEQ ID NO:2, and measuring an effect of the
candidate compound or drug modulator on the biological activity of
the BGS-19 polypeptide or peptide. Such measurable effects include,
for example, physical binding interaction; the ability to cleave a
suitable immunoglobulin domain containing polypeptide substrate;
effects on native and cloned BGS-19-expressing cell line; and
effects of modulators or other immunoglobulin domain containing
polypeptide-mediated physiological measures.
[0447] Another method of identifying compounds that modulate the
biological activity of the novel BGS-19 polypeptides of the present
invention comprises combining a potential or candidate compound or
drug modulator of a immunoglobulin domain containing polypeptide
biological activity with a host cell that expresses the BGS-19
polypeptide and measuring an effect of the candidate compound or
drug modulator on the biological activity of the BGS-19
polypeptide. The host cell can also be capable of being induced to
express the BGS-19 polypeptide, e.g., via inducible expression.
Physiological effects of a given modulator candidate on the BGS-19
polypeptide can also be measured. Thus, cellular assays for
particular immunoglobulin domain containing polypeptide modulators
may be either direct measurement or quantification of the physical
biological activity of the BGS-19 polypeptide, or they may be
measurement or quantification of a physiological effect. Such
methods preferably employ a BGS-19 polypeptide as described herein,
or an overexpressed recombinant BGS-19 polypeptide in suitable host
cells containing an expression vector as described herein, wherein
the BGS-19 polypeptide is expressed, overexpressed, or undergoes
upregulated expression.
[0448] Another aspect of the present invention embraces a method of
screening for a compound that is capable of modulating the
biological activity of a BGS-19 polypeptide, comprising providing a
host cell containing an expression vector harboring a nucleic acid
sequence encoding a BGS-19 polypeptide, or a functional peptide or
portion thereof (e.g., SEQ ID NOS:2); determining the biological
activity of the expressed BGS-19 polypeptide in the absence of a
modulator compound; contacting the cell with the modulator compound
and determining the biological activity of the expressed BGS-19
polypeptide in the presence of the modulator compound. In such a
method, a difference between the activity of the BGS-19 polypeptide
in the presence of the modulator compound and in the absence of the
modulator compound indicates a modulating effect of the
compound.
[0449] Essentially any chemical compound can be employed as a
potential modulator or ligand in the assays according to the
present invention. Compounds tested as immunoglobulin domain
containing polypeptide modulators can be any small chemical
compound, or biological entity (e.g., protein, sugar, nucleic acid,
lipid). Test compounds will typically be small chemical molecules
and peptides. Generally, the compounds used as potential modulators
can be dissolved in aqueous or organic (e.g., DMSO-based)
solutions. The assays are designed to screen large chemical
libraries by automating the assay steps and providing compounds
from any convenient source. Assays are typically run in parallel,
for example, in microtiter formats on microtiter plates in robotic
assays. There are many suppliers of chemical compounds, including
Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich
(St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland), for example. Also, compounds may be synthesized by
methods known in the art.
[0450] High throughput screening methodologies are particularly
envisioned for the detection of modulators of the novel BGS-19
polynucleotides and polypeptides described herein. Such high
throughput screening methods typically involve providing a
combinatorial chemical or peptide library containing a large number
of potential therapeutic compounds (e.g., ligand or modulator
compounds). Such combinatorial chemical libraries or ligand
libraries are then screened in one or more assays to identify those
library members (e.g., particular chemical species or subclasses)
that display a desired characteristic activity. The compounds so
identified can serve as conventional lead compounds, or can
themselves be used as potential or actual therapeutics.
[0451] A combinatorial chemical library is a collection of diverse
chemical compounds generated either by chemical synthesis or
biological synthesis, by combining a number of chemical building
blocks (i.e., reagents such as amino acids). As an example, a
linear combinatorial library, e.g., a polypeptide or peptide
library, is formed by combining a set of chemical building blocks
in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide or peptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks.
[0452] The preparation and screening of combinatorial chemical
libraries is well known to those having skill in the pertinent art.
Combinatorial libraries include, without limitation, peptide
libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept.
Prot. Res., 37:487-493; and Houghton et al., 1991, Nature,
354:84-88). Other chemistries for generating chemical diversity
libraries can also be used. Nonlimiting examples of chemical
diversity library chemistries include, peptoids (PCT Publication
No. WO 91/019735), encoded peptides (PCT Publication No. WO
93/20242), random bio-oligomers (PCT Publication No. WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993,
Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides
(Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., 1992,
J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of
small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc.,
116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303),
and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem.,
59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook,
all supra), peptide nucleic acid libraries (U.S. Pat. No.
5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature
Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate
libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and
U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g.,
benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S.
Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; and the like).
[0453] Devices for the preparation of combinatorial libraries are
commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,
Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, a large number of combinatorial libraries are
commercially available (e.g., ComGenex, Princeton, N.J.; Asinex,
Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd.,
Moscow, Russia; 3 D Pharmaceuticals, Exton, Pa.; Martek
Biosciences, Columbia, Md., and the like).
[0454] In one embodiment, the invention provides solid phase based
in vitro assays in a high throughput format, where the cell or
tissue expressing an ion channel is attached to a solid phase
substrate. In such high throughput assays, it is possible to screen
up to several thousand different modulators or ligands in a single
day. In particular, each well of a microtiter plate can be used to
perform a separate assay against a selected potential modulator,
or, if concentration or incubation time effects are to be observed,
every 5-10 wells can test a single modulator. Thus, a single
standard microtiter plate can assay about 96 modulators. If 1536
well plates are used, then a single plate can easily assay from
about 100 to about 1500 different compounds. It is possible to
assay several different plates per day; thus, for example, assay
screens for up to about 6,000-20,000 different compounds are
possible using the described integrated systems.
[0455] In another of its aspects, the present invention encompasses
screening and small molecule (e.g., drug) detection assays which
involve the detection or identification of small molecules that can
bind to a given protein, i.e., a BGS-19 polypeptide or peptide.
Particularly preferred are assays suitable for high throughput
screening methodologies.
[0456] In such binding-based detection, identification, or
screening assays, a functional assay is not typically required. All
that is needed is a target protein, preferably substantially
purified, and a library or panel of compounds (e.g., ligands,
drugs, small molecules) or biological entities to be screened or
assayed for binding to the protein target. Preferably, most small
molecules that bind to the target protein will modulate activity in
some manner, due to preferential, higher affinity binding to
functional areas or sites on the protein.
[0457] An example of such an assay is the fluorescence based
thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP,
Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920
to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News,
20(8)). The assay allows the detection of small molecules (e.g.,
drugs, ligands) that bind to expressed, and preferably purified,
ion channel polypeptide based on affinity of binding determinations
by analyzing thermal unfolding curves of protein-drug or ligand
complexes. The drugs or binding molecules determined by this
technique can be further assayed, if desired, by methods, such as
those described herein, to determine if the molecules affect or
modulate function or activity of the target protein.
[0458] To purify a BGS-19 polypeptide or peptide to measure a
biological binding or ligand binding activity, the source may be a
whole cell lysate that can be prepared by successive freeze-thaw
cycles (e.g., one to three) in the presence of standard protease
inhibitors. The BGS-19 polypeptide may be partially or completely
purified by standard protein purification methods, e.g., affinity
chromatography using specific antibody described infra, or by
ligands specific for an epitope tag engineered into the recombinant
BGS-19 polypeptide molecule, also as described herein. Binding
activity can then be measured as described.
[0459] Compounds which are identified according to the methods
provided herein, and which modulate or regulate the biological
activity or physiology of the BGS-19 polypeptides according to the
present invention are a preferred embodiment of this invention. It
is contemplated that such modulatory compounds may be employed in
treatment and therapeutic methods for treating a condition that is
mediated by the novel BGS-19 polypeptides by administering to an
individual in need of such treatment a therapeutically effective
amount of the compound identified by the methods described
herein.
[0460] In addition, the present invention provides methods for
treating an individual in need of such treatment for a disease,
disorder, or condition that is mediated by the BGS-19 polypeptides
of the invention, comprising administering to the individual a
therapeutically effective amount of the BGS-19-modulating compound
identified by a method provided herein.
Gene Therapy
[0461] Gene therapy approaches may also be used in accordance with
the present invention to modulate the expression of a BGS-19
polynucleotide. Gene therapy refers to therapy performed by the
administration to a subject of an expressed or expressible
polynucleotide. Accordingly, the present invention provides for a
method for treating or preventing a BGS-19-related disorder
comprising administering to a patient in need thereof an effective
amount of a mammalian expression vector comprising a BGS-19
polynucleotide or a polynucleotide encoding a BGS-19 polypeptide,
BGS-19 agonist, BGS-19 antagonist, inhibitor of a BGS-19 agonist,
inhibitor of a BGS-19 antagonist, or a variant thereof.
[0462] Any of the methods for polynucleotide therapy available in
the art can be used in accordance with the present invention (See,
e.g., Goldspiel et al., 1993, Clinical Pharmacy 12:488-505;
Grossman and Wilson, 1993, Curr Opin Genet Devel. 3:110-114;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; Morgan
and Anderson, 1993, Ann Rev Biochem. 62:191-217; Mulligan, 1993,
Science 260:926-932; Tolstoshev, 1993, Ann Rev Pharmacol Toxicol.
32:573-596; Clowes et al., 1994, J Clin Invest. 93:644-651; Kiem et
al., 1994, Blood 83:1467-1473, each of which is incorporated herein
by reference). Gene therapy vectors can be administered to a
subject systemically or locally by, for example, intravenous
injection (See, e.g., U.S. Pat. No. 5,328,470) or by stereotactic
injection (See, e.g., Chen et al., 1994, Proc Natl Acad. Sci.
91:3054-57). Synthetic polynucleotides, the in vitro or in vivo
transcription and translation of which results in the production of
a BGS-19 antagonist, for example, may be constructed by techniques
well known in the art. For example, antisense, ribozyme, triple
helix molecules, and/or recombinant antibodies may be used to
target by polynucleotide therapy a BGS-19 polynucleotide of the
invention, resulting in a decrease in the respective BGS-19
polynucleotide expression and/or BGS-19 protein levels. Techniques
for the production and use of antisense, ribozyme, and/or triple
helix molecules are well known to those of skill in the art, and in
accordance with the present invention, can be applied to a
nucleotide sequence encoding a BGS-19 polypeptide of the
invention.
[0463] A pharmaceutical preparation of the polynucleotide therapy
vector can comprise a polynucleotide therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the polynucleotide delivery vehicle is embedded. Alternatively,
where the complete polynucleotide delivery vector can be produced
intact from recombinant cells, e.g., retroviral vectors, the
pharmaceutical preparation can include one or more cells which
produce the polynucleotide delivery system.
[0464] The present invention encompasses vectors comprising a
polynucleotide encoding a BGS-19 polypeptide of the invention, or
the complement thereof. In one embodiment, a BGS-19 polynucleotide
of the invention to be introduced for purposes of polynucleotide
therapy comprises an inducible promoter operably linked to the
coding region in the antisense orientation, such that expression of
the polynucleotide can be controlled using an appropriate inducer
or inhibitor of transcription. In another embodiment, the vector
comprises a promoter which expresses the cloned construct
constitutively. In a further embodiment, the promoter can be
downregulated by a suppressor molecule. Alternatively, the vector
comprises a promoter, such that an inducing molecule initiates or
increases expression of the cloned antisense BGS-19 polynucleotide.
In a preferred embodiment, the vector comprises a cell-specific
promoter. In another preferred embodiment, the vector comprises a
disease-specific promoter, such that expression is largely limited
to diseased tissues or tissues surrounding diseased tissues. In
another particular embodiment, a BGS-19 antisense oligonucleotide
is placed within a mammalian expression vector such that a BGS-19
antisense construct comprises the entire cDNA sequence.
[0465] Gene therapy involves introducing a polynucleotide construct
to cells in tissue culture or in vivo. Methods for introduction of
polynucleotides of the invention to cells in vitro include, but are
not limited to, electroporation, lipofection, calcium
phosphate-mediated transfection, and viral infection. Usually, the
method of transfer includes the transfer of a selectable marker to
the cells, after which the cells are placed under selection to
isolate the cells which have taken up and express the transferred
polynucleotide. The transfected cells then can be administered to a
subject.
[0466] An expression construct can be delivered directly into a
subject. In one embodiment, the polynucleotides of the invention
can be injected directly into the target tissue or cell derivation
site. Alternatively, a subject's cells are first transfected with
an expression construct in vitro, after which the transfected cells
are administered back into the subject (i.e., ex vivo
polynucleotide therapy). Accordingly, the polynucleotides of the
invention can be delivered in vivo or ex vivo to target cells.
Several methods have been developed for delivering the
polynucleotides of the invention to target cells or target
tissues.
[0467] Another approach to polynucleotide therapy involves
transferring a polynucleotide to cells in tissue culture by such
methods as electroporation, lipofection, calcium phosphate mediated
transfection, or viral infection. Usually, the method of transfer
includes the transfer of a selectable marker to the cells. The
cells are then placed under selection to isolate those cells that
have taken up and are expressing the transferred polynucleotide.
Those cells are then delivered to a subject. In another embodiment,
the polynucleotides of the invention can be introduced into the
target tissue as an implant, for example, in a polymer formulation
(See, e.g., U.S. Pat. No. 5,702,717). In another embodiment, the
polynucleotides of the invention can be targeted to the desired
cells or tissues.
[0468] In one embodiment, a polynucleotide of the invention is
administered to inhibit BGS-19 activity using polynucleotide
therapy.
[0469] In a particular embodiment, a vector is introduced in vivo
such that it is taken up by a cell and directs the transcription of
an antisense BGS-19 polynucleotide of the invention. Such a vector
can remain episomal or can become chromosomally integrate.
Expression vectors can be plasmid, viral, or others known in the
art, that can be used to replicate and/or express the cloned
nucleotide sequence encoding a BGS-19 antisense polynucleotide in a
target mammalian cell. A variety of expression vectors useful for
introducing into cells the polynucleotides of the inventions are
well known in the art (See, e.g., Promega.TM. catalogue, 2001;
Stratapolynucleotide.TM. catalogue, 2001). Expression constructs
can be introduced into target cells and/or tissues of a subject
using vectors which include, but are not limited to adenovirus,
adeno-associated virus, retrovirus and herpes virus vectors, in
addition to other particles that introduce DNA into cells, such as
liposomes.
[0470] A polynucleotide of the invention can be expressed using any
promoter known in the art capable of expression in mammalian,
preferably human cells. Such promoters can be inducible or
constitutive. These promoters include, but are not limited to, a
casein promoter (Cerdan et al., 1998, "Accurate spatial and
temporal transpolynucleotide expression driven by a 3.8-kilobase
promoter of the bovine beta-casein polynucleotide in the lactating
mouse mammary gland", Mol Reprod Dev. 49:236-45), whey acid
promoter (Doppler et al., 1991, "Lactogenic hormone and cell
type-specific control of the whey acidic protein polynucleotide
promoter in transfected mouse cells", Mol Endocrinol. 5:1624-1632),
SV40 early promoter region (Bernoist and Chambon, 1981, Nature
290:304-310), the promoter comprised in the 3' long terminal repeat
of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the
herpes thymidine kinase promoter (Wagner et al., 1981, "Nucleotide
sequence of the thymidine kinase polynucleotide of herpes simplex
virus type 1", Proc Natl Acad. Sci. 78:1441-1445) and the
regulatory sequences of the metallothionein polynucleotide
(Brinster et al., 1982, "Regulation of metallothionein--thymidine
kinase fusion plasmids injected into mouse eggs", Nature
296:39-42).
[0471] In one embodiment in which recombinant cells are used in
polynucleotide therapy, nucleotides complementary to
polynucleotides encoding polypeptides of the invention are
introduced into the cells such that they are expressible by the
cells or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific
embodiment, stem or progenitor cells are used. Any stem and/or
progenitor cells which can be isolated and maintained in vitro can
potentially be used in accordance with this embodiment of the
present invention (See, e.g., PCT Publication WO 94/08598; Stemple
and Anderson, 1992, Cell 71:973-985; Pittelkow and Scott, 1986,
Mayo Clinic Proc. 61:771; Rheinwald, 1980, Meth Cell Bio.
21A:229).
[0472] In a specific embodiment, the polynucleotide to be
introduced for purposes of polynucleotide therapy comprises an
inducible promoter operably linked to the coding region, such that
expression of the polynucleotide is controllable by controlling the
presence or absence of the appropriate inducer of
transcription.
[0473] A polynucleotide encoding a biologically active portion of a
polypeptide of the invention can be prepared, expressing the
encoded portion of the polypeptide protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the polypeptide.
[0474] In another embodiment, an antisense BGS-19 polynucleotide
comprises an appended group such as a peptide (e.g., for targeting
host cell receptors in vivo), or an agent that facilitates
transport across the cell membrane (See, e.g., Letsinger et al.,
1989, Proc Natl Acad. Sci. 86:6553-6556; Lemaitre et al., 1987,
Proc Natl Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810)
or the blood-brain barrier (See, e.g., PCT Publication No. WO
89/10134). In another embodiment, an antisense BGS-19
polynucleotide can be modified with hybridization-triggered
cleavage agents (See, e.g., Krol et al., 1988, Biotechniques
6:958-976) or intercalating agents (See, e.g., Zon, 1988, Pharm
Res. 5:539-549). To this end, an antisense BGS-19 polynucleotide
may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, or
hybridization-triggered cleavage agent.
[0475] Any type of plasmid, cosmid, YAC or viral vector can be used
to prepare the recombinant construct. Alternatively, vectors can be
used which selectively target a tissue or cell type, e.g., viruses
which infect cells of the immune system. Further specificity can be
realized by using a tissue-specific or cell-specific promoter in
the expression vector.
[0476] In a specific embodiment, an expression vector is
administered directly in vivo, where the vector is expressed to
produce the encoded product. This can be accomplished by any of
numerous methods known in the art, e.g., by placing a
polynucleotide of the invention in an appropriate expression vector
such that, upon administration, the vector becomes intracellular
and expresses a BGS-19 antisense oligonucleotide. Such vectors can
be internalized by using, for example, a defective or attenuated
retroviral vector or other viral vectors that can infect mammalian
cells (See, e.g., U.S. Pat. No. 4,980,286).
[0477] Alternatively, an expression construct comprising a
polynucleotide of the invention can be injected directly into a
target tissue as naked DNA. In another embodiment, an expression
construct comprising a polynucleotide of the invention can be
introduced intracellularly using microparticle bombardment, for
example, by using a Biolistic polynucleotide gun (Dupont.TM.). In
another embodiment, an expression construct comprising a
polynucleotide of the invention can be coated with lipids, or
cell-surface receptors, or transfecting agents, such that
encapsulation in liposomes, microparticles, or microcapsules
facilitates access to target tissues and/or entry into target
cells. In yet another embodiment, an expression construct
comprising a polynucleotide of the invention is linked to a
polypeptide that is internalized in a subset of cells or is
targeted to a particular cellular compartment. In a further
embodiment, the linked polypeptide is a nuclear targeting sequence
which targets the vector to the cell nucleus. In another further
embodiment, the linked polypeptide is a ligand that is internalized
by receptor-mediated endocytosis in cells expressing the respective
receptor for the ligand (See, e.g., Wu and Wu, 1987, J. Biol. Chem.
262:4429-4432).
[0478] In another embodiment, nucleic acid-ligand complexes can be
formed such that the ligand comprises a fusogenic viral peptide
which disrupts endosomes, thereby allowing the nucleic acid to
avoid lysosomal degradation. In another embodiment, a
polynucleotide of the invention can be targeted in vivo via a
cell-specific receptor resulting in cell-specific uptake and
expression (See, e.g., International Patent Publications WO
92/06180, WO 92/22635, WO 92/20316, WO 93/14188, and WO 93/2022. In
yet another embodiment, a polynucleotide of the invention is
introduced intracellularly and, by homologous recombination, can
transiently or stably be incorporated within the host cell DNA,
which then allows for its expression, (Koller and Smithies, 1989,
Proc Natl Acad. Sci. 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0479] In one embodiment, viral vectors are used that comprise
nucleic acids encoding compounds that activate cytokine receptors
(i.e., cytokines or antibodies), or compounds that activate
molecules expressed on activated immune cells (See, e.g., Miller et
al., 1993, Meth. Enzymol. 217:581-599). In a specific embodiment, a
viral vector that comprises polynucleotides encoding 4-1 BB ligand,
or anti-4-1 BB immunoglobulin, and/or IL-12 are used. For example,
a retroviral vector can be used in which sequences not necessary
for packaging of the viral genome and integration into host cell
DNA have been deleted, and polynucleotide sequences encoding 4-1 BB
ligand, or anti-4- 1 BB immunoglobulin, or IL-12 are cloned into
the vector, thereby facilitating delivery of the
transpolynucleotide into a subject. Greater detail about retroviral
vectors is available in Boesen et al., 1994, Biotherapy 6:291-302,
which describes the use of a retroviral vector to deliver the mdr1
polynucleotide to hematopoietic stem cells.
[0480] Other viral vectors can be used for polynucleotide therapy
approaches in accordance with the invention. For example,
adenoviruses are useful for delivering polynucleotide constructs to
respiratory epithelia. Other targets for adenovirus-based delivery
systems are the liver, the central nervous system, endothelial
cells, and muscle cells. Moreover, adenoviruses are able to infect
non-dividing cells (See, e.g., Rosenfeld et al., 1991, Science
252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli
et al., 1993, J. Clin. Invest. 91:225-234; Kozarsky and Wilson,
1993, Curr. Opin. Genetics Develop. 3:499-503; Bout et al., 1994,
Human Gene Therapy 5:3-10; PCT Publication No. WO 94/12649; and
Wang et al., 1995, Gene Therapy 2:775-783).
[0481] Adeno-associated virus can also be used in accordance with
the polynucleotide therapy approaches of the present invention
(See, e.g., Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0482] In this embodiment, the polynucleotide is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including, but not limited to, transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector comprising the polynucleotides, cell fusion,
chromosome-mediated polynucleotide transfer, microcell-mediated
polynucleotide transfer, and spheroplast fusion. Numerous
techniques are known in the art for the introduction of foreign
polynucleotides into cells (See, e.g., Maniatis et al., 1989;
Current Protocols in Molecular Biology, John Wiley & Sons,
2000; Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993, Meth Enzymol. 217:618-644; Cline, 1985, Pharmacol Ther.
29:69-92) and can be used in accordance with the present invention.
In a preferred embodiment, the technique stably transfers a
polynucleotide of the invention to a target cell, such that the
polynucleotide is inherited by the cell's progeny.
[0483] The resulting recombinant cells can be delivered to a
subject by various methods known in the art, and the skilled
artisan would appreciate appropriate modes of administration. For
example, intravenous administration may be the preferred mode of
administration for recombinant hematopoietic stem cells. Similarly,
the number of recombinant cells to be administered to a subject can
be determined by one skilled in the art, and would include a
consideration of factors such as the desired effect, the disease
state, and the mode of administration.
[0484] Cells into which a polynucleotide of the invention can be
introduced for purposes of polynucleotide therapy include, but are
not limited to pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratapolynucleotide; pSVK3, pBPV, pMSG and pSVL available from
Pharmacia; and pEF1V5, pcDNA3.1, and pRc/CMV2 available from
Invitrogen. Other suitable vectors will be readily apparent to the
skilled artisan.
[0485] One skilled in the art will appreciate that many different
promoters can be used to drive expression of a BGS-19 antisense
construct. In one embodiment, the promoter comprises
hormone-sensitive elements. For example, a promoter comprising an
androgen-sensitive enhancer would be activated to greater degree in
androgen-producing cells or adjacent tissues. Such an expression
construct may be beneficial for targeting tissues secreting
abnormally high levels of androgen. In another embodiment, the
promoter comprises elements of a fibroblast-specific promoter. In a
further embodiment, the fibroblast-specific promoter comprises
promoter elements from synovial fibroblasts. In another embodiment,
the promoter is derived from an imprinted polynucleotide, many of
which are known in the art. In another embodiment, the promoter is
derived from a tumor-specific promoter, many of which are known in
the art.
[0486] Alternatively, the promoter comprises elements of promoters
that are activated in aggressive rheumatoid arthritis synovial
fibroblasts. In a particular embodiment, the promoter comprises a
portion of a BGS-19 promoter. In a non-limiting example, a viral
vector is used in which the viral promoter is replaced fully, or in
part, with at least parts of a BGS-19 promoter. Such an expression
construct would more specifically be expressed in BGS-19-expressing
cells, and higher expression of a BGS-19 antisense oligonucleotide
would occur in cells expressing above-normal levels of BGS-19.
[0487] Gene therapy approaches may also be used in accordance with
the present invention to inhibit BGS-19. For example, ribozyme and
triple helix molecules may be used to target a BGS-19
polynucleotide products resulting in a decrease in BGS-19 protein.
Techniques for the production and use of antisense ribozyme and/or
triple helix molecules are well known to those of skill in the art
and can be designed with respect to the nucleotide sequence
encoding the amino acid sequence of BGS-19.
Antisense Gene Therapy
[0488] Antisense approaches to polynucleotide therapy involve the
use of riboprobes that may hybridize to a portion of the target
mRNA. The skilled artisan will recognize that absolute
complementarity is not required, such that some degree of mismatch
can result in, at least, transitory duplex formation. In one
non-limiting example, the antisense riboprobe binds to the target
mRNA transcript and prevents its translation.
[0489] Riboprobes that are complementary to the 5' untranslated
sequences, up to and including the AUG initiation codon, can be
used effectively to inhibit translation of a BGS-19 mRNA.
Additionally, riboprobes complementary to the 3' untranslated
sequences of mRNAs also can be effective at inhibiting BGS-19 mRNA
translation (See, e.g., Wagner, 1994, Nature 372:333-335).
Moreover, antisense riboprobes complementary to mRNA coding regions
can be used in accordance with the invention.
[0490] Preferably, in vitro studies are performed to assess the
ability of an antisense riboprobe to inhibit polynucleotide
expression. These studies typically use controls which distinguish
between antisense-mediated inhibition of polynucleotide expression
and nonspecific biological effects of riboprobes. Preferably, these
studies compare antisense-mediated changes in the levels of the
target RNA or target protein with levels of an internal control RNA
or protein.
[0491] In one embodiment, a recombinant DNA construct that has a
BGS-19 antisense riboprobe under the control of a pol III or pol II
promoter is used to polynucleotiderate BGS-19 antisense riboprobes
in a cell. The use of such a construct to transfect target cells in
the subject can result in the transcription of sufficient amounts
of a BGS-19 riboprobe to reduce or inhibit BGS-19 mRNA and/or
protein expression. Low transfection rates or low transcription
activity of the DNA construct can nevertheless polynucleotiderate
sufficient BGS-19 antisense molecules to demonstrate clinical
effectiveness.
[0492] In another embodiment, a BGS-19 antisense polynucleotide is
cloned into an expression vector, preferably a mammalian expression
vector. In a specific embodiment, the BGS-19 antisense
polynucleotide comprises the sequence of a full-length BGS-19 DNA.
In another specific embodiment, the BGS-19 antisense polynucleotide
comprises the sequence of a 5' untranslated region, which
optionally can include the sequence, AUG, indicating the start of
the coding region.
[0493] In another embodiment, antisense polynucleotides of the
invention are cloned into a vector, which is designed to target the
vector (and thereby target expression of the antisense riboprobe)
to specific tissues or cell-types. For example, an antisense
riboprobe can be linked to peptides or antibodies that specifically
bind receptors or antigens expressed on the target cell surface,
thereby targeting the vector to cells) can be administered.
[0494] In another embodiment, the vector comprises a promoter that
is more highly activated in diseased cells or tissues, as compared
to normal cells or tissues.
[0495] A preferred approach to achieve intracellular concentrations
of the antisense sufficient to suppress translation of endogenous
mRNAs involves the use of a recombinant DNA construct in which the
antisense oligonucleotide is placed under the control of a strong
pol III or pol III promoter. The use of such a construct to
transfect target cells in a patient will result in the
transcription of sufficient amounts of single stranded RNAs that
can form complementary base pairs with the endogenous BGS-19
polynucleotide transcripts and thereby prevent translation of the
BGS-19 polynucleotide mRNA. For example, a vector can be introduced
in vivo such that the vector is taken up by a cell and directs the
transcription of an antisense RNA. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in mammalian cells.
[0496] Expression of the sequence encoding the antisense RNA can be
by any promoter known in the art to act in mammalian, preferably
human cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter
region (Bernoist and Chambon, 1981, Nature 290:304-310), the
promoter comprised in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:1441-1445), and the regulatory sequences of the
metallothionein polynucleotide (Brinster et al., 1982, Nature
296:39-42). Any type of plasmid, cosmid, YAC (yeast artificial
chromosome), or viral vector can be used to prepare the recombinant
DNA construct which can be introduced directly into the tissue
site. Alternatively, viral vectors that selectively infect the
desired tissue can be used.
[0497] A construct designed for polynucleotide therapy approaches
can also be used in contexts no involving polynucleotide
therapy.
Ribozyme Therapy
[0498] The invention also provides for ribozymes targeting BGS-19
expression. Ribozymes are enzymatic RNA molecules capable of
catalyzing the specific cleavage of a single-stranded nucleic acid,
such as an mRNA (See, e.g., Rossi, 1994, Current Biology
4:469-471). The mechanism of a trans-acting ribozyme action
involves sequence-specific hybridization of the ribozyme molecule
to a complementary target, followed by an endonucleolytic cleavage.
The composition of ribozyme molecules include one or more sequences
complementary to the target polynucleotide mRNA, and catalytic
sequences responsible for mRNA cleavage (See, e.g., U.S. Pat. No.
5,093,246 which is incorporated by reference in its entirety).
Thus, ribozymes, e.g., hammerhead ribozymes (Haselhoff and Gerlach,
1988, Nature 334:585-591), can be used to catalytically cleave mRNA
transcripts thereby inhibiting the expression of a protein encoded
by a particular mRNA. A trans-acting ribozyme having specificity
for a polynucleotide encoding a polypeptide of the invention can be
designed based upon the nucleotide sequence of the polynucleotides
of the invention. Accordingly, in one embodiment, an engineered
hammerhead motif ribozyme molecule specifically and efficiently
catalyzes endonucleolytic cleavage of RNA sequences encoding a
BGS-19 polypeptide of the invention.
[0499] In another embodiment, an mRNA encoding a polypeptide of the
invention is used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules (See, e.g.,
Bartel and Szostak, 1993, Science 261:1411-1418).
[0500] Specific ribozyme cleavage sites within a potential RNA
target are identified by scanning the molecule of interest for
ribozyme cleavage sites, which include the sequences GUA, GUU and
GUC. Once identified, short RNA sequences of approximately 15 to 20
ribonucleotides corresponding to a cleavage site of a target
polynucleotide are evaluated for predicted structural features,
such as secondary structure, that may make the oligonucleotide
suitable. The suitability of candidate sequences also can be
evaluated by testing their ability to hybridize with complementary
oligonucleotides, using for example, ribonuclease protection
assays.
[0501] In one embodiment, a ribozyme in the form of an antisense
riboprobe is polynucleotiderated from a mammalian expression
vector. In another embodiment, a ribozyme in the form of an
oligonucleotide administered directly to the patient. In a further
embodiment, the ribozyme is administered systemically. In another
further embodiment, the ribozyme is administered directly to the
cells or tissue, in vivo or ex vivo.
[0502] The ribozymes of the present invention also include RNA
endoribonucleases, such as the ribozyme which occurs naturally in
Tetrahymena thermophila (also known as the IVS, or L-19 IVS RNA)
and has been extensively described (Zaug et al., 1984, Science
224:574-578; Been and Cech, 1986, Cell 47:207-216; Zaug and Cech,
1986, Science 231:470-475; Zaug et al., 1986, Nature 324:429-433;
published International Patent Publication No. WO 88/04300). These
ribozymes have an 8 bp active site which hybridizes to a target RNA
sequence to cause cleavage of the target RNA. Accordingly, the
invention encompasses ribozymes that target active sites comprising
8 bp, which are present in a BGS-19 polynucleotide.
[0503] As discussed for antisense approaches, supra, the ribozymes
of the invention can be composed of modified oligonucleotides (e.g.
for improved stability or targeting) and should be delivered to
cells that express a BGS-19 polynucleotide in vivo. A preferred
method of delivery involves using a DNA construct "encoding" the
ribozyme under the control of a strong constitutive pol III or pol
II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to cause degradation of an endogenous
BGS-19 mRNA and thereby inhibit translation. Because ribozymes
unlike antisense molecules, are catalytic, a relatively low
intracellular concentration is required for efficiency.
[0504] Ribozymes of the invention can be prepared by any method
known in the art for the synthesis of DNA and RNA molecules. For
example, chemical synthesis can be achieved by synthesizing
oligodeoxyribonucleotides and oligoribonucleotides using solid
phase phosphoramidite chemical synthesis. Alternatively, ribozyme
polynucleotides can be polynucleotiderated by in vitro or in vivo
transcription of DNA sequences. Such DNA sequences can be
incorporated into a wide variety of vectors which incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters. Alternatively, antisense cDNA constructs can be
introduced stably into cell lines, such that the synthesize
ribozyrnes are expressed constitutively or inducibly, depending on
the promoter used.
Triple Helix Therapy
[0505] The invention also encompasses polynucleotides which form
triple helical structures. For example, expression of a polypeptide
of the invention can be inhibited by targeting nucleotide sequences
complementary to the regulatory region of the polynucleotide
encoding the polypeptide (e.g., the promoter and/or enhancer) to
form triple helical structures that prevent transcription of the
polynucleotide in target cells (see, e.g., Helene, 1991, Anticancer
Drug Des. 6:569-584; Helene, 1992, Ann NY Acad. Sci. 660:27-36;
Maher, 1992, Bioassays 14:807-815).
[0506] Polynucleotides to be used to inhibit transcription by
triple helix formation can be single stranded oligonucleotides. The
base composition of these oligonucleotides can be designed to
promote triple helix formation via Hoogsteen base pairing rules,
preferably with long stretches of purines or pyrimidines on one
strand of the duplex. Nucleotide sequences can be pyrimidine-based
thereby resulting in TAT and CGC+ triplets] across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. Purine-rich polynucleotides also can be
chosen, for example, comprising a stretch of guanine residues.
These molecules can form a triple helix with a DNA duplex that is
rich in GC pairs, in which most of the purine residues are located
on a single strand of the targeted duplex, resulting in GGC
triplets across the three strands in the triplex.
[0507] Additionally, the number of potential sequences that can be
targeted for triple helix formation can be increased by creating a
"switchback" polynucleotide. Switchback molecules are synthesized
in an alternating 5'-3',3'-5' manner, such that the molecule first
hybridizes with one strand of a duplex, followed by hybridization
with another strand, thus eliminating the requirement for a stretch
of purines or pyrimidines on one strand of a duplex.
[0508] Ribozyme and triple helix molecules of the invention can be
prepared by any method known in the art for the synthesis of DNA or
RNA molecules (e.g., oligodeoxyribonucleotides or
oligoribonucleotides). Such methods include, for example, solid
phase phosphoramidite chemical synthesis. For further examples of
methods of synthesis, see Section 5.6.14 regarding methods for
synthesis of antisense oligonucleotides, supra.
[0509] These oligonucleotides can be administered directly, for
example, via injection. Alternatively, RNA molecules can be
polynucleotiderated in vitro or in vivo by transcription of DNA
sequences. Such DNA sequences may be incorporated into a wide
variety of vectors known in the art that feature a suitable RNA
polymerase promoter such as, for example, a T7 or SP6 polymerase
promoter.
Antibody Gene Therapy
[0510] In one embodiment, polynucleotides comprising sequences
encoding antibodies that bind to a BGS-19 are administered via
polynucleotide therapy. In a particular embodiment, recombinant
cells are used that comprise polynucleotides encoding antibodies to
BGS-19 polypeptides of the invention. The polynucleotide construct
is expressed such that the recombinant antibody is secreted or
expressed on the cell surface. The recombinant cells are then
administered in vivo for therapeutic effect.
[0511] Antibodies of the invention, including antibodies conjugated
to therapeutic moieties, can be administered to an individual alone
or in combination with a chemotherapeutic drug, cytotoxic factor,
and/or cytokine. In one embodiment, an antibodies directed to a
BGS-19 polypeptide is administered first, followed by
chemotherapeutic drug, cytotoxic factor, and/or cytokine within 24
hours. The treatment cycle can be repeated if warranted by the
clinical response of the patient. Furthermore, the antibody,
chemotherapeutic drug, cytotoxic factor, and/or cytokine can be
administered via separate routes, such as for example, by
intravenous and intramuscular administration. Cytotoxic factors
include, but are not limited to, TNF-.alpha., TNF-.beta., IL-1,
IFN-.gamma., and IL-2. Chemotherapeutic drugs include, but are not
limited to, 5-fluorouracil (5FU), vinblastine, actinomycin D,
etoposide, cisplatin, methotrexate, and doxorubicin. Cytokines
include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, and IL-12.
Vaccine Therapy
[0512] The nucleotides of the invention, including variants and
derivatives, can be used as vaccines, e.g., by polynucleotidetic
immunization. Genetic immunization is particularly advantageous as
it stimulates a cytotoxic T-cell response but does not utilize live
attenuated vaccines, which can revert to a virulent form and infect
the host causing the very infection sought to be prevented. As used
herein, polynucleotidetic immunization comprises inserting the
nucleotides of the invention into a host, such that the nucleotides
are taken up by cells of the host and the proteins encoded by the
nucleotides are translated. These translated proteins are then
either secreted or processed by the host cell for presentation to
immune cells and an immune reaction is stimulated. Preferably, the
immune reaction is a cytotoxic T cell response, however, a humoral
response or macrophage stimulation is also useful in preventing
future infections. The skilled artisan will appreciate that there
are various methods for introducing foreign nucleotides into a host
animal and subsequently into cells for polynucleotidetic
immunization, for example, by intramuscular injection of about 50
mg of plasmid DNA encoding the proteins of the invention
solubilized in 50 ml of sterile saline solution, with a suitable
adjuvant (See, e.g., Weiner and Kennedy, 1999, Scientific American
7:50-57; Lowrie et al., 1999, Nature 400:269-271).
Combination Therapies
[0513] The administration of a BGS-19 antagonist can potentiate the
effect of anti-cancer agents. In a preferred embodiment, the
invention further encompasses the use of combination therapy to
prevent or treat cancer.
[0514] In one embodiment, breast cancer can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with 5-fluorouracil, cisplatin, docetaxel, doxorubicin,
Herceptin.RTM., gemcitabine (Seidman, 2001, "Gemcitabine as
single-agent therapy in the management of advanced breast cancer",
Oncology 15:11-14), IL-2, paclitaxel, and/or VP-16 (etoposide).
[0515] In another embodiment, prostate cancer can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with paclitaxel, docetaxel, mitoxantrone, and/or an
androgen receptor antagonist (e.g., flutamide).
[0516] In another embodiment, leukemia can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with fludarabine, cytosine arabinoside, gemtuzumab
(MYLOTARG), daunorubicin, methotrexate, vincristine,
6-mercaptopurine, idarubicin, mitoxantrone, etoposide,
asparaginase, prednisone and/or cyclophosphamide. As another
example, myeloma can be treated with a pharmaceutical composition
comprising a BGS-19 antagonist in combination with
dexamethasone.
[0517] In another embodiment, melanoma can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with dacarbazine.
[0518] In another embodiment, colorectal cancer can be treated with
a pharmaceutical composition comprising a BGS-19 antagonist in
combination with irinotecan.
[0519] In another embodiment, lung cancer can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with paclitaxel, docetaxel, etoposide and/or
cisplatin.
[0520] In another embodiment, non-Hodgkin's lymphoma can be treated
with a pharmaceutical composition comprising a BGS-19 antagonist in
combination with cyclophosphamide, CHOP, etoposide, bleomycin,
mitoxantrone and/or cisplatin.
[0521] In another embodiment, gastric cancer can be treated with a
pharmaceutical composition comprising a BGS-19 antagonist in
combination with cisplatin.
[0522] In another embodiment, pancreatic cancer can be treated with
a pharmaceutical composition comprising a BGS-19 antagonist in
combination with gemcitabine.
[0523] These combination therapies can also be used to prevent
cancer, prevent the recurrence of cancer, or prevent the spread or
metastasis or cancer.
[0524] Combination therapy also includes, in addition to
administration of a BGS-19 antagonist, the use of one or more
molecules, compounds or treatments that aid in the prevention or
treatment of cancer (i.e., cancer therapeutics), which molecules,
compounds or treatments includes, but is not limited to,
chemoagents, immunotherapeutics, cancer vaccines, anti-angiogenic
agents, cytokines, hormone therapies, polynucleotide therapies, and
radiotherapies.
[0525] In one embodiment, one or more chemoagents, in addition to a
BGS-19 antagonist, is administered to treat a cancer patient. A
chemoagent (or "anti-cancer agent" or "anti-tumor agent" or "cancer
therapeutic") refers to any molecule or compound that assists in
the treatment of tumors or cancer. Examples of chemoagents
contemplated by the present invention include, but are not limited
to, cytosine arabinoside, taxoids (e.g., paclitaxel, docetaxel),
anti-tubulin agents (e.g., paclitaxel, docetaxel, epothilone B, or
its analogues), macrolides (e.g., rhizoxin) cisplatin, carboplatin,
adriamycin, tenoposide, mitozantron, discodermolide, eleutherobine,
2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide,
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin), antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol,
5-fluorouracil, fludarabine, gemcitabine, dacarbazine,
temozolamide), asparaginase, Bacillus Calmette and Guerin,
diphtheria toxin, hexamethylmelamine, hydroxyurea, LYSODREN.RTM.,
nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel,
camptothecin, topotecan, irinotecan (CAMPTOSAR, CPT-11),
vincristine, vinca alkyloids such as vinblastine), podophyllotoxin
(including derivatives such as epipodophyllotoxin, VP-16
(etoposide), VM-26 (teniposide)), cytochalasin B, colchicine,
gramicidin D, ethidium bromide, emetine, mitomycin, procarbazine,
mechlorethamine, anthracyclines (e.g., daunorubicin (formerly
daunomycin), doxorubicin, doxorubicin liposomal),
dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D,
procaine, tetracaine, lidocaine, propranolol, puromycin,
anti-mitotic agents, abrin, ricin A, pseudomonas exotoxin, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, aldesleukin, allutamine, anastrozle, bicalutamide,
biaomycin, busulfan, capecitabine, carboplain, chlorabusil,
cladribine, cylarabine, daclinomycin, estramusine, floxuridhe,
gemcitabine, gosereine, idarubicin, itosfamide, lauprolide acetate,
levamisole, lomusline, mechlorethamine, magestrol, acetate,
mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin,
picamycin, riuxlmab, campath-1, straplozocin, thioguanine,
tretinoin, vinorelbine, or any fragments, family members, or
derivatives thereof, including pharmaceutically acceptable salts
thereof. Compositions comprising one or more chemoagents (e.g.,
FLAG, CHOP) are also contemplated by the present invention. FLAG
comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP
comprises cyclophosphamide, vincristine, doxorubicin, and
prednisone.
[0526] In one embodiment, said chemoagent is gemcitabine at a dose
ranging from 100 to 1000 mg/m.sup.2/cycle. In one embodiment, said
chemoagent is dacarbazine at a dose ranging from 200 to 4000
mg/m.sup.2/cycle. In a preferred embodiment, said dose ranges from
700 to 1000 mg/m.sup.2/cycle. In another embodiment, said
chemoagent is fludarabine at a dose ranging from 25 to 50
mg/m.sup.2/cycle. In another embodiment, said chemoagent is
cytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000
mg/m.sup.2/cycle. In another embodiment, said chemoagent is
docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle. In another
embodiment, said chemoagent is paclitaxel at a dose ranging from 5
to 15 mg/kg/cycle. In yet another embodiment, said chemoagent is
cisplatin at a dose ranging from 5 to 20 mg/kg/cycle. In yet
another embodiment, said chemoagent is 5-fluorouracil at a dose
ranging from 5 to 20 mg/kg/cycle. In yet another embodiment, said
chemoagent is doxorubicin at a dose ranging from 2 to 8
mg/kg/cycle. In yet another embodiment, said chemoagent is
epipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle. In
yet another embodiment, said chemoagent is cyclophosphamide at a
dose ranging from 50 to 200 mg/kg/cycle. In yet another embodiment,
said chemoagent is irinotecan at a dose ranging from 50 to 75, 75
to 100, 100 to 125, or 125 to 150 mg/m.sup.2/cycle. In yet another
embodiment, said chemoagent is vinblastine at a dose ranging from
3.7 to 5.4, 5.5 to 7.4, 7.5 to 11, or 11 to 18.5 mg/m.sup.2/cycle.
In yet another embodiment, said chemoagent is vincristine at a dose
ranging from 0.7 to 1.4, or 1.5 to 2 mg/m.sup.2/cycle. In yet
another embodiment, said chemoagent is methotrexate at a dose
ranging from 3.3 to 5, 5 to 10, 10 to 100, or 100 to 1000
mg/m.sup.2/cycle.
[0527] In a preferred embodiment, the invention further encompasses
the use of low doses of chemoagents when administered as part of a
BGS-19 antagonist treatment regimen. For example, initial treatment
with a BGS-19 antagonist increases the sensitivity of a tumor to
subsequent challenge with a dose of chemoagent, which dose is near
or below the lower range of dosages when the chemoagent is
administered without a BGS-19 antagonist. In one embodiment, a
BGS-19 antagonist and a low dose (e.g., 6 to 60 mg/m.sup.2/day or
less) of docetaxel are administered to a cancer patient. In another
embodiment, a BGS-19 antagonist and a low dose (e.g., 10 to 135
mg/m.sup.2/day or less) of paclitaxel are administered to a cancer
patient. In yet another embodiment, a BGS-19 antagonist and a low
dose (e.g., 2.5 to 25 mg/m.sup.2/day or less) of fludarabine are
administered to a cancer patient. In yet another embodiment, a
BGS-19 antagonist and a low dose (e.g., 0.5 to 1.5 g/m.sup.2/day or
less) of cytosine arabinoside (Ara-C) are administered to a cancer
patient.
[0528] The invention, therefore, contemplates the use of one or
more BGS-19 antagonists, which is administered prior to,
subsequently, or concurrently with low doses of chemoagents, for
the prevention or treatment of cancer.
[0529] In one embodiment, said chemoagent is gemcitabine at a dose
ranging from 10 to 100 mg/m.sup.2/cycle.
[0530] In one embodiment, said chemoagent is cisplatin, e.g.,
PLATINOL or PLATINOL-AQ (Bristol Myers), at a dose ranging from 5
to 10, 10 to 20,20 to 40, or 40 to 75 mg/m.sup.2/cycle. In another
embodiment, a dose of cisplatin ranging from 7.5 to 75
mg/m.sup.2/cycle is administered to a patient with ovarian cancer.
In another embodiment, a dose of cisplatin ranging from 5 to 50
mg/m.sup.2/cycle is administered to a patient with bladder
cancer.
[0531] In another embodiment, said chemoagent is carboplatin, e.g.,
PARAPLATIN (Bristol Myers), at a dose ranging from 2 to 4, 4 to 8,
8 to 16, 16 to 35, or 35 to 75 mg/m.sup.2/cycle. In another
embodiment, a dose of carboplatin ranging from 7.5 to 75
mg/m.sup.2/cycle is administered to a patient with ovarian cancer.
In another embodiment, a dose of carboplatin ranging from 5 to 50
mg/m.sup.2/cycle is administered to a patient with bladder cancer.
In another embodiment, a dose of carboplatin ranging from 2 to 20
mg/m.sup.2/cycle is administered to a patient with testicular
cancer.
[0532] In another embodiment, said chemoagent is docetaxel, e.g.,
TAXOTERE (Rhone Poulenc Rorer) at a dose ranging from 6 to 10, 10
to 30, or 30 to 60 mg/m.sup.2/cycle.
[0533] In another embodiment, said chemoagent is paclitaxel, e.g.,
TAXOL (Bristol Myers Squibb), at a dose ranging from 10 to 20, 20
to 40, 40 to 70, or 70 to 135 mg/kg/cycle.
[0534] In another embodiment, said chemoagent is 5-fluorouracil at
a dose ranging from 0.5 to 5 mg/kg/cycle.
[0535] In another embodiment, said chemoagent is doxorubicin, e.g.,
ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX (Bristol
Myers Squibb), at a dose ranging from 2 to 4, 4 to 8, 8 to 15, 15
to 30, or 30 to 60 mg/kg/cycle.
[0536] In another embodiment, a BGS-19 antagonist is administered
in combination with one or more immunotherapeutic agents, such as
antibodies and immunomodulators, which includes, but is not limited
to, Herceptin.RTM., Retuxan.RTM., OvaRex, Panorex, BEC2, IMC-C225,
Vitaxin, Campath I/H, Smart M195, LymphoCide, Smart I D10, and
Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.
[0537] In another embodiment, a BGS-19 antagonist is administered
in combination with one or more anti-angiogenic agents, which
includes, but is not limited to, angiostatin, thalidomide, kringle
5, endostatin, Serpin (Serine Protease Inhibitor) anti-thrombin, 29
kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of
fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa
proteolytic fragment of platelet factor-4, a 13-amino acid peptide
corresponding to a fragment of platelet factor-4 (Maione et al.,
1990, Cancer Res. 51:2077-2083), a 14-amino acid peptide
corresponding to a fragment of collagen I (Tolma et al., 1993, J.
Cell Biol. 122:497-511), a 19 amino acid peptide corresponding to a
fragment of Thrombospondin I (Tolsma et al., 1993, J. Cell Biol.
122:497-511), a 20-amino acid peptide corresponding to a fragment
of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-1334), or
any fragments, family members, or derivatives thereof, including
pharmaceutically acceptable salts thereof.
[0538] Other peptides that inhibit angiopolynucleotidesis and
correspond to fragments of laminin, fibronectin, procollagen, and
EGF have also been described (see, e.g., Cao, 1998, Prog Mol
Subcell Biol. 20:161-176). Monoclonal antibodies and cyclic
pentapeptides, which block certain integrins that bind RGD proteins
(i.e., possess the peptide motif Arg-Gly-Asp), have been
demonstrated to have anti-vascularization activities (Brooks et
al., 1994, Science 264:569-571; Hammes et al., 1996, Nature
Medicine 2:529-533). Moreover, inhibition of the urokinase
plasminogen activator receptor by receptor antagonists inhibits
angiopolynucleotidesis, tumor growth and metastasis (Min et al.,
1996, Cancer Res. 56: 2428-33; Crowley et al., 1993, Proc Natl
Acad. Sci. 90:5021-25). Use of such anti-angiogenic agents is also
contemplated by the present invention.
[0539] In another embodiment, a BGS-19 antagonist is administered
in combination with a regimen of radiation.
[0540] In another embodiment, a BGS-19 antagonist is administered
in combination with one or more cytokines, which includes, but is
not limited to, lymphokines, tumor necrosis factors, tumor necrosis
factor-like cytokines, lymphotoxin-.alpha., lymphotoxin-.beta.,
interferon-.alpha., interferon-.beta., macrophage inflammatory
proteins, granulocyte monocyte colony stimulating factor,
interleukins (including, but not limited to, interleukin-1,
interleukin-2, interleukin-6, interleukin-12, interleukin-15,
interleukiri-18), OX40, CD27, CD30, CD40 or CD137 ligands, Fas-Fas
ligand, 4-1BBL, endothelial monocyte activating protein or any
fragments, family members, or derivatives thereof, including
pharmaceutically acceptable salts thereof.
[0541] In yet another embodiment, a BGS-19 antagonist is
administered in combination with a cancer vaccine. Examples of
cancer vaccines include, but are not limited to, autologous cells
or tissues, non-autologous cells or tissues, carcinoembryonic
antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live
vaccine, melanocyte lineage proteins (e.g., gp100, MART-1/MelanA,
TRP-1 (gp75), tyrosinase, widely shared tumor-specific antigens
(e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3,
N-acetylglucosaminyltransferase-V, p15), mutated antigens that are
tumor-specific (.beta.-catenin, MUM-1, CDK4), nonmelanoma antigens
(e.g., HER-2/neu (breast and ovarian carcinoma), human
papillomavirus-E6, E7 (cervical carcinoma), MUC-1 (breast, ovarian
and pancreatic carcinoma)). For human tumor antigens recognized by
T cells, see polynucleotiderally Robbins and Kawakami, 1996, Curr.
Opin. Immunol. 8:628-36. Cancer vaccines may or may not be purified
preparations.
[0542] In yet another embodiment, a BGS-19 antagonist is used in
association with a hormonal treatment. Hormonal therapeutic
treatments comprise hormonal agonists, hormonal antagonists (e.g.,
flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH
antagonists), inhibitors of hormone biosynthesis and processing,
and steroids (e.g., dexamethasone, retinoids, betamethasone,
cortisol, cortisone, prednisone, dehydrotestosterone,
glucocorticoids, mineralocorticoids, estrogen, testosterone,
progestins), antigestagens (e.g., mifepristone, onapristone), and
antiandrogens (e.g., cyproterone acetate).
[0543] In yet another embodiment, a BGS-19 antagonist is used in
association with a polynucleotide therapy program in the treatment
of cancer. In one embodiment, polynucleotide therapy with
recombinant cells secreting interleukin-2 is administered in
combination with a BGS-19 antagonist to prevent or treat cancer,
particularly lymphoma (See, e.g., Deshmukh et al., 2001, J.
Neurosurg. 94:287-292).
[0544] In one embodiment, a BGS-19 antagonist is administered, in
combination with at least one cancer therapeutic agent, for a short
treatment cycle to a cancer patient to treat cancer. The duration
of treatment with the cancer therapeutic agent may vary according
to the particular cancer therapeutic agent used. The invention also
contemplates discontinuous administration or daily doses divided
into several partial administrations. An appropriate treatment time
for a particular cancer therapeutic agent will be appreciated by
the skilled artisan, and the invention contemplates the continued
assessment of optimal treatment schedules for each cancer
therapeutic agent.
[0545] The present invention contemplates at least one cycle,
preferably more than one cycle during which a single therapeutic or
sequence of therapeutics is administered. An appropriate period of
time for one cycle will be appreciated by the skilled artisan, as
will the total number of cycles, and the interval between cycles.
The invention contemplates the continued assessment of optimal
treatment schedules for each BGS-19 antagonist and cancer
therapeutic agent.
Pharmaceutical Compositions
[0546] Since inhibition of expression of a BGS-19 polynucleotide or
inhibition of a BGS-19 protein can have significant therapeutic
responses in a patient with a BGS-19-related disorder, the
invention provides useful pharmaceutical compositions, treatment
courses, and modes of delivery. Accordingly, in one embodiment, a
pharmaceutical composition comprises a polynucleotide or
polypeptide of the invention, and derivatives thereof, which refers
to any pharmaceutically acceptable homolog, analogue, or fragment
corresponding to the pharmaceutical composition of the invention.
In another embodiment, the present invention provides for a
pharmaceutical composition that comprises a BGS-19 antagonist and a
pharmaceutically acceptable carrier.
[0547] The carrier can be a diluent, adjuvant, excipient, or
vehicle with which the therapeutic is administered. Such carriers
can be sterile liquids, such as saline solutions in water, or oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. A saline solution is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The carrier, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations
and the like. The composition can be formulated as a suppository,
with traditional binders and carriers such as triglycerides.
Examples of suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences by E. W. Martin. Examples of
suitable pharmaceutical carriers are a variety of cationic lipids,
including, but not limited to
N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTMA") and diolesylphosphotidylethanolamine ("DOPE"). Liposomes
are also suitable carriers for the antisense oligonucleotides of
the invention. Such compositions should comprise a therapeutically
effective amount of the compound, together with a suitable amount
of carrier so as to provide the form for proper administration to
the patient. The formulation should suit the mode of
administration.
[0548] Pharmaceutically acceptable salts are prepared from
pharmaceutically acceptable, essentially nontoxic, acids and bases,
including inorganic and organic acids and bases. Pharmaceutically
acceptable salts include those formed with free amino groups such
as those derived from hydrochloric, phosphoric, acetic, oxalic,
tartaric acids, etc., and those formed with free carboxyl groups
such as those derived from sodium, potassium, ammonium, calcium,
ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol, histidine, procaine, etc.
[0549] Suitable pharmaceutically acceptable carriers include
essentially chemically inert and nontoxic compositions that do not
interfere with the effectiveness of the biological activity of the
pharmaceutical composition. Examples of suitable pharmaceutical
carriers include, but are not limited to, saline solutions,
glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)-
N,N,N-trimethylammonium chloride ("DOTMA"),
diolesylphosphotidylethanolamine ("DOPE") and liposomes. Such
compositions should comprise a therapeutically effective amount of
the compound, together with a suitable amount of carrier so as to
provide an appropriate formulation for administration to a patient.
For example, oral administration requires enteric coatings to
protect the antagonist from degradation within the gastrointestinal
tract. In another example, the antagonist may be administered in a
liposomal formulation to facilitate transport in circulatory
system, effect delivery across cell membranes to intracellular
sites, and shield the antagonist from degradative enzymes.
[0550] In another embodiment, a pharmaceutical composition
comprises a BGS-19 antagonist and one or more therapeutic agents
and a pharmaceutically acceptable carrier. In a particular
embodiment, the pharmaceutical composition comprises a BGS-19
antagonist and one or more cancer therapeutic agents and a
pharmaceutically acceptable carrier.
[0551] In a further embodiment, a pharmaceutical composition,
comprising a BGS-19 antagonist, with or without other therapeutic
agents, and a pharmaceutically acceptable carrier, is at an
effective dose.
[0552] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups, such as for example, those
derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric
acids, and those formed with free carboxyl groups, such as for
example, those derived from sodium, potassium, ammonium, calcium,
ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol, histidine, and procaine.
[0553] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for subcutaneous injection or intravenous administration to
humans. Typically, compositions for subcutaneous injection or
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lidocaine to ease
pain at the site of the injection. Generally, the ingredients are
supplied either separately or mixed together in unit dosage form,
for example, as a dry lyophilized powder or water-free concentrate
in a hermetically sealed container such as an ampule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle, bag, or other acceptable container, containing
sterile pharmaceutical grade water, saline, or other acceptable
diluents. Where the composition is administered by injection, an
ampule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0554] The polynucleotides, polypeptides, and antibodies of the
invention can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the polynucleotide, protein, or antibody, and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
[0555] The invention includes methods for preparing pharmaceutical
compositions for modulating the expression or activity of a
polypeptide or polynucleotide of the invention. Such methods
comprise formulating a pharmaceutically acceptable carrier with an
agent which modulates expression or activity of a polypeptide or
polynucleotide of the invention. Such compositions can further
include additional active agents. Thus, the invention further
includes methods for preparing a pharmaceutical composition by
formulating a pharmaceutically acceptable carrier with an agent
which modulates expression or activity of a polypeptide or
polynucleotide of the invention and additional polynucleotides,
polypeptides, and antibodies of the invention.
[0556] Selection of a preferred effective dose can be determined by
a skilled artisan based upon the consideration of factors which
will be known to one of ordinary skill in the art. Such factors
include the particular form of a BGS-19 antagonist and its
pharmacokinetic parameters such as bioavailability, metabolism and
half-life, which is established during the development procedures
typically employed in obtaining regulatory approval of a
pharmaceutical compound. Further factors that can be used to
determine an effective dose include the disease to be treated, the
benefit to be achieved in a patient, the patient's body mass, the
patient's immune status, the route of administration, whether
administration of a BGS-19 antagonist and/or combination
therapeutic agent is acute or chronic, concomitant medications, and
other factors known by the skilled artisan to affect the efficacy
of administered pharmaceutical agents.
[0557] In one embodiment, the pharmaceutical composition comprises
a BGS-19 antisense oligonucleotide at a dose of about 0.01 to 0.1,
0.1 to 0.9, 1 to 5, or 6 to 10 mg/kg/day; and a pharmaceutically
acceptable carrier. The actual amount of any particular antisense
oligonucleotide administered can depend on several factors, such as
the type of disease, the toxicity of the antisense oligonucleotide
to normal cells of the body, the rate of uptake of the antisense
oligonucleotide by tumor cells, and the weight and age of the
individual to whom the antisense oligonucleotide is administered.
The skilled artisan will appreciate the factors that may interfere
with the action or biological activity of the antisense
oligonucleotide in vivo, an effective amount of the antisense
oligonucleotide can be determined empirically by routine
procedures, including, for example, via clinical trials.
[0558] In another embodiment, the pharmaceutical compositions of
the invention comprise a BGS-19 antisense oligonucleotide at a
particularly high dose, which ranges from about 10 to 50 mg/kg/day.
In a specific embodiment a particularly high dose of BGS-19
antisense oligonucleotide, ranging from 11 to 15, 16 to 20, 21 to
25,26 to 30, 31 to 35, 36 to 40,41 to 45, or 46 to 50 mg/kg/day, is
administered during a treatment cycle.
[0559] A preferred effective dose of a BGS-19 antisense
oligonucleotide can be determined by a skilled artisan, especially
given that several antisense oligonucleotide compounds are
currently undergoing clinical trials. These routine trials can
establish the particular form of antisense oligonucleotide to be
administered, an appropriate delivery route, and a particular
antisense oligonucleotide's pharmacokinetic parameters such as
bioavailability, metabolism, and half-life. Other factors typically
considered during the course of a clinical trial are the patient's
body mass, the patient's immune status, the disease to be treated,
the benefit to be achieved in a patient, the route of
administration, whether administration of an antisense
oligonucleotide and/or combination therapeutic agent is acute or
chronic, concomitant medications, and other factors known by the
skilled artisan to affect the efficacy of administered
pharmaceutical agents.
Modes of Administration
[0560] Administration of the pharmaceutical compositions of the
invention includes, but is not limited to, oral, intravenous
infusion, subcutaneous injection, intramuscular, topical, depo
injection, implantation, time-release mode, intracavitary,
intranasal, inhalation, intratumor, intraocular, and controlled
release. A pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intramuscular, intraperitoneal,
intraorbital, intracapsular, intraspinal, intrasternal,
intra-arterial, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration. The
skilled artisan can appreciate the specific advantages and
disadvantages to be considered in choosing a mode of
administration.
[0561] Multiple modes of administration are encompassed by the
invention. For example, a BGS-19 antagonist is administered by
subcutaneous injection, whereas a combination therapeutic agent is
administered by intravenous infusion.
[0562] A BGS-19 antagonist can be administered before, during,
and/or after the administration of one or more therapeutic agents.
In one embodiment, a BGS-19 antagonist can first be administered to
cancer patient to reduce the expression of BGS-19, which increases
the tumor's sensitivity to subsequent challenge with a cancer
therapeutic agent. In another embodiment, a BGS-19 antagonist can
be administered after administration of a cancer therapeutic agent
to reduce tumor expression of BGS-19, which can deter tumor
resistance, and thereby prevent relapse or minimization of response
to the cancer therapeutic agent. In yet another embodiment, there
can be a period of overlap between the administration of BGS-19
antagonist and one or more cancer therapeutic agents.
[0563] Moreover, administration of one or more species of BGS-19
antagonist, with or without other therapeutic agents, may occur
simultaneously (i.e., co-administration) or sequentially. In one
embodiment, a BGS-19 antagonist is first administered to increase
sensitivity of a tumor to subsequent administration of a cancer
therapeutic agent or irradiation therapy. In another embodiment,
the periods of administration of one or more species of a BGS-19
antagonist, with or without other therapeutic agents may overlap.
For example, a BGS-19 antagonist is administered for 14 days, and a
second therapeutic agent is introduced beginning on the seventh day
of BGS-19 antagonist treatment, and treatment with the second
therapeutic agent continues beyond the 14-day BGS-19 antagonist
treatment.
[0564] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide, for example. The parenteral preparation
can be enclosed in ampules, disposable syringes or multiple dose
vials made of glass or plastic.
[0565] Pharmaceutical compositions adapted for parenteral
administration include, but are not limited to, aqueous and
non-aqueous sterile injectable solutions or suspensions, which may
contain antioxidants, buffers, bacteriostats and solutes that
render the compositions substantially isotonic with the blood of an
intended recipient. Such compositions may also comprise water,
alcohols, polyols, glycerine and vegetable oils, for example.
Compositions adapted for parenteral administration can be packaged
in unit-dose or multi-dose containers (e.g., sealed ampules and
vials). These compositions can be stored in a freeze-dried
(lyophilized) condition, which requires the addition of a sterile
liquid carrier, e.g., sterile saline solution for injections, prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets. Such
compositions should comprise a therapeutically effective amount of
a BGS-19 antagonist and/or other therapeutic agent, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0566] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Penetrants for transmucosal administration are
polynucleotiderally known in the art, and include, for example,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
polynucleotiderally known in the art.
[0567] Pharmaceutical compositions adapted for transdermal
administration can be provided as discrete patches intended to
remain in intimate contact with the epidermis for a prolonged
period of time. Pharmaceutical compositions adapted for topical
administration may be provided as, for example, ointments, creams,
suspensions, lotions, powders, solutions, pastes, gels, sprays,
aerosols, or oils. A topical ointment or cream is preferably used
for topical administration to the skin, mouth, eye or other
external tissues. When formulated in an ointment, the active
ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
maybe formulated in a cream with an oil-in-water base or a
water-in-oil base.
[0568] Pharmaceutical compositions adapted for topical
administration to the eye include, for example, eye drops or
injectable compositions. In these compositions, the active
ingredient can be dissolved or suspended in a suitable carrier,
which includes, for example, an aqueous solvent with or without
carboxymethylcellulose. Pharmaceutical compositions adapted for
topical administration in the mouth include, for example, lozenges,
pastilles and mouthwashes.
[0569] Pharmaceutical compositions adapted for oral administration
may be provided, for example, as capsules, tablets, powders,
granules, solutions, syrups, suspensions (in aqueous or non-aqueous
liquids), edible foams, whips, or emulsions. Tablets or hard
gelatine capsules may comprise, for example, lactose, starch or
derivatives thereof, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, stearic acid or salts thereof. Soft
gelatin capsules may comprise, for example, vegetable oils, waxes,
fats, semi-solid, or liquid polyols. Solutions and syrups may
comprise, for example, water, polyols and sugars.
[0570] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, and troches can contain any of the following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic
acid, Primogel, or corn starch; a lubricant such as magnesium
stearate or Sterotes; a glidant such as colloidal silicon dioxide;
a sweetening agent such as sucrose or saccharin; or a flavoring
agent such as peppermint, methyl salicylate, or orange
flavoring.
[0571] An active agent intended for oral administration may be
coated with or admixed with a material (e.g., glyceryl monostearate
or glyceryl distearate) that delays disintegration or affects
absorption of the active agent in the gastrointestinal tract. Thus,
for example, the sustained release of an active agent may be
achieved over many hours and, if necessary, the active agent can be
protected from being degraded within the gastrointestinal tract.
Taking advantage of the various pH and enzymatic conditions along
the gastrointestinal tract, pharmaceutical compositions for oral
administration may be formulated to facilitate release of an active
agent at a particular gastrointestinal location. Oral formulations
preferably comprise 10% to 95% active ingredient by weight.
[0572] Pharmaceutical compositions adapted for nasal administration
can comprise solid carriers such as powders (preferably having a
particle size in the range of 20 to 500 microns). Powders can be
administered in the manner in which snuff is taken, i.e., by rapid
inhalation through the nose from a container of powder held close
to the nose. Alternatively, compositions adopted for nasal
administration may comprise liquid carriers such as, for example,
nasal sprays or nasal drops. These compositions may comprise
aqueous or oil solutions of the active ingredient. Compositions for
administration by inhalation may be supplied in specially adapted
devices including, but not limited to, pressurized aerosols,
nebulizers, or insufflators, which can be constructed so as to
provide predetermined dosages of the active ingredient.
[0573] Pharmaceutical compositions adapted for rectal
administration can be prepared in the form of suppositories (e.g.,
with conventional suppository bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery. Pharmaceutical
compositions adapted for vaginal administration may be provided,
for example, as pessaries, tampons, creams, gels, pastes, foams, or
spray formulations.
[0574] In one embodiment, a pharmaceutical composition of the
invention is delivered by a controlled-release system. For example,
the pharmaceutical composition may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used (See, e.g., Langer, 1990, Science
249:1527-1533; Sefton, 1987, CRC Crit Ref Biomed Eng. 14:201;
Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N Engl
J. Med. 321:574). In another embodiment, the compound can be
delivered in a vesicle, in particular a liposome (See, e.g.,
Langer, 1990, Science 249:1527-1533; Treat et al., 1989, in
Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.) Liss, New York, pp. 353-365;
Lopez-Berestein, ibid., pp. 317-327; International Patent
Publication No. WO 91/04014; U.S. Pat. No. 4,704,355). In another
embodiment, polymeric materials can be used (See, e.g., Medical
Applications of Controlled Release, Langer and Wise (eds.) CRC
Press: Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.) Wiley:
New York (1984); Ranger and Peppas, 1953, J. Macromol Sci Rev
Macromol Chem. 23:61; Levy et al., 1985, Science 228:190; During et
al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg.
71:105).
[0575] In one embodiment, the active compounds, which comprise
polynucleotides, polypeptides, or antibodies of the invention, are
prepared with carriers that will protect the compound from rapid
elimination from the body. Such carriers can be a controlled
release formulation, which includes, but is not limited to,
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0576] In a particular embodiment, polypeptides of the invention
can be administered using a biodegradable polymer having reverse
thermal gelatin properties (See, e.g., U.S. Pat. No.
5,702,717).
[0577] In yet another embodiment, a controlled release system can
be placed in proximity of the target. For example, a micropump may
deliver controlled doses directly into the axillary lymph node
region, thereby requiring only a fraction of the systemic dose
(See, e.g., Goodson, 1984, in Medical Applications of Controlled
Release, vol. 2, pp. 115-138).
[0578] In one embodiment, it may be desirable to administer a
pharmaceutical composition of the invention locally to the area in
need of treatment; this may be achieved, for example, by local
infusion during surgery, topical application (e.g., in conjunction
with a wound dressing after surgery), injection, by means of a
catheter, by means of a suppository, or by means of an implant. An
implant can be of a porous, non-porous, or gelatinous material,
including membranes, such as sialastic membranes, or fibers.
Suppositories polynucleotiderally comprise active ingredients in
the range of 0.5% to 10% by weight.
[0579] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions, or dispersions, or sterile
powders (for the extemporaneous preparation of sterile injectable
solutions or dispersions). For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF; Parsippany, N.J.) or phosphate buffered
saline (PBS). The carrier can be a solvent or dispersion medium
comprising, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion, or by the use of a surfactant. Prevention of
the action of microorganisms can be achieved by various
antibacterial and antifungal agents, such as for example, parabens,
chlorobutanol, phenol, ascorbic acid, and thimerosal. It can be
preferable to include in the composition isotonic agents, such as
for example, sugars, polyalcohols (e.g., mannitol), sorbitol, and
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, such as for example, aluminum
monostearate and gelatin.
[0580] Sterile injectable solutions can be prepared by
incorporating the required amount of an active compound (e.g., a
polypeptide or antibody) in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
comprises a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder comprising the active
ingredient.
[0581] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which comprises a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0582] Oral or parenteral compositions can be formulated in dosage
unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for the subject to be treated, such that
each unit contains a predetermined quantity of active compound,
which is calculated to produce the desired therapeutic effect, and
a pharmaceutical carrier. The skilled artisan will appreciate that
dosage unit forms are dependent on the unique characteristics of
the active compound, the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for human administration.
[0583] For antibodies, the preferred dosage is 0.1 mg/kg to 100
mg/kg of body weight (polynucleotiderally 10 mg/kg to 20 mg/kg).
For example, if the antibody is to act in the brain, a dosage of 50
mg/kg to 100 mg/kg is usually appropriate. Since partially human
antibodies and fully human antibodies polynucleotiderally have a
longer half-life in a patient than other antibodies, lower dosages
and less frequent administration is possible. Modifications, such
as lipidation, can be used to stabilize antibodies and to enhance
uptake and tissue penetration (See, e.g., Cruikshank et al., 1997,
J Acquir Immune Defic Syndr Hum Retrovirol. 14:193-203).
[0584] In one embodiment, a therapeutically effective amount of a
polypeptide of the invention ranges from about 0.001 to 30 mg/kg
body weight. In another embodiment, a therapeutically effective
amount of a polypeptide of the invention ranges from about 0.01 to
25 mg/kg body weight. In another embodiment, a therapeutically
effective amount of a polypeptide of the invention ranges from
about 0.1 to 20 mg/kg body weight. In yet another embodiment, a
therapeutically effective amount of a polypeptide of the invention
ranges from about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7
mg/kg, or 5 to 6 mg/kg body weight.
[0585] The skilled artisan will appreciate that certain factors may
influence the dose necessary to effectively treat a subject, which
factors include, but are not limited to, previous treatment
regimens, severity of the disease or disorder, polynucleotideral
health and/or age of the subject, and concurrent diseases.
Moreover, treatment of a subject with a therapeutically effective
amount of a protein, polypeptide, or antibody can include a single
treatment or, preferably, can include a series of treatments. In a
preferred example, a subject is treated with antibody, protein, or
polypeptide in the range of between about 0.1 to 20 mg/kg body
weight, one time per week for between about 1 to 10 weeks,
preferably between 2 to 8 weeks, more preferably between about 3 to
7 weeks, and even more preferably for about 4, 5, or 6 weeks. It
will also be appreciated that the effective dosage of antibody,
protein, or polypeptide used for treatment may increase or decrease
over the course of a particular treatment. Changes in dosage may
result and become apparent from the results of diagnostic assays as
described herein.
Kits
[0586] The invention also encompasses kits for detecting the
presence of a BGS-19 polypeptide or polynucleotide of the invention
in a biological sample (a test sample). Such kits can be used to
determine if a subject is suffering from or is at increased risk of
developing a disorder associated with aberrant expression of a
polypeptide of the invention as discussed, for example, in sections
above relating to uses of the sequences of the invention.
[0587] In an exemplary embodiment, a kit comprises, in a first
container, a purified BGS-19 polynucleotide, BGS-19 polypeptide,
BGS-19 agonist, BGS-19 antagonist, and in a second container, a
molecule that binds to the BGS-19 polynucleotide, BGS-19
polypeptide, BGS-19 agonist, BGS-19 antagonist when bound to an
analyte in a biological sample. The molecule can be, for example, a
detectable tag that recognizes a complex comprising BGS-19 and the
analyte such that the interaction between BGS-19 and the analyte is
identified.
[0588] For example, kits can be used to determine if a subject is
suffering from or is at increased risk of disorders such as cancer,
in particular hormone-sensitive cancers, such as but not limited to
cancer of the breast, ovary, uterus, prostate, testis, skin and
brain.
[0589] In another example, kits can be used to determine if a
subject is suffering from or is at risk for a disorder associated
with aberrant expression of a polypeptide of the invention.
[0590] The kit, for example, can comprise a labeled compound or
agent capable of detecting the BGS-19 polypeptide or BGS-19 mRNA
encoding the polypeptide in a biological sample and means for
determining the amount of the polypeptide or mRNA in the sample
(e.g., an antibody which binds the polypeptide or an
oligonucleotide probe which binds to DNA or mRNA encoding the
polypeptide). Kits can also include instructions for observing that
the tested subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of the polypeptide if
the amount of the polypeptide or mRNA encoding the polypeptide is
above or below a normal level.
[0591] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide of the invention; and,
[0592] optionally, (2) a second, different antibody which binds to
either the BGS-19 polypeptide or the first antibody and is
conjugated to a detectable agent.
[0593] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a BGS-19 polynucleotide
sequence encoding a polypeptide of the invention or (2) a pair of
primers useful for amplifying a polynucleotide encoding a
polypeptide of the invention. The kit can also comprise, e.g., a
buffering agent, a preservative, or a protein stabilizing agent.
The kit can also comprise components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The kit can also
comprise a control sample or a series of control samples which can
be assayed and compared to the test sample contained. Each
component of the kit is usually enclosed within an individual
container and all of the various containers are within a single
package along with instructions for observing whether the tested
subject is suffering from or is at risk of developing a disorder
associated with aberrant expression of the polypeptide.
[0594] The invention provides a kit containing an antibody of the
invention conjugated to a detectable substance, and instructions
for use. Still another aspect of the invention is a pharmaceutical
composition comprising an antibody of the invention and a
pharmaceutically acceptable carrier. In preferred embodiments, the
pharmaceutical composition comprises an antibody of the invention,
a therapeutic moiety, and a pharmaceutically acceptable
carrier.
[0595] The pharmaceutical compositions of the invention can be
included in a container, pack, or dispenser together with
instructions for administration.
EXAMPLES
Example 1
Identification of BGS-19 Gene
[0596] IgSF protein sequences with immunoglobulin domains from
several different species, were used as probes to search the human
genomic sequence database. The search program used was Gapped BLAST
(Altschul et al., 1997, "Gapped BLAST and PSI-BLAST: a new
polynucleotideration of protein database search programs", Nucleic
Acids Res. 25:3389-3402). The best genomic exon matches from the
BLAST results were then searched against the non-redundant protein
and patent sequence databases. From this analysis, exons encoding
potential novel open reading frames (ORF) were identified based on
sequence homology. The protein sequences with the greatest
similarity were then used as templates to predict additional exons
using the GENEWISEDB program (Birney and Durbin, 2000, "Using
GeneWise in the Drosophila annotation experiment", Genome Res.
10:547-548). The final predicted exons were assembled and consensus
sequences of polynucleotides were obtained using the predicted exon
sequences. With these analyses, a predicted partial sequence of a
novel human cell surface IgSF protein, named BGS-19 was identified
directly from a human genomic sequence ACO11452. The
computationally predicted nucleotide and amino acid sequences of
BGS-19 are depicted in FIGS. 1-4.
[0597] The isolated full-length of BGS-19 is a 1985-bp cDNA that
encodes a 385 amino acid protein (see, e.g., FIGS. 3-4). A search
of BGS-19 against protein databases identified Siglec-6, Siglec-7,
and Siglec-10 as the three closest homologs of BGS-19. Using the
global alignment program, GAP, from the GCG bioinformatics software
package, BGS-19 was found to have 41.4% identity and 48.0%
similarity to Siglec-6, 44.7% identity and 50.8% similarity to
Siglec-7 protein, and 46.6% identity and 51.7% similarity to
Siglec-10 protein (FIGS. 4A-E). Similar to Siglec-6, Siglec-7 and
Siglec-10, BGS-19 has a predicted signal peptide cleavage site
between residues 15 and 16 (predicted by SPScan program from GCG
software package). There is a predicted single transmembrane domain
between residues 250 and 275 (SEQ ID NO:8). BGS-19 is a type I cell
surface receptor. Two predicted Ig domains are in the extracellular
region at residues 16-113 and residues 140-241, (HMM Pfam search,
Bateman et al., 2000).
[0598] BGS-19 has a typical ITIM (immuno-receptor tyrosine-based
inhibitory motif, residues 329-334=LHYASL). Tyr 331 is part of a
typical ITIM signature. Many studies have shown that all of the
inhibitory receptors identified thus far are characterized by one
or more ITIMs in their cytoplasmic tail. Upon tyrosine
phosphorylation, ITIM binds the src homology (SH2) domains of
phophatases such as SHP-1 and SHP-2, resulting in the
downregulation of natural killer cell triggering and cytotoxicity
(Renard et al., 1997, "Transduction of cytotoxic signals in natural
killer cells: a polynucleotideral model of fine tuning between
activatory and inhibitory pathways in lymphocytes", Immunol Rev.
155:205-221). Recently, Siglec-7 (p75/AIRM 1), was found to
function as an inhibitory receptor in human natural killer cells
(Falco et al., 1999, "Identification and molecular cloning of
p75/AIRM1, a novel member of the sialoadhesin family that functions
as an inhibitory receptor in human natural killer cells", J Exp
Med. 190:793-802). As such, BGS-19 can be useful as an inhibitory
receptor.
[0599] BGS-19 is highly expressed in lung and lymphocyte-rich
tissues, such as spleen, lymph node and bone marrow (FIG. 6).
Therefore, BGS-19 can be useful for studies and manipulations
involving cells of the immune system. See, e.g., Whitney et al.,
2001, "A new siglec family member, Siglec-10, is expressed in cells
of the immune system and has signaling properties similar to CD33",
Eur J. Biochem. 268:6083-6096 (demonstrating high expression of
Siglec-10 in asthmatic eosinophils). As such, BGS-19 can be useful
as a signaling molecule.
Example 2
Cloning of the Novel Human BGS-19
[0600] A multiplex cloning method was used to extend the
bioinformatic polynucleotide prediction for BGS 19 into full length
cDNA. The multiplex cloning method is used to extend large numbers
of bioinformatic polynucleotide predictions into full length
sequences by multiplexing probes and cDNA libraries to minimize the
overall effort typically required for cDNA cloning. Plasmid-based
directionally cloned cDNA libraries are converted into a population
of pure, covalently-closed, circular, single-stranded molecules.
Long biotinylated DNA oligo probes are designed from predicted
polynucleotide sequences. Probes and libraries are hybridized in
solution in formamide buffer rather than in the aqueous buffers
recommended in other biotin/strepavidin cDNA capture methods (i.e.,
GeneTrapper). Information on the representation of clones in the
libraries is not required to perform the hybridization. The
hybridization is carried out twice. After the first selection, the
isolated sequences are screened with PCR primers specific for the
targeted clones. The second hybridization is carried out with only
those oligo probes whose polynucleotide-specific PCR assays gave
positive results. The secondary hybridization serves to `normalize*
the selected library thereby decreasing the amount of screening
needed to identify particular clones. The method is robust and
sensitive. Typically, dozens of cDNAs are isolated for any one
particular polynucleotide, thereby increasing the chances of
obtaining a full-length cDNA. The entire complexity of any cDNA
library is screened in the solution hybridization process, which is
advantageous for finding rare sequences. Although 50 oligo probes
per experiment are currently being used, larger numbers of probes
can also be used.
General Cloning Strategy
[0601] Using bioinformatic predicted polynucleotide sequence, the
following types of polynucleotide-specific PCR primers and cloning
oligos are designed:
[0602] A) PCR primer pairs that reside within a single predicted
exon;
[0603] B) PCR primer pairs that cross putative exon/intron
boundaries; and
[0604] C) an 80mer antisense and sense oligo with a biotin moiety
on the 5' end.
[0605] The primer pairs from the A type primer are optimized on
human genomic DNA; the B type primers are optimized on a mixture of
first strand cDNAs made with and without reverse transcriptase,
from brain and testis poly A.sup.+RNA. The information obtained
with the B type primers is used to assess which putative expressed
sequences exhibit reverse transcriptase dependent expression. The A
type primer pairs are less stringent for identifying expressed
sequences, because they amplify genomic DNA as well as cDNA.
However, because the A type primers can amplify genomic DNA, they
provide the necessary positive control for the primer pair.
Negative results using the B type primer are only valid upon
confirmation, with the positive control, that the sequence is
indeed expressed in the first strand.
[0606] The biotinylated 80mer oligos are added en masse to pools of
single stranded cDNA libraries. Up to 50 probes have been
successfully used on pools for 15 different libraries. The
orientation of the oligo depends on the orientation of the cDNA in
its vector. Antisense 80mer oligos are used for those libraries and
cloned into pCMVSPORT and pSPORT whereas sense 80mer oligos are
used for cDNA libraries cloned into pSPORT2. After the primary
selection is carried out, all of the captured DNA is repaired to
double stranded form using the T7 primer for the commercial
libraries in pCMVSPORT, and the Sp6 primer for in-house constructed
libraries in pSPORT. The resulting DNA is electroporated into E.
coli DH12 S and plated onto 150 mm plates with nylon filters. The
cells are scraped from the plate and a frozen stock is made. This
is the primary selected library. One-fifth of the library is
converted into single strand form and the DNA assayed with the
polynucleotide specific primers pairs (GSPs). The second round of
hybridization is carried out with 80mer oligos for only those
sequences that were positive with the
polynucleotide-specific-primers. After the second round, the
captured single strand DNAs are repaired with a pool of GSPs, where
only the primer complementary to the single-stranded circular DNA
is used (the antisense primer for pCMVSPORT and pSPORT1 and the
sense primer for pSPORT2). The resulting colonies are screened by
PCR using the GSPs. Typically, greater than 80% of the clones are
positive for any given GSP. DNA from each well of the entire 96
well block of clones was prepared and each of the clones sized by
either PCR or restriction enzyme digestion. A selection of clones
for each targeted sequence are chosen for transposon-hopping and
DNA sequencing.
[0607] Success of the method, like any cDNA cloning method, depends
of on the quality of the libraries employed. High complexity and
large average insert size are required. HPLC is used to fractionate
cDNA to construct libraries.
Example 3
Construction of a Size Fractionated cDNA Library for the Isolation
of Large Insert Clones
[0608] Poly A.sup.+ RNA from Clontech is treated with DNase I to
remove genomic DNA contamination. The RNA is converted into double
stranded cDNA using the SuperScript.TM. Plasmid System for cDNA
Synthesis and Plasmid Cloning (Life Technologies). The cDNA is size
fractionated on a TransGenomics HPLC size exclusion column
(TosoHass) with dimensions of 7.8 mm.times.30 cm and a particle
size of 10 .mu.m. Tris buffered saline is used as the mobile phase,
and the column is run at a flow rate of 0.5 ml/min. The system is
calibrated using a 1 kb ladder to determine which fractions are to
be pooled to obtain the largest cDNA library. Generally, fractions
that eluted in the range of 12 to 15 minutes are used. The cDNA is
precipitated, concentrated and then ligated into the Sal I/Not I
sites in pSPORT. Following electroporation of the cDNA into DH12S,
DNA from the resulting colonies is prepared and subjected to Sal
I/Not I restriction enzyme digestion. Generally, the average insert
size of libraries made by this procedure is greater than 3.5 Kb and
the overall complexity of the library is greater than 10.sup.7
independent clones. The library is amplified in semi-solid agar for
2 days at 30 C. An aliquot (200 microliters) of the amplified
library is inoculated into a 200 ml culture for single-stranded DNA
isolation by super-infection with a fi helper phage. The
single-stranded circular DNA is concentrated by ethanol
precipitation, resuspended at a concentration of one microgram per
microliter and used for the cDNA capture experiments.
Example 4
Conversion of Double Stranded cDNA Libraries Into Single Strand
Circular Form
I. Preparation of culture.
[0609] LB medium (200 mL+400 .mu.l carb) is inoculated with 0.2 to
1 ml of thawed cDNA library. The culture is incubated, shaking at
250 rpm at 37.degree. C. for 45 min. The optical density of the
culture is measured. The OD600 is preferably between 0.025 and
0.040. One mL M13K07 helper phage is added to the culture and grown
for 2 hours. At that time, 500 .mu.L Kanamycin (30 mg/mL) is added
and incubation continued for 15-18 hours.
II. Preparation of cells for precipitation.
[0610] Cultures are poured into six 50 mL tubes. Cells are
centrifuged at 10000 rpm in an HB-6 rotor for 15 minutes at
4.degree. C. The supernatant is retrieved and cells discarded. The
supernatant is filtered through a 0.2 .mu.m filter. DNase 1 (12000
units from Gibco) is added and incubated at room temperature for 90
minutes.
Ill. PEG precipitation of DNA.
[0611] Fifty mL of ice-cold 40% PEG 8000, 2.5 M NaCl, 10 mM
MgSO.sub.4 is added to the cell pellets. The solution is mixed and
distributed into 6 centrifuge tubes and covered with parafilm. The
tubes are incubated on wet ice for 1 hour (or at 4.degree. C.
overnight).
[0612] Phage are pelleted at 10000 rpm in an HB-6 rotor for 20
minutes at 4.degree. C. The supernatant is discarded and the sides
of the tubes wiped dry. The pellets are resuspended in 1 mL TE, pH
8.
[0613] The resuspended pellets are placed in a 14 mL Sarstedt tube
(6 mL total). SDS is added to 0.1% (60 .mu.L of stock 10% SDS).
Proteinase K (60 .mu.L of 20 mg/mL) is then added and incubated at
42 C for 1 hour.
[0614] DNA is extracted with phenol/chloroform by first adding 1 mL
of 5M NaCl followed by an equal volume of phenol/chloroform (6 mL).
The mixture is vortexed and centrifuged at 5 K in an HB-6 rotor for
5 minutes at 4.degree. C. The aqueous (top) phase is transferred to
a new Sarstedt tube. Extractions are repeated until no interface is
visible.
[0615] The DNA is precipitated in ethanol by adding 2 volumes of
100% ethanol and precipitating overnight at -20.degree. C. The DNA
is centrifuged at 10000 rpm in HB-6 rotor for 20 minutes at
4.degree. C. The ethanol is discarded and the pellets resuspended
in 700 .mu.L 70% ethanol. The resuspended pellets are centrifuged
at 14000 rpm for 10 minutes at 4.degree. C. The ethanol is
discarded and the pellets dried by vacuum.
[0616] Oligosaccharides are then removed by resuspending the pellet
in 50 .mu.L TE, pH 8. The solutions are frozen on dry ice for 10
minutes and centrifuged at 14000 rpm for 15 minutes at 4.degree. C.
The supernatant is transferred to a new tube and the volume
recorded.
[0617] The concentration of DNA is determined by measuring
absorbance at 260/280. DNA is diluted 1:100 in a quartz cuvette (3
.mu.L DNA+297 .mu.L TE). The following equation is used to
calculate DNA concentration: (32 .mu.g/mL*OD)(mL/100
.mu.L)(100)(OD260)=DNA concentration The preferred purity ratio is
1.7-2.0. The DNA is diluted to 1 .mu.g/uL with TB, pH 8 and stored
at 4.degree. C.
[0618] To test the quality of single-stranded DNA (ssDNA) the
following reaction mixtures are prepared:
[0619] 1. DNA mix per reaction [0620] a. 1 .mu.L of 5 ng/.mu.L
ssDNA (1:200 dilution of VI.D.2 above) [0621] b. 11 .mu.L dH.sub.2O
[0622] c. 1.5 .mu.L 10 .mu.M T7 SPORT primer (fresh dilution of
stock) [0623] d. 1.5 .mu.L 10.times.Precision-Taq buffer
[0624] 2. Repair mix per reaction [0625] a. 4 .mu.L 5 mM dNTPs
(1.25 mM each) [0626] b. 1.5 .mu.L 10.times.Precision-Taq buffer
[0627] c. 9.25 .mu.L dH.sub.2O [0628] d. 0.25 .mu.L Precision-Taq
polymerase [0629] e. Preheat cocktail at 70.degree. C. until middle
of thermal cycle The DNA mixes are aliquoted into PCR tubes and
thermal cycle carried out as follows:
[0630] 1. 95.degree. C., 20 sec
[0631] 2. 59.degree. C., 1 min; add 15 .mu.L repair mix
[0632] 3. 73.degree. C., 23 min
[0633] Ethanol precipitation of the ssDNA is performed by adding 15
.mu.g glycogen, 16 .mu.L 7.5 M NH.sub.4OAc, 125 .mu.L 100% ethanol.
The sample is centrifuged at 14000 rpm for 30 minutes at 4.degree.
C. and the pellet washed with 125 .mu.L 70% ethanol. The ethanol is
discarded and pellet dried by vacuum. The pellet is resuspended in
10 .mu.L TB, pH 8.
[0634] The DNA is electroporated into DH10B or DH12S cells. A DNA
mixture consisting of:
[0635] 1. 2 .mu.L repaired library (=1.0.times.10-3 .mu.g)
[0636] 2. 1 .mu.L 1 ng/.mu.L unrepaired library (=1.0.times.10-3
.mu.g)
[0637] 3. 1 .mu.L 0.01 .mu.g/uL pUC19 positive control DNA
(=1.times.10-5 .mu.g) is aliquoted to Eppendorf tubes. Cells are
thawed on ice-water. Forty .mu.L of cells are added to each DNA
aliquot by pipetting into a chilled cuvette placed between metal
plates. Electroporation is carried out at 1.8 kV. Immediately
following electroporation, 1 mL SOC(SOB+glucose +Mg.sup.++) media
is added to the cuvette, then transferred to a 15 mL tube. Cells
are allowed to recover for 1 hr at 37.degree. C. with shaking (225
rpm). Cells are then plated according to the following dilution
scheme:
A. Dilutions of Culture
[0638] 1. Serial dilutions of culture in 1:10 increments (20 .mu.L
into 180 .mu.L LB broth)
[0639] 2. Repaired dilutions [0640] a. 1:100 [0641] b. 1:1K [0642]
c. 1:10K
[0643] 3. Unrepaired dilutions [0644] a. 1:10 [0645] b. 1:100
[0646] 4. Positive control dilutions [0647] a. 1:10 [0648] b. 1:100
100 .mu.L of each dilution is plated on small LB+carb plates and
incubated at 37.degree. C. overnight. Colonies are counted to
calculate titer as follows:
[0649] 1. use smallest countable dilution
[0650] 2. (# of colonies)(dilution factor)(200 .mu.L/100
.mu.L)(1000 .mu.L/20 .mu.L)=CFUs
[0651] 3. CFUs/.mu.g DNA used .dbd.CFU/.mu.g
% Background=(unrepaired CFU/.mu.g/repaired
CFU/.mu.g).times.100%
Example 5
Solution Hybridization and DNA Capture
[0652] One microliter of anti-sense biotinylated oligos (or sense
oligos when annealing to single stranded DNA from pSPORT2 vector),
containing one hundred and fifty nanograms of 1 to 50 different
80mer oligo probes, is added to six microliters (six micrograms) of
a mixture of up to 15 single-stranded covalently closed circular
cDNA libraries and seven microliters of 100% formamide in a 0.5 ml
PCR tube. The sequence of the 80mer oligos used is as follows:
TGGGCTGGTCCGTCCTTTGAACC
AGTAGCCATAAGCAGCAGTAGACTCGTCCCAGCCATCCCGGGGGTAGGAGA GGTTGC (SEQ ID
NO:57). The mixture is heated in a thermal cycler to 95.degree. C.
for 2 min. Fourteen microliters of 2.times. hybridization buffer
(50% formamide, 1.5 M NaCl, 0.04 M NaPO.sub.4, pH 7.2, 5 min EDTA,
0.2% SDS) is added to the heated probe/cDNA library mixture and
incubated at 42.degree. C. for 26 hours. Hybrids between the
biotinylated oligo and the circular cDNA are isolated by diluting
the hybridization mixture to 220 microliters solution containing 1
M NaCl, 10 mm Tris-HCl pH 7.5, 1 mM EDTA, pH 8.0 and adding 125
microliters of streptavidin magnetic beads. This solution is
incubated at 42.degree. C. for 60 min, and mixed every 5 min to
re-suspend the beads. The beads are separated from the solution
with a magnet and washed three times in 200 microliters of
0.1.times.SSPE, 0.1% SDS at 45.degree. C.
[0653] The single stranded cDNA is released from the biotinylated
oligo/streptavidin magnetic bead complex by adding 50 microliters
of 0.1 N NaOH and incubating at room temperature for 10 min. Six
microliters of 3 M sodium acetate is added along with 15 micrograms
of glycogen and the solution ethanol precipitated with 120
microliters of 100% ethanol. The precipitated DNA is resuspended in
12 microliters of TB (10 min Tris HCl, pH 8.0), 1 mM EDTA, pH 8.0).
The single-stranded cDNA is converted into double-stranded DNA in a
thermal cycler by mixing 5 microliters of the captured DNA with 1.5
microliters of 10 micromolar standard SP6 primer for libraries in
pSPORT 1 and 2 and 17 primer for libraries in pCMVSPORT and 1.5
microliters of 10.times.PCR buffer.
[0654] Sequences of primers used to repair single-stranded circular
DNA isolated from the primary selection are as follows:
TABLE-US-00002 T7Sport 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 58)
SP6Sport 5'-ATTTAGGTGACACTATAG-3' (SEQ ID NO: 59)
[0655] The mixture is heated to 95.degree. C. for 20 seconds and
the temperature gradually brought down to 59.degree. C. Fifteen
microliters of a repair mix, that was preheated to 70.degree. C. is
added to the DNA (repair mix contains 4 microliters of 5 mM dNTPs
(1.25 mM each), 1.5 microliters of 10.times.PCR buffer, 9.25
microliters of water, and 0.25 microliters of Taq polymerase). The
solution incubation temperature is raised back to 73.degree. C. and
incubated for 23 mm. The repaired DNA is ethanol precipitated and
resuspended in 10 microliters of TB. Electroporation is carried out
using two microliters DNA per 40 microliters of E. coli DH12S
cells. Three hundred and thirty three microliters are plated onto
one 150-mm plate of LB agar plus 100 micrograms/milliliter of
ampicillin. After overnight incubation at 37.degree. C., the
colonies from all plates are harvested by scraping into 10 ml of LB
medium +50 micrograms/milliliter of ampicillin and 2 ml of sterile
glycerol.
[0656] The second round of selection is initiated by making
single-stranded circular DNA from the primary selected library
using the method listed above. The purified single-stranded
circular DNA is then assayed with polynucleotide-specific primers
for each of the targeted sequences using standard PCR
conditions.
[0657] The sequences of the Gene-Specific-Primer ("GSP") pairs used
to identify the various targeted cDNAs in the primary selected
single stranded cDNA libraries are as follows: TABLE-US-00003 Left
CATCGTGTCTTGCAACCTCT (SEQ ID NO: 60) Primer 1: Right
CTCTCTCCACCCGAAAGAAG (SEQ ID NO: 61) Primer 1:
[0658] The secondary hybridization is carried out using only those
80mer biotinylated probes whose targeted sequences were positive
with the GSPs. The resulting single-stranded circular DNA is
converted to double strands using the antisense oligo for each
target sequence as the repair primer (the sense primer is used for
material captured from pSPORT2 libraries. The resulting double
stranded DNA is electroporated into DH10B and the resulting
colonies inoculated into 96 deep well blocks. Following overnight
growth, DNA is prepared and sequentially screened for each of the
targeted sequences using the GSPs. The DNA is also cut with Sal I
and Not I and the inserts sized by agarose gel electrophoresis.
Example 6
Expression Profile of BGS-19
[0659] The same PCR primer pair that was used to identify BGS-19
cDNA clones was used to measure the steady state levels of mRNA by
quantitative PCR (SEQ ID NO:60 and 61). Briefly, first strand cDNA
was made from commercially available mRNA (Clontech) and subjected
to real time quantitative PCR using a PE 5700 instrument (Applied
Biosystems, Foster City, Calif.) which detects the amount of DNA
amplified during each cycle by the fluorescent output of SYBR
green, a DNA binding dye specific for double strands. The
specificity of the primer pair for its target is verified by
performing a thermal denaturation profile at the end of the run
which gives an indication of the number of different DNA sequences
present by determining melting Tm. In the case of the BGS-19 primer
pair, only one DNA fragment was detected having a
homopolynucleotideous melting point. Contributions of contaminating
genomic DNA to the assessment of tissue abundance is controlled for
by performing the PCR with first strand made with and without
reverse transcriptase. In all cases, the contribution of material
amplified in the no reverse transcriptase controls was
negligible.
[0660] Small variations in the amount of cDNA used in each tube was
determined by performing a parallel experiment using a primer pair
for a polynucleotide expressed in equal amounts in all tissues,
cyclophilin. These data were used to normalize the data obtained
with the BGS-19 primer pair. The PCR data was converted into a
relative assessment of the difference in transcript abundance
amongst the tissues tested and the data are presented in bar graph
form. Transcripts corresponding to BGS-19 are found in highest
concentration in RNA isolated from the spleen and lung.
TABLE-US-00004 Primer pair used for profiling BGS19.2s
AAGAACCAGACCAAGCACCT (SEQ ID NO: 80) BGS19.2a CCCTTTCTGGAGAAGTCCAC
(SEQ ID NO: 81)
Detail Methods
DNase the RNA
I. Dilute 5 .mu.g of poly A+ RNA to 77 uL w/DEPC H.sub.2O
[0661] II. Rxn mix--make cocktail for samples +1 extra reaction for
pipetting errors TABLE-US-00005 Components vol/rxn 10X PCR Buffer
10 .lamda. 25 mM MgCl2 8 .lamda. RNase-Out 40 U/uL 2.5 .lamda.
RNase-Free Dnase (B-M) 2.5 .lamda. 23 .lamda.
[0662] A. Add 23.lamda. mix to each sample
[0663] B. Incubate @ RT, 15'
[0664] C. Add 1 micorliters 250 mM EDTA
[0665] D. Incubate @ 6 degrees C., 15 minutes on ice
III. Clean-Up
[0666] A. Extract w/100.lamda. Phenol:Chloroform:Isoamyl Alcohol
[0667] 1. Vortex 1 minute [0668] 2. Spin @ 12 K rpm 2 minute [0669]
3. Remove 90-95.lamda. of the top aqueous phase, transfer to new
tube
[0670] B. Ethanol precipitation [0671] 1. Add 1.lamda. 20
ug/.lamda. glycogen, 15.lamda. 2 M NaAcetate, 290.lamda. 100% EtOH
[0672] 2. ppt @-2 degrees C. for 1 h [0673] 3. Pellet @ 4 degrees
C., for 30 minutes [0674] 4. Wash in 500.times.70% EtOH, dry [0675]
5. Resuspend in 22 uL RNase-free WATER
First Strand cDNA Synthesis
[0675] I. Split volume of RNA above into 2 tubes (RT+/RT-)
II. Prime
[0676] A. Add 1.lamda. Oligo(dT) to each
[0677] B. Incubate @ 7 degrees C., 10 minutes on ice
[0678] III. Rxn mix--make cocktail for samples +1 extra reaction
for pipetting errors TABLE-US-00006 Components Vol/rxn 10X PCR
Buffer 2 .lamda. 25 mM MgCl2 2 .lamda. 10 mM dNTP mix 1 .lamda.
0.1M DTT 2 .lamda. 7 .lamda.
[0679] A. Add 7.lamda. mix to each sample
[0680] B. Incubate @ 4 degrees C., 5 minutes
[0681] C. Add 1.lamda. SuperScript II RT to RT+ and 1.times.DEPC
WATER to RT- samples
[0682] D. Incubate @ 4 degrees C., 50 minutes
IV. Terminate rxn @ 7 degrees C., 15 minutes on ice
V. Add 1.lamda. RNase H, incubate @ 3 degrees C., 20 minutes
VI. Add 79.lamda. water final conc.=2.5 ng/uL cDNA (assuming 100%
conversion)
Quantitative PCR
I. Determine number of rxns and amount of mix needed
[0683] A. all samples run in triplicate, so sample tubes need 3.5
rxns worth of mix
[0684] B. =(2.times.# tissue samples +1 no template control +1 for
pipetting error)(3.5)
[0685] II. Rxn mix TABLE-US-00007 Components vol/rxn 2X SybrGreen
Master Mix 25 .lamda. water 23.5 .lamda. primer mix (10 uM ea.) 0.5
.lamda. cDNA (2.5 ng/uL) 1 .lamda.
[0686] A. Make mix minus cDNA for enough reactions as determined
above
[0687] B. Aliquot 171.5.lamda. of mix to sample tubes
[0688] C. Add 3.5.lamda. of cDNA to each sample tube
[0689] D. Mix gently and spin down to collect
[0690] E. Aliquot 3.times.50.lamda. to optical plate
III. Set up 5700
[0691] A. Enter primer and sample set-up
[0692] B. Save (plate) As . . .
[0693] C. Run default program including dissociation protocol
[0694] 1) Hold, 2 min, 50.degree. C. [0695] 2) Hold, 10 min,
95.degree. C. [0696] 3) Cycle 40 cycles [0697] Melt, 15 sec,
96.degree. C. [0698] Anneal/Extend, 1 min, 60.degree. C.
Example 7
Method of Assessing the Expression Profile of the Novel BGS-19
Polypeptides of the Present Invention Using Expanded mRNA Tissue
and Cell Sources
[0699] Total RNA from tissues was isolated using the TriZol
protocol (Invitrogen) and quantified by determining its absorbance
at 260 nM. An assessment of the 18 s and 28 s ribosomal RNA bands
was made by denaturing gel electrophoresis to determine RNA
integrity.
[0700] The specific sequence to be measured was aligned with
related genes found in GenBank to identity regions of significant
sequence divergence to maximize primer and probe specificity.
Gene-specific primers and probes were designed using the ABI primer
express software to amplify small amplicons (150 base pairs or
less) to maximize the likelihood that the primers function at 100%
efficiency. All primer/probe sequences were searched against Public
Genbank databases to ensure target specificity. Primers and probes
were obtained from ABI.
[0701] For BGS-19, the primer probe sequences were as follows
TABLE-US-00008 Forward Primer 5'-CAGGGATGGTTCCAAAGTGAA-3' (SEQ ID
NO: 86) Reverse Primer 5'-GTGCGACTCCCACACACTTG-3' (SEQ ID NO: 87)
TaqMan Probe 5'-AGGTCTCCATGGCAACAGGACACCA-3' (SEQ ID NO: 88)
DNA Contamination
[0702] To access the level of contaminating genomic DNA in the RNA,
the RNA was divided into 2 aliquots and one half was treated with
Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated
and non-treated were then subjected to reverse transcription
reactions with (RT+) and without (RT-) the presence of reverse
transcriptase. TaqMan assays were carried out with gene-specific
primers (see above) and the contribution of genomic DNA to the
signal detected was evaluated by comparing the threshold cycles
obtained with the RT+/RT- non-Dnase treated RNA to that on the
RT+/RT- Dnase treated RNA. The amount of signal contributed by
genomic DNA in the Dnased RT- RNA must be less that 10% of that
obtained with Dnased RT+RNA. If not the RNA was not used in actual
experiments.
Reverse Transcription Reaction and Sequence Detection
[0703] 100 ng of Dnase-treated total RNA was annealed to 2.5 .mu.M
of the respective gene-specific reverse primer in the presence of
5.5 mM Magnesium Chloride by heating the sample to 72.degree. C.
for 2 min and then cooling to 55.degree. C. for 30 min. 1.25
U/.mu.l of MuLv reverse transcriptase and 500 .mu.M of each dNTP
was added to the reaction and the tube was incubated at 37.degree.
C. for 30 min. The sample was then heated to 90.degree. C. for 5
min to denature enzyme.
[0704] Quantitative sequence detection was carried out on an ABI
PRISM 7700 by adding to the reverse transcribed reaction 2.5 .mu.M
forward and reverse primers, 2.0 .mu.M of the TaqMan probe, 500
.mu.M of each dNTP, buffer and 5 U AmpliTaq Gold.TM.. The PCR
reaction was then held at 94.degree. C. for 12 min, followed by 40
cycles of 94.degree. C. for 15 sec and 60.degree. C. for 30
sec.
Data Handling
[0705] The threshold cycle (Ct) of the lowest expressing tissue
(the highest Ct value) was used as the baseline of expression and
all other tissues were expressed as the relative abundance to that
tissue by calculating the difference in Ct value between the
baseline and the other tissues and using it as the exponent in
2.sup.(.DELTA.Ct)
[0706] The expanded expression profile of the BGS-19 polypeptide is
provided in FIGS. 7 and 8, and described elsewhere herein.
Example 8
Method of Creating N- and C-Terminal Deletion Mutants Corresponding
to the BGS-19 Polypeptide of the Present Invention
[0707] As described elsewhere herein, the present invention
encompasses the creation of N- and C-terminal deletion mutants, in
addition to any combination of N- and C-terminal deletions thereof,
corresponding to the BGS-19 polypeptide of the present invention. A
number of methods are available to one skilled in the art for
creating such mutants. Such methods may include a combination of
PCR amplification and gene cloning methodology. Although one of
skill in the art of molecular biology, through the use of the
teachings provided or referenced herein, and/or otherwise known in
the art as standard methods, could readily create each deletion
mutant of the present invention, exemplary methods are described
below.
[0708] Briefly, using the isolated cDNA clone encoding the
full-length BGS-19 polypeptide sequence (as described in herein),
appropriate primers of about 15-25 nucleotides derived from the
desired 5' and 3' positions of SEQ ID NO:1 may be designed to PCR
amplify, and subsequently clone, the intended N- and/or C-terminal
deletion mutant. Such primers could comprise, for example, an
initiation and stop codon for the 5' and 3' primer, respectively.
Such primers may also comprise restriction sites to facilitate
cloning of the deletion mutant post amplification. Moreover, the
primers may comprise additional sequences, such as, for example,
flag-tag sequences, kozac sequences, or other sequences discussed
and/or referenced herein.
[0709] For example, in the case of the L16 to K385 N-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant: TABLE-US-00009
5' Primer (SEQ ID NO: 82) 5'-GCAGGA GCGGCCGC
CTGAACAAGGATCCCAGTTACAGTC-3' NotI 3' Primer (SEQ ID NO: 83)
5'-GCAGCA GTCGAC CTTTGGAAGCATCCCTGACATCTCC-3' SalI
[0710] For example, in the case of the M1 to E228 C-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant: TABLE-US-00010
5' Primer (SEQ ID NO: 84) 5'-GCAGCA GCGGCCGC
ATGCTGCTGCTGCCCCTGCTGGTGC-3' NotI 3' Primer (SEQ ID NO: 85)
5'-GCAGCA GTCGAC CTGGGAGCCCAGAGGGTGCTGAGCG-3' SalI
[0711] Representative PCR amplification conditions are provided
below, although the skilled artisan would appreciate that other
conditions may be required for efficient amplification. A 100 ul
PCR reaction mixture may be prepared using long of the template DNA
(cDNA clone of BGS-19), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA
polymerase (PE), and standard Taq DNA polymerase buffer. Typical
PCR cycling condition are as follows:
[0712] 20-25 cycles: 45 sec, 93 degrees [0713] 2 min, 50 degrees
[0714] 2 min, 72 degrees
[0715] 1 cycle: 10 min, 72 degrees
[0716] After the final extension step of PCR, 5 U Klenow Fragment
may be added and incubated for 15 min at 30 degrees.
[0717] Upon digestion of the fragment with the NotI and SalI
restriction enzymes, the fragment could be cloned into an
appropriate expression and/or cloning vector which has been
similarly digested (e.g., pSport1, among others). The skilled
artisan would appreciate that other plasmids could be equally
substituted, and may be desirable in certain circumstances. The
digested fragment and vector are then ligated using a DNA ligase,
and then used to transform competent E. coli cells using methods
provided herein and/or otherwise known in the art.
[0718] The 5' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula: (S+(X*3)) to ((S+(X*3))+25), wherein `S` is
equal to the nucleotide position of the initiating start codon of
the BGS-19 gene (SEQ ID NO:1), and `X` is equal to the most
N-terminal amino acid of the intended N-terminal deletion mutant.
The first term will provide the start 5' nucleotide position of the
5' primer, while the second term will provide the end 3' nucleotide
position of the 5' primer corresponding to sense strand of SEQ ID
NO:1. Once the corresponding nucleotide positions of the primer are
determined, the final nucleotide sequence may be created by the
addition of applicable restriction site sequences to the 5' end of
the sequence, for example. As referenced herein, the addition of
other sequences to the 5' primer may be desired in certain
circumstances (e.g., kozac sequences, etc.).
[0719] The 3' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula: (S+(X*3)) to ((S+(X*3))-25), wherein `S` is
equal to the nucleotide position of the initiating start codon of
the BGS-19 gene (SEQ ID NO:1), and `X` is equal to the most
C-terminal amino acid of the intended N-terminal deletion mutant.
The first term will provide the start 5' nucleotide position of the
3' primer, while the second term will provide the end 3' nucleotide
position of the 3' primer corresponding to the anti-sense strand of
SEQ ID NO:1. Once the corresponding nucleotide positions of the
primer are determined, the final nucleotide sequence may be created
by the addition of applicable restriction site sequences to the 5'
end of the sequence, for example. As referenced herein, the
addition of other sequences to the 3' primer may be desired in
certain circumstances (e.g., stop codon sequences, etc.). The
skilled artisan would appreciate that modifications of the above
nucleotide positions may be necessary for optimizing PCR
amplification.
[0720] The same general formulas provided above may be used in
identifying the 5' and 3' primer sequences for amplifying any
C-terminal deletion mutant of the present invention. Moreover, the
same general formulas provided above may be used in identifying the
5' and 3' primer sequences for amplifying any combination of
N-terminal and C-terminal deletion mutant of the present invention.
The skilled artisan would appreciate that modifications of the
above nucleotide positions may be necessary for optimizing PCR
amplification.
Example 9
Method of Enhancing the Biological Activity Functional
Characteristics of Invention Through Molecular Evolution
[0721] Although many of the most biologically active proteins known
are highly effective for their specified function in an organism,
they often possess characteristics that make them undesirable for
transgenic, therapeutic, pharmaceutical, and/or industrial
applications. Among these traits, a short physiological half-life
is the most prominent problem, and is present either at the level
of the protein, or the level of the proteins mRNA. The ability to
extend the half-life, for example, would be particularly important
for a proteins use in gene therapy, transgenic animal production,
the bioprocess production and purification of the protein, and use
of the protein as a chemical modulator among others. Therefore,
there is a need to identify novel variants of isolated proteins
possessing characteristics which enhance their application as a
therapeutic for treating diseases of animal origin, in addition to
the proteins applicability to common industrial and pharmaceutical
applications.
[0722] Thus, one aspect of the present invention relates to the
ability to enhance specific characteristics of invention through
directed molecular evolution. Such an enhancement may, in a
non-limiting example, benefit the inventions utility as an
essential component in a kit, the inventions physical attributes
such as its solubility, structure, or codon optimization, the
inventions specific biological activity, including any associated
enzymatic activity, the proteins enzyme kinetics, the proteins Ki,
Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding
activity, antagonist/inhibitory activity (including direct or
indirect interaction), agonist activity (including direct or
indirect interaction), the proteins antigenicity (e.g., where it
would be desirable to either increase or decrease the antigenic
potential of the protein), the immunogenicity of the protein, the
ability of the protein to form dimers, trimers, or multimers with
either itself or other proteins, the antigenic efficacy of the
invention, including its subsequent use a preventative treatment
for disease or disease states, or as an effector for targeting
diseased genes. Moreover, the ability to enhance specific
characteristics of a protein may also be applicable to changing the
characterized activity of an enzyme to an activity completely
unrelated to its initially characterized activity. Other desirable
enhancements of the invention would be specific to each individual
protein, and would thus be well known in the art and contemplated
by the present invention.
[0723] For example, an engineered immunoglobulin domain containing
protein may be constitutively active upon binding of its cognate
ligand. Alternatively, an engineered immunoglobulin domain
containing protein may be constitutively active in the absence of
ligand binding. In yet another example, an engineered
immunoglobulin domain containing protein may be capable of being
activated with less than all of the regulatory factors and/or
conditions typically required for immunoglobulin domain containing
protein activation (e.g., ligand binding, phosphorylation,
conformational changes, etc.). Such immunoglobulin domain
containing proteins would be useful in screens to identify
immunoglobulin domain containing protein modulators, among other
uses described herein.
[0724] Directed evolution is comprised of several steps. The first
step is to establish a library of variants for the gene or protein
of interest. The most important step is to then select for those
variants that entail the activity you wish to identify. The design
of the screen is essential since your screen should be selective
enough to eliminate non-useful variants, but not so stringent as to
eliminate all variants. The last step is then to repeat the above
steps using the best variant from the previous screen. Each
successive cycle, can then be tailored as necessary, such as
increasing the stringency of the screen, for example.
[0725] Over the years, there have been a number of methods
developed to introduce mutations into macromolecules. Some of these
methods include, random mutagenesis, "error-prone" PCR, chemical
mutagenesis, site-directed mutagenesis, and other methods well
known in the art (for a comprehensive listing of current
mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)).
Typically, such methods have been used, for example, as tools for
identifying the core functional region(s) of a protein or the
function of specific domains of a protein (if a multi-domain
protein). However, such methods have more recently been applied to
the identification of macromolecule variants with specific or
enhanced characteristics.
[0726] Random mutagenesis has been the most widely recognized
method to date. Typically, this has been carried out either through
the use of "error-prone" PCR (as described in Moore, J., et al,
Nature Biotechnology 14:458, (1996), or through the application of
randomized synthetic oligonucleotides corresponding to specific
regions of interest (as described by Derbyshire, K. M. et al, Gene,
46:145-152, (1986), and Hill, D E, et al, Methods Enzymol.,
55:559-568, (1987). Both approaches have limits to the level of
mutagenesis that can be obtained. However, either approach enables
the investigator to effectively control the rate of mutagenesis.
This is particularly important considering the fact that mutations
beneficial to the activity of the enzyme are fairly rare. In fact,
using too high a level of mutagenesis may counter or inhibit the
desired benefit of a useful mutation.
[0727] While both of the aforementioned methods are effective for
creating randomized pools of macromolecule variants, a third
method, termed "DNA Shuffling", or "sexual PCR" (WPC, Stemmer,
PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling
has also been referred to as "directed molecular evolution",
"exon-shuffling", "directed enzyme evolution", "in vitro
evolution", and "artificial evolution". Such reference terms are
known in the art and are encompassed by the invention. This new,
preferred, method apparently overcomes the limitations of the
previous methods in that it not only propagates positive traits,
but simultaneously eliminates negative traits in the resulting
progeny.
[0728] DNA shuffling accomplishes this task by combining the
principal of in vitro recombination, along with the method of
"error-prone" PCR. In effect, you begin with a randomly digested
pool of small fragments of your gene, created by Dnase I digestion,
and then introduce said random fragments into an "error-prone" PCR
assembly reaction. During the PCR reaction, the randomly sized DNA
fragments not only hybridize to their cognate strand, but also may
hybridize to other DNA fragments corresponding to different regions
of the polynucleotide of interest--regions not typically accessible
via hybridization of the entire polynucleotide. Moreover, since the
PCR assembly reaction utilizes "error-prone" PCR reaction
conditions, random mutations are introduced during the DNA
synthesis step of the PCR reaction for all of the
fragments--further diversifying the potential hybridation sites
during the annealing step of the reaction.
[0729] A variety of reaction conditions could be utilized to
carry-out the DNA shuffling reaction. However, specific reaction
conditions for DNA shuffling are provided, for example, in PNAS,
91:10747, (1994). Briefly:
[0730] Prepare the DNA substrate to be subjected to the DNA
shuffling reaction. Preparation may be in the form of simply
purifying the DNA from contaminating cellular material, chemicals,
buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and
may entail the use of DNA purification kits as those provided by
Qiagen, Inc., or by the Promega, Corp., for example.
[0731] Once the DNA substrate has been purified, it would be
subjected to Dnase I digestion. About 2-4 ug of the DNA
substrate(s) would be digested with 0.0015 units of Dnase I (Sigma)
per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20
min. at room temperature. The resulting fragments of 10-50 bp could
then be purified by running them through a 2% low-melting point
agarose gel by electrophoresis onto DE81 ion-exchange paper
(Whatman) or could be purified using Microcon concentrators
(Amicon) of the appropriate molecular weight cuttoff, or could use
oligonucleotide purification columns (Qiagen), in addition to other
methods known in the art. If using DE81 ion-exchange paper, the
10-50 bp fragments could be eluted from said paper using 1 M NaCL,
followed by ethanol precipitation.
[0732] The resulting purified fragments would then be subjected to
a PCR assembly reaction by re-suspension in a PCR mixture
containing: 2 mM of each dNTP, 2.2 mM MgC12, 50 mM KCl, 10 mM
Tris.cndot.HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment
concentration of 10-30 ng/ul. No primers are added at this point.
Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul
of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s,
50-55 C for 30 s, and 72 C for 30 s using 30-45 cycles, followed by
72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150
thermocycler. After the assembly reaction is completed, a 1:40
dilution of the resulting primerless product would then be
introduced into a PCR mixture (using the same buffer mixture used
for the assembly reaction) containing 0.8 um of each primer and
subjecting this mixture to 15 cycles of PCR (using 94 C for 30 s,
50 C for 30 s, and 72 C for 30 s). The referred primers would be
primers corresponding to the nucleic acid sequences of the
polynucleotide(s) utilized in the shuffling reaction. Said primers
could consist of modified nucleic acid base pairs using methods
known in the art and referred to else where herein, or could
contain additional sequences (i.e., for adding restriction sites,
mutating specific base-pairs, etc.).
[0733] The resulting shuffled, assembled, and amplified product can
be purified using methods well known in the art (e.g., Qiagen PCR
purification kits) and then subsequently cloned using appropriate
restriction enzymes.
[0734] Although a number of variations of DNA shuffling have been
published to date, such variations would be obvious to the skilled
artisan and are encompassed by the invention. The DNA shuffling
method can also be tailered to the desired level of mutagenesis
using the methods described by Zhao, et al. (Nucl Acid Res., 25(6):
1307-1308, (1997).
[0735] As described above, once the randomized pool has been
created, it can then be subjected to a specific screen to identify
the variant possessing the desired characteristic(s). Once the
variant has been identified, DNA corresponding to the variant could
then be used as the DNA substrate for initiating another round of
DNA shuffling. This cycle of shuffling, selecting the optimized
variant of interest, and then re-shuffling, can be repeated until
the ultimate variant is obtained. Examples of model screens applied
to identify variants created using DNA shuffling technology may be
found in the following publications: J. C., Moore, et al., J. Mol.
Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol.,
18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech.,
15:436-438, (1997).
[0736] DNA shuffling has several advantages. First, it makes use of
beneficial mutations. When combined with screening, DNA shuffling
allows the discovery of the best mutational combinations and does
not assume that the best combination contains all the mutations in
a population. Secondly, recombination occurs simultaneously with
point mutagenesis. An effect of forcing DNA polymerase to
synthesize full-length genes from the small fragment DNA pool is a
background mutagenesis rate. In combination with a stringent
selection method, enzymatic activity has been evolved up to 16000
fold increase over the wild-type form of the enzyme. In essence,
the background mutagenesis yielded the genetic variability on which
recombination acted to enhance the activity.
[0737] A third feature of recombination is that it can be used to
remove deleterious mutations. As discussed above, during the
process of the randomization, for every one beneficial mutation,
there may be at least one or more neutral or inhibitory
mutations.
[0738] Such mutations can be removed by including in the assembly
reaction an excess of the wild-type random-size fragments, in
addition to the random-size fragments of the selected mutant from
the previous selection. During the next selection, some of the most
active variants of the polynucleotide/polypeptide/enzyme, should
have lost the inhibitory mutations.
[0739] Finally, recombination enables parallel processing. This
represents a significant advantage since there are likely multiple
characteristics that would make a protein more desirable (e.g.
solubility, activity, etc.). Since it is increasingly difficult to
screen for more than one desirable trait at a time, other methods
of molecular evolution tend to be inhibitory. However, using
recombination, it would be possible to combine the randomized
fragments of the best representative variants for the various
traits, and then select for multiple properties at once.
[0740] DNA shuffling can also be applied to the polynucleotides and
polypeptides of the present invention to decrease their
immunogenicity in a specified host, particularly if the
polynucleotides and polypeptides provide a therapeutic use. For
example, a particular variant of the present invention may be
created and isolated using DNA shuffling technology. Such a variant
may have all of the desired characteristics, though may be highly
immunogenic in a host due to its novel intrinsic structure.
Specifically, the desired characteristic may cause the polypeptide
to have a non-native structure which could no longer be recognized
as a "self" molecule, but rather as a "foreign", and thus activate
a host immune response directed against the novel variant. Such a
limitation can be overcome, for example, by including a copy of the
gene sequence for a xenobiotic ortholog of the native protein in
with the gene sequence of the novel variant gene in one or more
cycles of DNA shuffling. The molar ratio of the ortholog and novel
variant DNAs could be varied accordingly. Ideally, the resulting
hybrid variant identified would contain at least some of the coding
sequence which enabled the xenobiotic protein to evade the host
immune system, and additionally, the coding sequence of the
original novel varient that provided the desired
characteristics.
[0741] Likewise, the invention encompasses the application of DNA
shuffling technology to the evolution of polynucletotides and
polypeptides of the invention, wherein one or more cycles of DNA
shuffling include, in addition to the gene template DNA,
oligonucleotides coding for known allelic sequences, optimized
codon sequences, known variant sequences, known polynucleotide
polymorphism sequences, known ortholog sequences, known homolog
sequences, additional homologous sequences, additional
non-homologous sequences, sequences from another species, and any
number and combination of the above.
[0742] In addition to the described methods above, there are a
number of related methods that may also be applicable, or desirable
in certain cases. Representative among these are the methods
discussed in PCT applications WO 98/31700, and WO 98/32845, which
are hereby incorporated by reference. Furthermore, related methods
can also be applied to the polynucleotide sequences of the present
invention in order to evolve invention for creating ideal variants
for use in gene therapy, protein engineering, evolution of whole
cells containing the variant, or in the evolution of entire enzyme
pathways containing polynucleotides of the invention as described
in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO
98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438,
(1997), respectively.
[0743] Additional methods of applying "DNA Shuffling" technology to
the polynucleotides and polypeptides of the present invention,
including their proposed applications, may be found in U.S. Pat.
No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No.
WO 97/20078; PCT Application No. WO 97/35966; and PCT Application
No. WO 98/42832; PCT Application No. The forgoing are hereby
incorporated in their entirety herein for all purposes.
[0744] One skilled in the art could easily modify the exemplified
studies to test the activity of polynucleotides of the invention
(e.g., gene therapy), agonists, and/or antagonists of
polynucleotides or polypeptides of the invention.
[0745] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
INCORPORATION BY REFERENCE
[0746] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, Detailed Description, and Examples is hereby
incorporated herein by reference. Further, the hard copy of the
sequence listing submitted herewith and the corresponding computer
readable form are both incorporated herein by reference in their
entireties.
EQUIVALENTS
[0747] Those skilled in the art will recognize, or through routine
experimentation, will be able to ascertain many equivalents to the
particular embodiments of the invention described herein. The
claimed invention intends to encompass all such equivalents.
[0748] Having herein above disclosed exemplary embodiments of the
present invention, those skilled in the art will recognize that
this disclosure is only exemplary such that various alternatives,
adaptations, and modifications are within the scope of the
invention, and are contemplated by the Applicants. Accordingly, the
present invention is not limited to the specific embodiments as
illustrated above, but is defined by the following claims.
Sequence CWU 1
1
88 1 1985 DNA Homo sapiens CDS (140)..(1294) 1 cggacgcgtg
ggcgaggctc ctcctctgtg gatggtcact gcccctccac caggcttcct 60
gctggaggag tttccttccc agccaggccg gcccagaagc cagatggtcc cgggacaggc
120 ccagccccag agcccagag atg ctg ctg ctg ccc ctg ctg ctg ccc gtg
ctg 172 Met Leu Leu Leu Pro Leu Leu Leu Pro Val Leu 1 5 10 ggg gcg
ggg tcc ctg aac aag gat ccc agt tac agt ctt caa gtg cag 220 Gly Ala
Gly Ser Leu Asn Lys Asp Pro Ser Tyr Ser Leu Gln Val Gln 15 20 25
agg cag gtg ccg gtg ccg gag ggc ctg tgt gtc atc gtg tct tgc aac 268
Arg Gln Val Pro Val Pro Glu Gly Leu Cys Val Ile Val Ser Cys Asn 30
35 40 ctc tcc tac ccc cgg gat ggc tgg gac gag tct act gct gct tat
ggc 316 Leu Ser Tyr Pro Arg Asp Gly Trp Asp Glu Ser Thr Ala Ala Tyr
Gly 45 50 55 tac tgg ttc aaa gga cgg acc agc cca aag acg ggt gct
cct gtg gcc 364 Tyr Trp Phe Lys Gly Arg Thr Ser Pro Lys Thr Gly Ala
Pro Val Ala 60 65 70 75 act aac aac cag agt cga gag gtg gaa atg agc
acc cgg gac cga ttc 412 Thr Asn Asn Gln Ser Arg Glu Val Glu Met Ser
Thr Arg Asp Arg Phe 80 85 90 cag ctc act ggg gat ccc ggc aaa ggg
agc tgc tcc ttg gtg atc aga 460 Gln Leu Thr Gly Asp Pro Gly Lys Gly
Ser Cys Ser Leu Val Ile Arg 95 100 105 gac gcg cag agg gag gat gag
gca tgg tac ttc ttt cgg gtg gag aga 508 Asp Ala Gln Arg Glu Asp Glu
Ala Trp Tyr Phe Phe Arg Val Glu Arg 110 115 120 gga agc cgt gtg aga
cat agt ttc ctg agc aat gcg ttc ttt cta aaa 556 Gly Ser Arg Val Arg
His Ser Phe Leu Ser Asn Ala Phe Phe Leu Lys 125 130 135 gta aca gcc
ctg act aag aag cct gat gtc tac atc ccc gag acc ctg 604 Val Thr Ala
Leu Thr Lys Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu 140 145 150 155
gag ccc ggg cag ccg gtg acg gtc atc tgt gtg ttt aac tgg gct ttc 652
Glu Pro Gly Gln Pro Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe 160
165 170 aag aaa tgt cca gcc cct tct ttc tcc tgg acg ggg gct gcc ctc
tcc 700 Lys Lys Cys Pro Ala Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu
Ser 175 180 185 cct aga aga acc aga cca agc acc tcc cag ccc tca gac
ccc ggg gtc 748 Pro Arg Arg Thr Arg Pro Ser Thr Ser Gln Pro Ser Asp
Pro Gly Val 190 195 200 ctg gag ctg cca ccc att caa atg gag cac gaa
gga gag ttc acc tgc 796 Leu Glu Leu Pro Pro Ile Gln Met Glu His Glu
Gly Glu Phe Thr Cys 205 210 215 cac gct cag cac cct ctg ggc tcc cag
cac gtc tct ctc agc ctc tcc 844 His Ala Gln His Pro Leu Gly Ser Gln
His Val Ser Leu Ser Leu Ser 220 225 230 235 gtg cac tgg aag ctg gag
cat ggg gga gga ctt ggc ctg ggg gct gcc 892 Val His Trp Lys Leu Glu
His Gly Gly Gly Leu Gly Leu Gly Ala Ala 240 245 250 ctg gga gct ggc
gtc gct gcc ctg ctc gct ttc tgt tcc tgc ctt gtc 940 Leu Gly Ala Gly
Val Ala Ala Leu Leu Ala Phe Cys Ser Cys Leu Val 255 260 265 gtc ttc
agg gtg aag atc tgc agg aag gaa gct cgc aag agg gca gca 988 Val Phe
Arg Val Lys Ile Cys Arg Lys Glu Ala Arg Lys Arg Ala Ala 270 275 280
gct gag cag gac gtg ccc tcc acc ctg gga ccc atc tcc cag ggt cac
1036 Ala Glu Gln Asp Val Pro Ser Thr Leu Gly Pro Ile Ser Gln Gly
His 285 290 295 cag cat gaa tgc tcg gca ggc agc tcc caa gac cac ccg
ccc cca ggt 1084 Gln His Glu Cys Ser Ala Gly Ser Ser Gln Asp His
Pro Pro Pro Gly 300 305 310 315 gca gcc acc tac acc ccg ggg aag ggg
gaa gag cag gag ctc cac tat 1132 Ala Ala Thr Tyr Thr Pro Gly Lys
Gly Glu Glu Gln Glu Leu His Tyr 320 325 330 gcc tcc ctc agc ttc cag
ggc ctg agg ctc tgg gag cct gcg gac cag 1180 Ala Ser Leu Ser Phe
Gln Gly Leu Arg Leu Trp Glu Pro Ala Asp Gln 335 340 345 gag gcc ccc
agc acc acc gag tac tcg gag atc aag atc cac aca gga 1228 Glu Ala
Pro Ser Thr Thr Glu Tyr Ser Glu Ile Lys Ile His Thr Gly 350 355 360
cag ccc ctg agg ggc cca ggc ttt ggg ctt caa ttg gag agg gag atg
1276 Gln Pro Leu Arg Gly Pro Gly Phe Gly Leu Gln Leu Glu Arg Glu
Met 365 370 375 tca ggg atg gtt cca aag tgaagaggtc tccatggcaa
caggacacca 1324 Ser Gly Met Val Pro Lys 380 385 gcaagtgtgt
gggagtcgca ctggtgtgac ggccagaact ggactcagat ttcagcccca 1384
tccccaatga agagcttgag tttgaagatt atactttttt tgagacaggg tctgactctg
1444 tcctccaggc cggagtccag tggtgcaatc tcggctcact gtagcctcaa
cctgccgggt 1504 tgaagtgagc ctcccatttc agcctcccaa gtagctggga
ctacaattgt gagccaccat 1564 gccaggctca ttgttgtatt tttggtagag
acggggtttt gccatgtttc cctggctggt 1624 ctcagactcc tgggctcaag
caatctgccc gcctctgcct cccagggtgc tgggattgca 1684 gacgtgagcc
accacagctg gctgaagatt atactttcaa ttcagagcga gtttgaagat 1744
gacactttga ggcatcgtgt ctatggttca ttactacaga agcttctctg gatgtgtaaa
1804 gcacaggaaa ccaggcagag gaggcacagg gtgctctcca gaacgagaag
ccagctcctg 1864 gagttgtttg ctgcaactgc cattccccgt tgatgaccat
gctcttcctt cagaagaggg 1924 agagtgagag gaccaagtcc aagtggttcc
catttgaaca tttaaaaaaa aaaaaaaaaa 1984 g 1985 2 385 PRT Homo sapiens
2 Met Leu Leu Leu Pro Leu Leu Leu Pro Val Leu Gly Ala Gly Ser Leu 1
5 10 15 Asn Lys Asp Pro Ser Tyr Ser Leu Gln Val Gln Arg Gln Val Pro
Val 20 25 30 Pro Glu Gly Leu Cys Val Ile Val Ser Cys Asn Leu Ser
Tyr Pro Arg 35 40 45 Asp Gly Trp Asp Glu Ser Thr Ala Ala Tyr Gly
Tyr Trp Phe Lys Gly 50 55 60 Arg Thr Ser Pro Lys Thr Gly Ala Pro
Val Ala Thr Asn Asn Gln Ser 65 70 75 80 Arg Glu Val Glu Met Ser Thr
Arg Asp Arg Phe Gln Leu Thr Gly Asp 85 90 95 Pro Gly Lys Gly Ser
Cys Ser Leu Val Ile Arg Asp Ala Gln Arg Glu 100 105 110 Asp Glu Ala
Trp Tyr Phe Phe Arg Val Glu Arg Gly Ser Arg Val Arg 115 120 125 His
Ser Phe Leu Ser Asn Ala Phe Phe Leu Lys Val Thr Ala Leu Thr 130 135
140 Lys Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro
145 150 155 160 Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe Lys Lys
Cys Pro Ala 165 170 175 Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser
Pro Arg Arg Thr Arg 180 185 190 Pro Ser Thr Ser Gln Pro Ser Asp Pro
Gly Val Leu Glu Leu Pro Pro 195 200 205 Ile Gln Met Glu His Glu Gly
Glu Phe Thr Cys His Ala Gln His Pro 210 215 220 Leu Gly Ser Gln His
Val Ser Leu Ser Leu Ser Val His Trp Lys Leu 225 230 235 240 Glu His
Gly Gly Gly Leu Gly Leu Gly Ala Ala Leu Gly Ala Gly Val 245 250 255
Ala Ala Leu Leu Ala Phe Cys Ser Cys Leu Val Val Phe Arg Val Lys 260
265 270 Ile Cys Arg Lys Glu Ala Arg Lys Arg Ala Ala Ala Glu Gln Asp
Val 275 280 285 Pro Ser Thr Leu Gly Pro Ile Ser Gln Gly His Gln His
Glu Cys Ser 290 295 300 Ala Gly Ser Ser Gln Asp His Pro Pro Pro Gly
Ala Ala Thr Tyr Thr 305 310 315 320 Pro Gly Lys Gly Glu Glu Gln Glu
Leu His Tyr Ala Ser Leu Ser Phe 325 330 335 Gln Gly Leu Arg Leu Trp
Glu Pro Ala Asp Gln Glu Ala Pro Ser Thr 340 345 350 Thr Glu Tyr Ser
Glu Ile Lys Ile His Thr Gly Gln Pro Leu Arg Gly 355 360 365 Pro Gly
Phe Gly Leu Gln Leu Glu Arg Glu Met Ser Gly Met Val Pro 370 375 380
Lys 385 3 1632 DNA Homo sapiens CDS (1)..(1632) 3 ggg tcc ctg aac
aag gat ccc agt tac agt ctt caa gtg cag agg cag 48 Gly Ser Leu Asn
Lys Asp Pro Ser Tyr Ser Leu Gln Val Gln Arg Gln 1 5 10 15 gtg ccg
gtg ccg gag ggc ctg tgt gtc atc gtg tct tgc aac ctc tcc 96 Val Pro
Val Pro Glu Gly Leu Cys Val Ile Val Ser Cys Asn Leu Ser 20 25 30
tac ccc cgg gat ggc tgg gac gag tct act gct gct tat ggc tac tgg 144
Tyr Pro Arg Asp Gly Trp Asp Glu Ser Thr Ala Ala Tyr Gly Tyr Trp 35
40 45 ttc aaa gga cgg acc agc cca aag acg ggt gct cct gtg gcc act
aac 192 Phe Lys Gly Arg Thr Ser Pro Lys Thr Gly Ala Pro Val Ala Thr
Asn 50 55 60 aac cag agt cga gag gtg gaa atg agc acc cgg gac cga
ttc cag ctc 240 Asn Gln Ser Arg Glu Val Glu Met Ser Thr Arg Asp Arg
Phe Gln Leu 65 70 75 80 act ggg gat ccc ggc aaa ggg agc tgc tcc ttg
gtg atc aga gac gcg 288 Thr Gly Asp Pro Gly Lys Gly Ser Cys Ser Leu
Val Ile Arg Asp Ala 85 90 95 cag agg gag gat gag gca tgg tac ttc
ttt cgg gtg gag aga gga agc 336 Gln Arg Glu Asp Glu Ala Trp Tyr Phe
Phe Arg Val Glu Arg Gly Ser 100 105 110 cgt gtg aga cat agt ttc ctg
agc aat gcg ttc ttt cta aaa gta aca 384 Arg Val Arg His Ser Phe Leu
Ser Asn Ala Phe Phe Leu Lys Val Thr 115 120 125 gcc ctg act aag aag
cct gat gtc tac atc ccc gag acc ctg gag ccc 432 Ala Leu Thr Lys Lys
Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro 130 135 140 ggg cag ccg
gtg acg gtc atc tgt gtg ttt aac tgg gct ttc aag aaa 480 Gly Gln Pro
Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe Lys Lys 145 150 155 160
tgt cca gcc cct tct ttc tcc tgg acg ggg gct gcc ctc tcc cct aga 528
Cys Pro Ala Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Pro Arg 165
170 175 aga acc aga cca agc acc tcc cac ttc tca gtg ctc agc ttc acg
ccc 576 Arg Thr Arg Pro Ser Thr Ser His Phe Ser Val Leu Ser Phe Thr
Pro 180 185 190 agc ccc cag gac cac gac acc gac ctc acc tgc cat gtg
gac ttc tcc 624 Ser Pro Gln Asp His Asp Thr Asp Leu Thr Cys His Val
Asp Phe Ser 195 200 205 aga aag ggt gtg agc gca cag agg acc gtc cga
ctc cgt gtg gcc tcc 672 Arg Lys Gly Val Ser Ala Gln Arg Thr Val Arg
Leu Arg Val Ala Ser 210 215 220 ctg agc tgc acg tcg att ctg cct ctt
cct tcc cta gtc ctg gaa aac 720 Leu Ser Cys Thr Ser Ile Leu Pro Leu
Pro Ser Leu Val Leu Glu Asn 225 230 235 240 ctc ggg aac ggc aca tcc
ctc ccg gtc ctg gag ggc caa agc ctg cgc 768 Leu Gly Asn Gly Thr Ser
Leu Pro Val Leu Glu Gly Gln Ser Leu Arg 245 250 255 ctg gtc tgt gtc
acc cac agc agc ccc cca gcc agg ctg agc tgg acc 816 Leu Val Cys Val
Thr His Ser Ser Pro Pro Ala Arg Leu Ser Trp Thr 260 265 270 cgg tgg
gga cag acc gtg ggc ccc tcc cag ccc tca gac ccc ggg gtc 864 Arg Trp
Gly Gln Thr Val Gly Pro Ser Gln Pro Ser Asp Pro Gly Val 275 280 285
ctg gag ctg cca ccc att caa atg gag cac gaa gga gag ttc acc tgc 912
Leu Glu Leu Pro Pro Ile Gln Met Glu His Glu Gly Glu Phe Thr Cys 290
295 300 cac gct cag cac cct ctg ggc tcc cag cac gtc tct ctc agc ctc
tcc 960 His Ala Gln His Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu
Ser 305 310 315 320 gtg cac tac cct cca cag ctg ctg ggc ccc tcc tgc
tcc tgg gag gct 1008 Val His Tyr Pro Pro Gln Leu Leu Gly Pro Ser
Cys Ser Trp Glu Ala 325 330 335 gag ggt ctg cac tgc agc tgc tcc tcc
cag gcc agc ccg gcc ccc tct 1056 Glu Gly Leu His Cys Ser Cys Ser
Ser Gln Ala Ser Pro Ala Pro Ser 340 345 350 ctg cgc tgg tgg ctt ggg
gag gag ctg ctg gag ggg aac agc agt cag 1104 Leu Arg Trp Trp Leu
Gly Glu Glu Leu Leu Glu Gly Asn Ser Ser Gln 355 360 365 ggc tcc ttc
gag gtc acc ccc agc tca gcc ggg ccc tgg gcc aac agc 1152 Gly Ser
Phe Glu Val Thr Pro Ser Ser Ala Gly Pro Trp Ala Asn Ser 370 375 380
tcc ctg agc ctc cat gga ggg ctc agc tcc ggc ctc agg ctc cgc tgt
1200 Ser Leu Ser Leu His Gly Gly Leu Ser Ser Gly Leu Arg Leu Arg
Cys 385 390 395 400 aag gcc tgg aac gtc cac ggg gcc cag agt ggc tct
gtc ttc cag ctg 1248 Lys Ala Trp Asn Val His Gly Ala Gln Ser Gly
Ser Val Phe Gln Leu 405 410 415 cta cca ggg aag ctg gag cat ggg gga
gga ctt ggc ctg ggg gct gcc 1296 Leu Pro Gly Lys Leu Glu His Gly
Gly Gly Leu Gly Leu Gly Ala Ala 420 425 430 ctg gga gct ggc gtc gct
gcc ctg ctc gct ttc tgt tcc tgc ctt gtc 1344 Leu Gly Ala Gly Val
Ala Ala Leu Leu Ala Phe Cys Ser Cys Leu Val 435 440 445 gtc ttc agg
aaa tac tca att tcc aga tcc tct tgt gca tcc tcc ttg 1392 Val Phe
Arg Lys Tyr Ser Ile Ser Arg Ser Ser Cys Ala Ser Ser Leu 450 455 460
ctc tcg ctt agc ccc cat gac cct aat ttg acc ccc ttt ctc ccc tgc
1440 Leu Ser Leu Ser Pro His Asp Pro Asn Leu Thr Pro Phe Leu Pro
Cys 465 470 475 480 att cag ggt cac cag cat gaa tgc tcg gca ggc agc
tcc caa gac cac 1488 Ile Gln Gly His Gln His Glu Cys Ser Ala Gly
Ser Ser Gln Asp His 485 490 495 ccg ccc cca ggt gca gcc acc tac acc
ccg ggg aag ggg gaa gag cag 1536 Pro Pro Pro Gly Ala Ala Thr Tyr
Thr Pro Gly Lys Gly Glu Glu Gln 500 505 510 gag ctc cac tat gcc tcc
ctc agc ttc cag ggc ctg agg ctc tgg gag 1584 Glu Leu His Tyr Ala
Ser Leu Ser Phe Gln Gly Leu Arg Leu Trp Glu 515 520 525 cct gcg gac
cag gag gcc ccc agc acc acc gag tac tcg gag atc aag 1632 Pro Ala
Asp Gln Glu Ala Pro Ser Thr Thr Glu Tyr Ser Glu Ile Lys 530 535 540
4 544 PRT Homo sapiens 4 Gly Ser Leu Asn Lys Asp Pro Ser Tyr Ser
Leu Gln Val Gln Arg Gln 1 5 10 15 Val Pro Val Pro Glu Gly Leu Cys
Val Ile Val Ser Cys Asn Leu Ser 20 25 30 Tyr Pro Arg Asp Gly Trp
Asp Glu Ser Thr Ala Ala Tyr Gly Tyr Trp 35 40 45 Phe Lys Gly Arg
Thr Ser Pro Lys Thr Gly Ala Pro Val Ala Thr Asn 50 55 60 Asn Gln
Ser Arg Glu Val Glu Met Ser Thr Arg Asp Arg Phe Gln Leu 65 70 75 80
Thr Gly Asp Pro Gly Lys Gly Ser Cys Ser Leu Val Ile Arg Asp Ala 85
90 95 Gln Arg Glu Asp Glu Ala Trp Tyr Phe Phe Arg Val Glu Arg Gly
Ser 100 105 110 Arg Val Arg His Ser Phe Leu Ser Asn Ala Phe Phe Leu
Lys Val Thr 115 120 125 Ala Leu Thr Lys Lys Pro Asp Val Tyr Ile Pro
Glu Thr Leu Glu Pro 130 135 140 Gly Gln Pro Val Thr Val Ile Cys Val
Phe Asn Trp Ala Phe Lys Lys 145 150 155 160 Cys Pro Ala Pro Ser Phe
Ser Trp Thr Gly Ala Ala Leu Ser Pro Arg 165 170 175 Arg Thr Arg Pro
Ser Thr Ser His Phe Ser Val Leu Ser Phe Thr Pro 180 185 190 Ser Pro
Gln Asp His Asp Thr Asp Leu Thr Cys His Val Asp Phe Ser 195 200 205
Arg Lys Gly Val Ser Ala Gln Arg Thr Val Arg Leu Arg Val Ala Ser 210
215 220 Leu Ser Cys Thr Ser Ile Leu Pro Leu Pro Ser Leu Val Leu Glu
Asn 225 230 235 240 Leu Gly Asn Gly Thr Ser Leu Pro Val Leu Glu Gly
Gln Ser Leu Arg 245 250 255 Leu Val Cys Val Thr His Ser Ser Pro Pro
Ala Arg Leu Ser Trp Thr 260 265 270 Arg Trp Gly Gln Thr Val Gly Pro
Ser Gln Pro Ser Asp Pro Gly Val 275 280 285 Leu Glu Leu Pro Pro Ile
Gln Met Glu His Glu Gly Glu Phe Thr Cys 290 295 300 His Ala Gln His
Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser 305 310 315 320 Val
His Tyr Pro Pro Gln Leu Leu Gly Pro Ser Cys Ser Trp Glu Ala 325 330
335 Glu Gly Leu His Cys Ser Cys Ser Ser Gln Ala Ser Pro Ala Pro Ser
340 345 350 Leu Arg Trp Trp Leu Gly Glu Glu Leu Leu Glu Gly Asn Ser
Ser Gln 355 360 365 Gly Ser Phe Glu Val Thr Pro Ser Ser Ala Gly Pro
Trp Ala Asn Ser 370 375 380 Ser Leu Ser Leu His Gly Gly Leu Ser Ser
Gly Leu Arg Leu Arg Cys 385 390 395 400 Lys Ala Trp Asn Val His
Gly
Ala Gln Ser Gly Ser Val Phe Gln Leu 405 410 415 Leu Pro Gly Lys Leu
Glu His Gly Gly Gly Leu Gly Leu Gly Ala Ala 420 425 430 Leu Gly Ala
Gly Val Ala Ala Leu Leu Ala Phe Cys Ser Cys Leu Val 435 440 445 Val
Phe Arg Lys Tyr Ser Ile Ser Arg Ser Ser Cys Ala Ser Ser Leu 450 455
460 Leu Ser Leu Ser Pro His Asp Pro Asn Leu Thr Pro Phe Leu Pro Cys
465 470 475 480 Ile Gln Gly His Gln His Glu Cys Ser Ala Gly Ser Ser
Gln Asp His 485 490 495 Pro Pro Pro Gly Ala Ala Thr Tyr Thr Pro Gly
Lys Gly Glu Glu Gln 500 505 510 Glu Leu His Tyr Ala Ser Leu Ser Phe
Gln Gly Leu Arg Leu Trp Glu 515 520 525 Pro Ala Asp Gln Glu Ala Pro
Ser Thr Thr Glu Tyr Ser Glu Ile Lys 530 535 540 5 364 PRT Homo
sapiens 5 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala
Leu Ala 1 5 10 15 Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser
Val Thr Val Gln 20 25 30 Glu Gly Leu Cys Val Leu Val Pro Cys Thr
Phe Phe His Pro Ile Pro 35 40 45 Tyr Tyr Asp Lys Asn Ser Pro Val
His Gly Tyr Trp Phe Arg Glu Gly 50 55 60 Ala Ile Ile Ser Gly Asp
Ser Pro Val Ala Thr Asn Lys Leu Asp Gln 65 70 75 80 Glu Val Gln Glu
Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro 85 90 95 Ser Arg
Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp 100 105 110
Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser 115
120 125 Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His
Arg 130 135 140 Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His
Ser Lys Asn 145 150 155 160 Leu Thr Cys Ser Val Ser Trp Ala Cys Glu
Gln Gly Thr Pro Pro Ile 165 170 175 Phe Ser Trp Leu Ser Ala Ala Pro
Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190 His Ser Ser Val Leu Ile
Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200 205 Asn Leu Thr Cys
Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu 210 215 220 Arg Thr
Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr 225 230 235
240 Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly
245 250 255 Leu Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu
Leu Ala 260 265 270 Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr
His Arg Arg Lys 275 280 285 Ala Ala Arg Thr Ala Val Gly Ser Asn Asp
Thr His Pro Thr Thr Gly 290 295 300 Ser Ala Ser Pro Lys His Gln Lys
Asn Ser Lys Leu His Gly Pro Thr 305 310 315 320 Glu Thr Ser Ser Cys
Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu 325 330 335 Glu Leu His
Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys 340 345 350 Asp
Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln 355 360 6 374 PRT Homo
sapiens 6 Met Leu Leu Leu Leu Leu Leu Pro Leu Leu Trp Gly Arg Glu
Arg Val 1 5 10 15 Glu Gly Gln Lys Ser Asn Arg Lys Asp Tyr Ser Leu
Thr Met Gln Ser 20 25 30 Ser Val Thr Val Gln Glu Gly Met Cys Val
His Val Arg Cys Ser Phe 35 40 45 Ser Tyr Pro Val Asp Ser Gln Thr
Asp Ser Asp Pro Val His Gly Tyr 50 55 60 Trp Phe Arg Ala Gly Asn
Asp Ile Ser Trp Lys Ala Pro Val Ala Thr 65 70 75 80 Asn Asn Pro Ala
Trp Ala Val Gln Glu Glu Thr Arg Asp Arg Phe His 85 90 95 Leu Leu
Gly Asp Pro Gln Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp 100 105 110
Ala Arg Met Ser Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly 115
120 125 Asn Ile Lys Trp Asn Tyr Lys Tyr Asp Gln Leu Ser Val Asn Val
Thr 130 135 140 Asp Pro Pro Gln Asn Leu Thr Val Thr Val Phe Gln Gly
Glu Gly Thr 145 150 155 160 Ala Ser Thr Ala Leu Gly Asn Ser Ser Ser
Leu Ser Val Leu Glu Gly 165 170 175 Gln Ser Leu Arg Leu Val Cys Ala
Val Asp Ser Asn Pro Pro Ala Arg 180 185 190 Leu Ser Trp Thr Trp Arg
Ser Leu Thr Leu Tyr Pro Ser Gln Pro Ser 195 200 205 Asn Pro Leu Val
Leu Glu Leu Gln Val His Leu Gly Asp Glu Gly Glu 210 215 220 Phe Thr
Cys Arg Ala Gln Asn Ser Leu Gly Ser Gln His Val Ser Leu 225 230 235
240 Asn Leu Ser Leu Gln Gln Glu Tyr Thr Gly Lys Met Arg Pro Val Ser
245 250 255 Gly Val Leu Leu Gly Ala Val Gly Gly Ala Gly Ala Thr Ala
Leu Val 260 265 270 Phe Leu Ser Phe Cys Val Ile Phe Ile Val Val Arg
Ser Cys Arg Lys 275 280 285 Lys Ser Ala Arg Pro Ala Ala Asp Val Gly
Asp Ile Gly Met Lys Asp 290 295 300 Ala Asn Thr Ile Arg Gly Ser Ala
Ser Gln Gly Asn Leu Thr Glu Ser 305 310 315 320 Trp Ala Asp Asp Asn
Pro Arg His His Gly Leu Ala Ala His Ser Ser 325 330 335 Gly Glu Glu
Arg Glu Ile Gln Tyr Ala Pro Leu Ser Phe His Lys Gly 340 345 350 Glu
Pro Gln Asp Leu Ser Gly Gln Glu Ala Thr Asn Asn Glu Tyr Ser 355 360
365 Glu Ile Lys Ile Pro Lys 370 7 697 PRT Homo sapiens 7 Met Leu
Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala 1 5 10 15
Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro 20
25 30 Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg
Gln 35 40 45 Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe
Lys Ala Val 50 55 60 Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr
Asn His Gln Ser Arg 65 70 75 80 Glu Val Glu Met Ser Thr Arg Gly Arg
Phe Gln Leu Thr Gly Asp Pro 85 90 95 Ala Lys Gly Asn Cys Ser Leu
Val Ile Arg Asp Ala Gln Met Gln Asp 100 105 110 Glu Ser Gln Tyr Phe
Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr 115 120 125 Asn Phe Met
Asn Asp Gly Phe Phe Leu Lys Val Thr Ala Leu Thr Gln 130 135 140 Lys
Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val 145 150
155 160 Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys Pro Pro
Pro 165 170 175 Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly
Thr Lys Pro 180 185 190 Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr
Pro Arg Pro Gln Asp 195 200 205 His Asn Thr Asp Leu Thr Cys His Val
Asp Phe Ser Arg Lys Gly Val 210 215 220 Ser Val Gln Arg Thr Val Arg
Leu Arg Val Ala Tyr Ala Pro Arg Asp 225 230 235 240 Leu Val Ile Ser
Ile Ser Arg Asp Asn Thr Pro Ala Leu Glu Pro Gln 245 250 255 Pro Gln
Gly Asn Val Pro Tyr Leu Glu Ala Gln Lys Gly Gln Phe Leu 260 265 270
Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro Pro Ala Thr Leu Ser Trp 275
280 285 Val Leu Gln Asn Arg Val Leu Ser Ser Ser His Pro Trp Gly Pro
Arg 290 295 300 Pro Leu Gly Leu Glu Leu Pro Gly Val Lys Ala Gly Asp
Ser Gly Arg 305 310 315 320 Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly
Ser Gln Gln Arg Ala Leu 325 330 335 Asp Leu Ser Val Gln Tyr Pro Pro
Glu Asn Leu Arg Val Met Val Ser 340 345 350 Gln Ala Asn Arg Thr Val
Leu Glu Asn Leu Gly Asn Gly Thr Ser Leu 355 360 365 Pro Val Leu Glu
Gly Gln Ser Leu Cys Leu Val Cys Val Thr His Ser 370 375 380 Ser Pro
Pro Ala Arg Leu Ser Trp Thr Gln Arg Gly Gln Val Leu Ser 385 390 395
400 Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu Leu Pro Arg Val Gln
405 410 415 Val Glu His Glu Gly Glu Phe Thr Cys His Ala Arg His Pro
Leu Gly 420 425 430 Ser Gln His Val Ser Leu Ser Leu Ser Val His Tyr
Ser Pro Lys Leu 435 440 445 Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu
Gly Leu His Cys Ser Cys 450 455 460 Ser Ser Gln Ala Ser Pro Ala Pro
Ser Leu Arg Trp Trp Leu Gly Glu 465 470 475 480 Glu Leu Leu Glu Gly
Asn Ser Ser Gln Asp Ser Phe Glu Val Thr Pro 485 490 495 Ser Ser Ala
Gly Pro Trp Ala Asn Ser Ser Leu Ser Leu His Gly Gly 500 505 510 Leu
Ser Ser Gly Leu Arg Leu Arg Cys Glu Ala Trp Asn Val His Gly 515 520
525 Ala Gln Ser Gly Ser Ile Leu Gln Leu Pro Asp Lys Lys Gly Leu Ile
530 535 540 Ser Thr Ala Phe Ser Asn Gly Ala Phe Leu Gly Ile Gly Ile
Thr Ala 545 550 555 560 Leu Leu Phe Leu Cys Leu Ala Leu Ile Ile Met
Lys Ile Leu Pro Lys 565 570 575 Arg Arg Thr Gln Thr Glu Thr Pro Arg
Pro Arg Phe Ser Arg His Ser 580 585 590 Thr Ile Leu Asp Tyr Ile Asn
Val Val Pro Thr Ala Gly Pro Leu Ala 595 600 605 Gln Lys Arg Asn Gln
Lys Ala Thr Pro Asn Ser Pro Arg Thr Pro Leu 610 615 620 Pro Pro Gly
Ala Pro Ser Pro Glu Ser Lys Lys Asn Gln Lys Lys Gln 625 630 635 640
Tyr Gln Leu Pro Ser Phe Pro Glu Pro Lys Ser Ser Thr Gln Ala Pro 645
650 655 Glu Ser Gln Glu Ser Gln Glu Glu Leu His Tyr Ala Thr Leu Asn
Phe 660 665 670 Pro Gly Val Arg Pro Arg Pro Glu Ala Arg Met Pro Lys
Gly Thr Gln 675 680 685 Ala Asp Tyr Ala Glu Val Lys Phe Gln 690 695
8 26 PRT Homo sapiens 8 Ala Ala Leu Gly Ala Gly Val Ala Ala Leu Leu
Ala Phe Cys Ser Cys 1 5 10 15 Leu Val Val Phe Arg Val Lys Ile Cys
Arg 20 25 9 100 PRT Homo sapiens 9 Gly Ser Leu Asn Lys Asp Pro Ser
Tyr Ser Leu Gln Val Gln Arg Gln 1 5 10 15 Val Pro Val Pro Glu Gly
Leu Cys Val Ile Val Ser Cys Asn Leu Ser 20 25 30 Tyr Pro Arg Asp
Gly Trp Asp Glu Ser Thr Ala Ala Tyr Gly Tyr Trp 35 40 45 Phe Lys
Gly Arg Thr Ser Pro Lys Thr Gly Ala Pro Val Ala Thr Asn 50 55 60
Asn Gln Ser Arg Glu Val Glu Met Ser Thr Arg Asp Arg Phe Gln Leu 65
70 75 80 Thr Gly Asp Pro Gly Lys Gly Ser Cys Ser Leu Val Ile Arg
Asp Ala 85 90 95 Gln Arg Glu Asp 100 10 102 PRT Homo sapiens 10 Val
Thr Ala Leu Thr Lys Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu 1 5 10
15 Glu Pro Gly Gln Pro Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe
20 25 30 Lys Lys Cys Pro Ala Pro Ser Phe Ser Trp Thr Gly Ala Ala
Leu Ser 35 40 45 Pro Arg Arg Thr Arg Pro Ser Thr Ser Gln Pro Ser
Asp Pro Gly Val 50 55 60 Leu Glu Leu Pro Pro Ile Gln Met Glu His
Glu Gly Glu Phe Thr Cys 65 70 75 80 His Ala Gln His Pro Leu Gly Ser
Gln His Val Ser Leu Ser Leu Ser 85 90 95 Val His Trp Lys Leu Glu
100 11 64 PRT Homo sapiens 11 Met Leu Leu Leu Pro Leu Leu Leu Pro
Val Leu Gly Ala Gly Ser Leu 1 5 10 15 Asn Lys Asp Pro Ser Tyr Ser
Leu Gln Val Gln Arg Gln Val Pro Val 20 25 30 Pro Glu Gly Leu Cys
Val Ile Val Ser Cys Asn Leu Ser Tyr Pro Arg 35 40 45 Asp Gly Trp
Asp Glu Ser Thr Ala Ala Tyr Gly Tyr Trp Phe Lys Gly 50 55 60 12 66
PRT Homo sapiens 12 Thr Ser Pro Lys Thr Gly Ala Pro Val Ala Thr Asn
Asn Gln Ser Arg 1 5 10 15 Glu Val Glu Met Ser Thr Arg Asp Arg Phe
Gln Leu Thr Gly Asp Pro 20 25 30 Gly Lys Gly Ser Cys Ser Leu Val
Ile Arg Asp Ala Gln Arg Glu Asp 35 40 45 Glu Ala Trp Tyr Phe Phe
Arg Val Glu Arg Gly Ser Arg Val Arg His 50 55 60 Ser Phe 65 13 7
PRT Homo sapiens 13 Leu Lys Val Thr Ala Leu Thr 1 5 14 13 PRT Homo
sapiens 14 Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly 1 5
10 15 68 PRT Homo sapiens 15 Arg Val Lys Ile Cys Arg Lys Glu Ala
Arg Lys Arg Ala Ala Ala Glu 1 5 10 15 Gln Asp Val Pro Ser Thr Leu
Gly Pro Ile Ser Gln Gly His Gln His 20 25 30 Glu Cys Ser Ala Gly
Ser Ser Gln Asp His Pro Pro Pro Gly Ala Ala 35 40 45 Thr Tyr Thr
Pro Gly Lys Gly Glu Glu Gln Glu Leu His Tyr Ala Ser 50 55 60 Leu
Ser Phe Gln 65 16 47 PRT Homo sapiens 16 Gly Leu Arg Leu Trp Glu
Pro Ala Asp Gln Glu Ala Pro Ser Thr Thr 1 5 10 15 Glu Tyr Ser Glu
Ile Lys Ile His Thr Gly Gln Pro Leu Arg Gly Pro 20 25 30 Gly Phe
Gly Leu Gln Leu Glu Arg Glu Met Ser Gly Met Val Pro 35 40 45 17 58
PRT Homo sapiens 17 Arg Val Lys Ile Cys Arg Lys Glu Ala Arg Lys Arg
Ala Ala Ala Glu 1 5 10 15 Gln Asp Val Pro Ser Thr Leu Gly Pro Ile
Ser Gln Gly His Gln His 20 25 30 Glu Cys Ser Ala Gly Ser Ser Gln
Asp His Pro Pro Pro Gly Ala Ala 35 40 45 Thr Tyr Thr Pro Gly Lys
Gly Glu Glu Gln 50 55 18 14 PRT Homo sapiens 18 Gly Pro Gly Phe Gly
Leu Gln Leu Glu Arg Glu Met Ser Gly 1 5 10 19 13 PRT Homo sapiens
19 His Tyr Ala Ser Leu Ser Phe Gln Gly Leu Arg Leu Trp 1 5 10 20 43
PRT Homo sapiens 20 Arg Val Lys Ile Cys Arg Lys Glu Ala Arg Lys Arg
Ala Ala Ala Glu 1 5 10 15 Gln Asp Val Pro Ser Thr Leu Gly Pro Ile
Ser Gln Gly His Gln His 20 25 30 Glu Cys Ser Ala Gly Ser Ser Gln
Asp His Pro 35 40 21 7 PRT Homo sapiens 21 Pro Gly Ala Ala Thr Tyr
Thr 1 5 22 11 PRT Homo sapiens 22 Gly Lys Gly Glu Glu Gln Glu Leu
His Tyr Ala 1 5 10 23 10 PRT Homo sapiens 23 Leu Leu Leu Pro Leu
Leu Leu Pro Val Leu 1 5 10 24 12 PRT Homo sapiens 24 Met Leu Leu
Leu Pro Leu Leu Leu Pro Val Leu Gly 1 5 10 25 6 PRT Homo sapiens 25
Val Pro Glu Gly Leu Cys 1 5 26 8 PRT Homo sapiens 26 Pro Val Pro
Glu Gly Leu Cys Val 1 5 27 7 PRT Homo sapiens 27 Ala Tyr Gly Tyr
Trp Phe Lys 1 5 28 9 PRT Homo sapiens 28 Ala Ala Tyr Gly Tyr Trp
Phe Lys Gly 1 5 29 7 PRT Homo sapiens 29 Gly Ala Pro Val Ala Thr
Asn 1 5 30 9 PRT Homo sapiens 30 Thr Gly Ala Pro Val Ala Thr Asn
Asn 1 5 31 19 PRT Homo sapiens 31 Gly Ala Pro Val Ala Thr Asn Asn
Asn Gln Ser Arg Glu Val Glu Met 1 5 10 15 Ser Thr Arg 32 28 PRT
Homo sapiens 32 Gly Ala Pro Val Ala Thr Asn Asn Asn Gln Ser Arg Glu
Val Glu Met 1 5 10 15 Ser Thr Arg Asp Arg Phe Gln Leu Thr Gly Asp
Pro 20 25 33 10 PRT Homo sapiens 33 Gln Ser Arg Glu Val Glu Met Ser
Thr Arg 1 5 10 34 12 PRT Homo sapiens 34 Asn Gln Ser Arg Glu Val
Glu Met Ser Thr Arg Asp 1 5 10 35 8 PRT Homo sapiens 35 Arg Phe Gln
Leu Thr Gly Asp Pro 1 5 36 10 PRT Homo sapiens 36 Asp Arg
Phe Gln Leu Thr Gly Asp Pro Gly 1 5 10 37 12 PRT Homo sapiens 37
Lys Gly Ser Cys Ser Leu Val Ile Arg Asp Ala Gln 1 5 10 38 14 PRT
Homo sapiens 38 Gly Lys Gly Ser Cys Ser Leu Val Ile Arg Asp Ala Gln
Arg 1 5 10 39 9 PRT Homo sapiens 39 Tyr Phe Phe Arg Val Glu Arg Gly
Ser 1 5 40 11 PRT Homo sapiens 40 Trp Tyr Phe Phe Arg Val Glu Arg
Gly Ser Arg 1 5 10 41 9 PRT Homo sapiens 41 Phe Phe Leu Lys Val Thr
Ala Leu Thr 1 5 42 11 PRT Homo sapiens 42 Ala Phe Phe Leu Lys Val
Thr Ala Leu Thr Lys 1 5 10 43 26 PRT Homo sapiens 43 Lys Pro Asp
Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val 1 5 10 15 Thr
Val Ile Cys Val Phe Asn Trp Ala Phe 20 25 44 28 PRT Homo sapiens 44
Lys Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro 1 5
10 15 Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe Lys 20 25 45 14
PRT Homo sapiens 45 Cys Pro Ala Pro Ser Phe Ser Trp Thr Gly Ala Ala
Leu Ser 1 5 10 46 16 PRT Homo sapiens 46 Lys Cys Pro Ala Pro Ser
Phe Ser Trp Thr Gly Ala Ala Leu Ser Pro 1 5 10 15 47 11 PRT Homo
sapiens 47 Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser 1 5 10 48 13
PRT Homo sapiens 48 Ala Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser
Pro 1 5 10 49 7 PRT Homo sapiens 49 Pro Pro Ile Gln Met Glu His 1 5
50 8 PRT Homo sapiens 50 Pro Pro Ile Gln Met Glu His Glu 1 5 51 9
PRT Homo sapiens 51 Leu Pro Pro Ile Gln Met Glu His Glu 1 5 52 12
PRT Homo sapiens 52 Gly Ala Ala Leu Gly Ala Gly Val Ala Ala Leu Leu
1 5 10 53 13 PRT Homo sapiens 53 Leu Gly Ala Ala Leu Gly Ala Gly
Val Ala Ala Leu Leu 1 5 10 54 14 PRT Homo sapiens 54 Leu Gly Ala
Ala Leu Gly Ala Gly Val Ala Ala Leu Leu Ala 1 5 10 55 9 PRT Homo
sapiens 55 Glu Leu His Tyr Ala Ser Leu Ser Phe 1 5 56 11 PRT Homo
sapiens 56 Gln Glu Leu His Tyr Ala Ser Leu Ser Phe Gln 1 5 10 57 80
DNA Homo sapiens 57 tgggctggtc cgtcctttga accagtagcc ataagcagca
gtagactcgt cccagccatc 60 ccgggggtag gagaggttgc 80 58 20 DNA
Bacteriophage T7 58 taatacgact cactataggg 20 59 18 DNA
Bacteriophage SP6 59 atttaggtga cactatag 18 60 20 DNA Homo sapiens
60 catcgtgtct tgcaacctct 20 61 20 DNA Homo sapiens 61 ctctctccac
ccgaaagaag 20 62 14 PRT Homo sapiens 62 Val Ile Val Ser Cys Asn Leu
Ser Tyr Pro Arg Asp Gly Trp 1 5 10 63 14 PRT Homo sapiens 63 Pro
Val Ala Thr Asn Asn Gln Ser Arg Glu Val Glu Met Ser 1 5 10 64 13
PRT Homo sapiens 64 Phe Lys Gly Arg Thr Ser Pro Lys Thr Gly Ala Pro
Val 1 5 10 65 13 PRT Homo sapiens 65 Arg Glu Val Glu Met Ser Thr
Arg Asp Arg Phe Gln Leu 1 5 10 66 13 PRT Homo sapiens 66 Lys Val
Thr Ala Leu Thr Lys Lys Pro Asp Val Tyr Ile 1 5 10 67 13 PRT Homo
sapiens 67 Thr Gly Ala Ala Leu Ser Pro Arg Arg Thr Arg Pro Ser 1 5
10 68 17 PRT Homo sapiens 68 Ile Arg Asp Ala Gln Arg Glu Asp Glu
Ala Trp Tyr Phe Phe Arg Val 1 5 10 15 Glu 69 14 PRT Homo sapiens 69
Arg Glu Val Glu Met Ser Thr Arg Asp Arg Phe Gln Leu Thr 1 5 10 70
14 PRT Homo sapiens 70 Glu Cys Ser Ala Gly Ser Ser Gln Asp His Pro
Pro Pro Gly 1 5 10 71 14 PRT Homo sapiens 71 Asp Gln Glu Ala Pro
Ser Thr Thr Glu Tyr Ser Glu Ile Lys 1 5 10 72 16 PRT Homo sapiens
72 Thr Ser Pro Lys Thr Gly Ala Pro Val Ala Thr Asn Asn Gln Ser Arg
1 5 10 15 73 16 PRT Homo sapiens 73 Glu Thr Leu Glu Pro Gly Gln Pro
Val Thr Val Ile Cys Val Phe Asn 1 5 10 15 74 16 PRT Homo sapiens 74
Trp Lys Leu Glu His Gly Gly Gly Leu Gly Leu Gly Ala Ala Leu Gly 1 5
10 15 75 16 PRT Homo sapiens 75 Leu Glu His Gly Gly Gly Leu Gly Leu
Gly Ala Ala Leu Gly Ala Gly 1 5 10 15 76 16 PRT Homo sapiens 76 His
Gly Gly Gly Leu Gly Leu Gly Ala Ala Leu Gly Ala Gly Val Ala 1 5 10
15 77 16 PRT Homo sapiens 77 Gly Gly Leu Gly Leu Gly Ala Ala Leu
Gly Ala Gly Val Ala Ala Leu 1 5 10 15 78 16 PRT Homo sapiens 78 Leu
Gly Ala Ala Leu Gly Ala Gly Val Ala Ala Leu Leu Ala Phe Cys 1 5 10
15 79 17 PRT Homo sapiens 79 Glu His Glu Gly Glu Phe Thr Cys His
Ala Gln His Pro Leu Gly Ser 1 5 10 15 Gln 80 20 DNA Homo sapiens 80
aagaaccaga ccaagcacct 20 81 20 DNA Homo sapiens 81 ccctttctgg
agaagtccac 20 82 39 DNA Homo sapiens 82 gcagcagcgg ccgcctgaac
aaggatccca gttacagtc 39 83 37 DNA Homo sapiens 83 gcagcagtcg
acctttggaa ccatccctga catctcc 37 84 39 DNA Homo sapiens 84
gcagcagcgg ccgcatgctg ctgctgcccc tgctgctgc 39 85 37 DNA Homo
sapiens 85 gcagcagtcg acctgggagc ccagagggtg ctgagcg 37 86 21 DNA
Homo sapiens 86 cagggatggt tccaaagtga a 21 87 20 DNA Homo sapiens
87 gtgcgactcc cacacacttg 20 88 25 DNA Homo sapiens 88 aggtctccat
ggcaacagga cacca 25
* * * * *
References