U.S. patent application number 10/105929 was filed with the patent office on 2002-09-26 for tango-73 nucleic acids.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc., a Delaware corporation. Invention is credited to Goodearl, Andrew D.J., Holtzman, Douglas A..
Application Number | 20020137142 10/105929 |
Document ID | / |
Family ID | 26733694 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137142 |
Kind Code |
A1 |
Holtzman, Douglas A. ; et
al. |
September 26, 2002 |
Tango-73 nucleic acids
Abstract
The invention relates to Tango-71, Tango-73, Tango-74, Tango-76,
and Tango-83, polypeptides, nucleic acid molecules encoding
Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83, and uses
thereof.
Inventors: |
Holtzman, Douglas A.;
(Jamaica Plain, MA) ; Goodearl, Andrew D.J.;
(Natick, MA) |
Correspondence
Address: |
ANITA L. MEIKLEJOHN, PH.D.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
Millennium Pharmaceuticals, Inc., a
Delaware corporation
|
Family ID: |
26733694 |
Appl. No.: |
10/105929 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10105929 |
Mar 25, 2002 |
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10057084 |
Jan 25, 2002 |
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10057084 |
Jan 25, 2002 |
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09130491 |
Aug 6, 1998 |
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6416974 |
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60054966 |
Aug 6, 1997 |
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60058108 |
Sep 5, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/70578 20130101; A61K 38/00 20130101; C12N 9/6489 20130101;
A01K 2217/05 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C07K 014/705; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 55% identical to the nucleotide sequence
of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:10, the cDNA insert of the plasmid deposited with ATCC as
Accession Number______, or a complement thereof; b) a nucleic acid
molecule comprising a fragment of at least 300 nucleotides of the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:10, the cDNA insert of the plasmid
deposited with ATCC as Accession Number______, or a complement
thereof; c) a nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, or an amino acid sequence encoded by the cDNA
insert of the plasmid deposited with ATCC as Accession
Number______; d) a nucleic acid molecule which encodes a fragment
of a polypeptide comprising the amino acid sequence of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, wherein the fragment
comprises at least 15 contiguous amino acids of SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, or the polypeptide encoded by the
cDNA insert of the plasmid deposited with ATCC as Accession
Number______; and e) a nucleic acid molecule which encodes a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:8, or an amino acid sequence encoded by the cDNA insert of
the plasmid deposited with ATCC as Accession Number______, wherein
the nucleic acid molecule hybridizes to a nucleic acid molecule
comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:10, or a complement thereof under stringent
conditions.
2. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) a nucleic acid comprising the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:10, the cDNA insert of the plasmid
deposited with ATCC as Accession Number______, or a complement
thereof; and b) a nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, or an amino acid sequence encoded by the cDNA
insert of the plasmid deposited with ATCC as Accession
Number______.
3. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of claim
1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide comprising the amino acid sequence
of SEQ ID NO:2 or SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, wherein
the fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2 or SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; b) a naturally
occurring allelic variant of a 9 polypeptide comprising the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, or an amino acid sequence encoded by the cDNA insert of the
plasmid deposited with ATCC as Accession Number______, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or a complement
thereof under stringent conditions; and c) a polypeptide which is
encoded by a nucleic acid molecule comprising a nucleotide sequence
which is at least 55% identical to a nucleic acid comprising the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or a complement thereof.
9. The isolated polypeptide of claim 8 comprising the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or
an amino acid sequence encoded by the cDNA insert of the plasmid
deposited with ATCC as Accession Number______.
10. The polypeptide of claim 8 further comprising heterologous
amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim
8.
12. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or an amino
acid sequence encoded by the cDNA insert of the plasmid deposited
with ATCC as Accession Number______; b) a polypeptide comprising a
fragment of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, or an amino acid sequence encoded by the
cDNA insert of the plasmid deposited with ATCC as Accession
Number______, wherein the fragment comprises at least 15 contiguous
amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
or an amino acid sequence encoded by the cDNA insert of the plasmid
deposited with ATCC as Accession Number______; and c) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:10, or an amino acid sequence encoded by the cDNA insert of the
plasmid deposited with ATCC as Accession Number______, wherein the
polypeptide is encoded by a nucleic acid molecule which hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or a complement
thereof under stringent conditions; comprising culturing the host
cell of claim 5 under conditions in which the nucleic acid molecule
is expressed.
13. A method for detecting the presence of a polypeptide of claim 8
in a sample, comprising: a) contacting the sample with a compound
which selectively binds to a polypeptide of claim 8; and b)
determining whether the compound binds to the polypeptide in the
sample.
14. The method of claim 13, wherein the compound which binds to the
polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a
polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule
of claim 1 in a sample, comprising the steps of: a) contacting the
sample with a nucleic acid probe or primer which selectively
hybridizes to the nucleic acid molecule; and b) determining whether
the nucleic acid probe or primer binds to a nucleic acid molecule
in the sample.
17. The method of claim 16, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds to a
polypeptide of claim 8 comprising the steps of: a) contacting a
polypeptide, or a cell expressing a polypeptide of claim 8 with a
test compound; and b) determining whether the polypeptide binds to
the test compound.
20. The method of claim 19, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detecting of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; c) detection of binding
using an assay for Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83-mediated signal transduction.
21. A method for modulating the activity of a polypeptide of claim
8 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 8 with a compound which binds to the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
22. A method for identifying a compound which modulates the
activity of a polypeptide of claim 8, comprising: a) contacting a
polypeptide of claim 8 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound which modulates the activity of the
polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Serial No.
60/054,966, filed Aug. 6, 1997 and U.S. Serial No. 60/058,108,
filed Sep. 5, 1997.
SUMMARY OF THE INVENTION
[0002] The invention relates to the discovery and characterization
of Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83. Tango-71
is a human protein which is approximately 90% identical to murine
ADAMTS-1. Tango-73 is a human protein that is 48% identical to rate
RVP.1 (Briehl et al., Mol. Endocrinol. 5:1381, 1991). Tango-74 is a
human protein with homology to TRAIL receptor (Pan et al., Science
276:111, 1997). Tango-76 is a rat protein which is approximately
40% identical to murine ADAMTS-1. Tango-83 is expressed by
stimulated human astrocytes.
[0003] The invention features isolated nucleic acid molecules
encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
polypeptides; isolated nucleic acid molecules encoding polypeptides
which are substantially similar to Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83; and isolated nucleic acid molecules which
hybridize under stringent conditions to a nucleic acid molecule
having the sequence of the protein coding portion of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.
[0004] The invention also features a host cell which includes an
isolated nucleic acid molecule encoding Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 and a nucleic acid vector (e.g., an
expression vector; a vector which includes a regulatory element; a
vector which includes a regulatory element selected from the group
consisting of the cytomegalovirus hCMV immediate early gene, the
early promoter of SV40 adenovirus, the late promoter of SV40
adenovirus, the lac system, the trp system, the TAC system, the TRC
system, the major operator and promoter regions of phage .lambda.,
the control regions of fd coat protein, the promoter for
3-phosphoglycerate kinase, the promoters of acid phosphatase, and
the promoters of the yeast .alpha.-mating factors; vector which
includes a regulatory element which directs tissue-specific
expression; a vector which includes a reporter gene; a vector which
includes a reporter gene selected from the group selected from the
group consisting of .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (neo.sup.r, G418.sup.r), dihydrofolate reductase
(DHFR), hygromycin-B-phosphotransfer- ase (HPH), thymidine kinase
(TK), lacZ (encoding .beta.-galactosidase), and xanthine guanine
phosphoribosyltransferase (XGPRT); a vector that is a plasmid, a
vector that is a virus; and a vector that is a retrovirus)
containing an isolated nucleic acid molecule encoding Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83.
[0005] The invention also features substantially pure Tango-71,
Tango-73, Tango-74, Tango-76, and Tango-83 polypeptides; a
substantially pure polypeptide which includes a first portion and a
second portion, the first portion including a Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 polypeptide and the second portion
including a detectable marker.
[0006] The invention also features an antibody that selectively
binds to a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
polypeptide (e.g., a monoclonal antibody).
[0007] The invention also features a pharmaceutical composition
which includes a Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 polypeptide.
[0008] The invention includes methods for diagnosing a disorder
associated with aberrant expression of a protein of the invention
(i.e., Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83), the
method including obtaining a biological sample from a patient and
measuring the expression of the protein in the biological sample,
wherein increased or decreased expression of the protein in the
biological sample compared to a control indicates that the patient
suffers from a disorder associated with aberrant expression of the
protein.
[0009] The invention encompasses isolated nucleic acid molecules
encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 or a
polypeptide fragment thereof; vectors containing these nucleic acid
molecules; cells harboring recombinant DNA encoding Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83; fusion proteins which
include all or a portion of Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83; transgenic animals which express Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83; and recombinant knock-out animals
which fail to express Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83.
[0010] The invention encompasses nucleic acids that have a sequence
that is substantially identical to a Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 nucleic acid sequence. A nucleic acid
molecule which is substantially identical to a given reference
nucleic acid molecule is hereby defined as a nucleic acid molecule
having a sequence that has at least 85%, preferably 90%, and more
preferably 95%, 98%, 99% or more identity to the sequence of the
given reference nucleic acid molecule.
[0011] The invention also includes polypeptides which are
substantially identical to Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 (e.g., polypeptides that are substantially identical to
the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, or SEQ ID NO:10).
[0012] A polypeptide which is "substantially identical" to a given
reference polypeptide molecule is a polypeptide having an amino
acid sequence that has at least 85%, preferably 90%, and more
preferably 95%, 98%, 99% or more identity to the amino acid
sequence of the given reference polypeptide.
[0013] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). Preferably, the two sequences are the same
length.
[0014] The determination of percent homology between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. Alternatively, PSI-Blast can be used to perform an
iterated search which detects distant relationships between
molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, (1988) CABIOS 4:11-17. Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0015] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0016] The nucleic acid molecules of the invention can be inserted
into vectors, as described below, which will facilitate expression
of the insert. The nucleic acid molecules and the polypeptides they
encode can be used directly as diagnostic or therapeutic agents, or
(in the case of a polypeptide) can be used to generate antibodies
that, in turn, are therapeutically useful. Accordingly, expression
vectors containing the nucleic acid molecules of the invention,
cells transfected with these vectors, the polypeptides expressed,
and antibodies generated, against either the entire polypeptide or
an antigenic fragment thereof, are among the preferred
embodiments.
[0017] A transformed cell is any cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a nucleic acid encoding a polypeptide of the invention
(e.g., a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
polypeptide).
[0018] An isolated nucleic acid molecule is a nucleic acid molecule
that is separated from the 5' and 3' coding sequences with which it
is immediately contiguous in the naturally occurring genome of an
organism. Isolated nucleic acid molecules include nucleic acid
molecule which are not naturally occurring, e.g., nucleic acid
molecules created by recombinant DNA techniques.
[0019] Nucleic acid molecules include both RNA and DNA, including
cDNA, genomic DNA, and synthetic (e.g., chemically synthesized)
DNA. Where single-stranded, the nucleic acid molecule may be a
sense strand or an antisense strand.
[0020] The invention also encompasses nucleic acid molecules that
hybridize, preferably under stringent conditions, to a nucleic acid
molecule encoding a Tango-71, Tango-73, Tango-74, Tango-76, or.
Tango-83 polypeptide (e.g., a nucleic acid molecule having the
sequence shown in SEQ ID NO:1, 3, 5, 7, or 9). Preferably the
hybridizing nucleic acid molecule consists of 400, more preferably
200 nucleotides. Preferred hybridizing nucleic acid molecules have
a biological activity possessed by Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83.
[0021] The invention also features substantially pure or isolated
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptides,
including those that correspond to various functional domains of
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83, or fragments
thereof.
[0022] The polypeptides of the invention can be produced
recombinantly, chemically synthesized, or purified from tissues in
which they are naturally expressed, according to standard
biochemical methods of purification.
[0023] Also included in the invention are functional polypeptides,
which possess one or more of the biological functions or activities
of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. These
functions include the ability to bind some or all of the proteins
which normally bind to Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83. A functional polypeptide is also considered within the
scope of the invention if it serves as an antigen for production of
antibodies that specifically bind to Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83. In many cases, functional polypeptides
retain one or more domains present in the naturally-occurring form
of the polypeptide.
[0024] The functional polypeptides may contain a primary amino acid
sequence that has been modified from those disclosed herein.
Preferably these modifications consist of conservative amino acid
substitutions, as described herein.
[0025] The terms "protein" and "polypeptide" are used herein to
describe any chain of amino acids, regardless of length or
post-translational modification (for example, glycosylation or
phosphorylation). Thus, the term "Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 polypeptides" includes full-length, naturally
occurring Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
protein, as well a recombinantly or synthetically produced
polypeptide that correspond to a full-length naturally occurring
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 protein or to
particular domains or portions of a naturally occurring protein.
The term also encompasses mature Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 which has an added amino-terminal methionine
(useful for expression in prokaryotic cells).
[0026] The term "purified" as used herein refers to a nucleic acid
or peptide that is substantially free of cellular material, viral
material, or culture medium when produced by recombinant DNA
techniques, or chemical precursors or other chemicals when
chemically synthesized.
[0027] Polypeptides or other compounds of interest are said to be
"substantially pure" when they are within preparations that are at
least 60% by weight (dry weight) the compound of interest.
Preferably, the preparation is at least 75%, more preferably at
least 90%, and most preferably at least 99%, by weight the compound
of interest. Purity can be measured by any appropriate standard
method, for example, by column chromatography, polyacrylamide gel
electrophoresis, or HPLC analysis.
[0028] The invention also features antibodies, e.g., monoclonal,
polyclonal, and engineered antibodies, which specifically bind
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. An antibody
which specifically binds to a given antigen is an antibody that
recognizes and binds to a particular antigen, but which does not
substantially recognize or bind to other molecules in a sample,
e.g., a biological sample, which includes Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83.
[0029] The invention also features antagonists and agonists of
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 that inhibit
one or more of the biological activities of Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83. Suitable antagonists can include
small molecules (i.e., molecules with a molecular weight below
about 500), large molecules (i.e., molecules with a molecular
weight above about 500), neutralizing antibodies, polypeptides
which compete with a native form of Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 for binding to a protein, and nucleic acid
molecules that interfere with transcription of Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 (for example, antisense nucleic
acid molecules and ribozymes). Agonists of Tango-71, Tango-73,
Tango-74, Tango-76, and Tango-83 also include small and large
molecules, and antibodies other than neutralizing antibodies.
[0030] The invention also features molecules which can increase or
decrease the expression of Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 (e.g., by influencing transcription or translation).
Small molecules (i.e., molecules with a molecular weight below
about 500), large molecules (i.e., molecules with a molecular
weight above about 500), and nucleic acid molecules that can be
used to inhibit the expression of Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 (for example, antisense and ribozyme
molecules) or to enhance their expression (for example, molecules
that bind to a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
transcription regulatory sequences and increase Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 transcription).
[0031] In addition, the invention features substantially pure
polypeptides that functionally interact with Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83, and the nucleic acid molecules
that encode them.
[0032] The invention encompasses methods for treating disorders
associated with aberrant expression or activity of a protein of the
invention (i.e., Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83). Thus, the invention includes methods for treating
disorders associated with excessive expression or activity of a
protein of the invention. Such methods entail administering a
compound which decreases the expression or activity of the protein.
The invention also includes methods for treating disorders
associated with insufficient expression or activity of a protein of
the invention. These methods entail administering a compound which
increases the expression or activity of the protein.
[0033] The invention also features methods for detecting a protein
of the invention (i.e., Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83). Such methods include: obtaining a biological sample;
contacting the sample with an antibody that specifically binds the
protein under conditions which permit specific binding; and
detecting any antibody-protein complexes formed.
[0034] In addition, the present invention encompasses methods and
compositions for the diagnostic evaluation, typing, and prognosis
of disorders associated with inappropriate expression or activity
of a protein of the invention. For example, the nucleic acid
molecules of the invention can be used as diagnostic hybridization
probes to detect, for example, inappropriate expression of a
protein of the invention or mutations in the gene encoding a
protein of the invention gene. Such methods may be used to classify
cells by the level of Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 expression.
[0035] The invention encompasses methods for diagnosing a disorder
associated with aberrant activity of a protein of the invention,
the methods including obtaining a biological sample from a patient
and measuring the activity of the protein in the biological sample,
wherein increased or decreased activity in the biological sample
compared to a control indicates that the patient suffers from a
disorder associated with aberrant activity of the protein.
[0036] The nucleic acid molecules of the invention can be used as
primers for diagnostic PCR analysis for the identification of gene
mutations, allelic variations and regulatory defects in the
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 gene. The
present invention further provides for diaghostic kits for the
practice of such methods.
[0037] The invention features methods of identifying compounds that
modulate the expression or activity of a protein of the invention
by assessing the expression or activity of the protein in the
presence and absence of a selected compound. A difference in the
level of expression or activity of the protein indicates that the
selected compound is capable of modulating expression or activity
of the protein. Expression can be assessed either at the level of
gene expression (e.g., by measuring mRNA) or protein expression by
techniques that are well known to skilled artisans. The activity of
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 can be assessed
functionally.
[0038] The preferred methods and materials are described below in
examples which are meant to illustrate, not limit, the invention.
Skilled artisans will recognize methods and materials that are
similar or equivalent to those described herein, and that can be
used in the practice or testing of the present invention.
[0039] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be limiting. other features and advantages of the invention will be
apparent from the detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 nucleotide sequence (SEQ ID NO:l) and deduced amino
acid sequence (SEQ ID NO:2) of Tango-71.
[0041] FIG. 2 Nucleotide acid sequence (SEQ ID NO:3) and deduced
amino acid sequence (SEQ ID NO:4) of Tango-73.
[0042] FIG. 3 Nucleotide acid sequence (SEQ ID NO:5) and deduced
amino acid sequence (SEQ ID NO:6) of Tango-74. The ATG encoding the
first Met is boxed as is the ATC encoding the final Ile.
[0043] FIG. 4 Nucleotide acid sequence of a 3' non-coding portion
of Tango-74 (SEQ ID NO:11).
[0044] FIG. 5 Alignment of a portion of the amino acid sequence of
Tango-74 (SEQ ID NO:6) and the amino acid sequence of TRAIL.
[0045] FIG. 6 Partial nucleotide sequence (SEQ ID NO:7) and deduced
amino acid sequence (SEQ ID NO:8) of Tango-76.
[0046] FIG. 7 Nucleotide sequence of a 5' portion of Tango-83 (SEQ
ID NO:9).
[0047] FIG. 8 Nucleotide sequence of a 3' portion of Tango-83 (SEQ
ID NO:10).
[0048] FIG. 9 Alignment of amino acid sequence of Tango-71 and the
amino acid sequence of ADAMTS-1.
[0049] FIG. 10 Alignment of the amino acid sequence of Tango-73 and
the amino acid sequence of RVPI.
[0050] FIG. 11 Alignment of the amino acid sequence of Tango-73 and
TMVCF.
[0051] FIG. 12 Northern blot analysis of Tango-73 mRNA.
[0052] FIG. 13 Northern blot analysis of Tango-83 mRNA.
[0053] FIG. 14 Alignment of amino acid sequence of Tango-76 and
ADAMTS-1.
DETAILED DESCRIPTION
Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83 Nucleic Acid
Molecules
[0054] The Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83
nucleic acid molecules of the invention can be cDNA, genomic DNA,
synthetic DNA, or RNA, and can be double-stranded or
single-stranded (i.e., either a sense or an antisense strand).
Fragments of these molecules are also considered within the scope
of the invention, and can be produced, for example, by the
polymerase chain reaction (PCR) or generated by treatment with one
or more restriction endonucleases. A ribonucleic acid (RNA)
molecule can be produced by in vitro transcription.
[0055] The nucleic acid molecules of the invention can contain
naturally occurring sequences, or sequences that differ from those
that occur naturally, but, due to the degeneracy of the genetic
code, encode the same polypeptide. In addition, these nucleic acid
molecules are not limited to sequences that only encode
polypeptides, and thus, can include some or all of the non-coding
sequences that lie upstream or downstream from a coding
sequence.
[0056] The nucleic acid molecules of the invention can be
synthesized (for example, by phosphoramidite-based synthesis) or
obtained from a biological cell, such as the cell of a mammal.
Thus, the nucleic acids can be those of a human, mouse, rat, guinea
pig, cow, sheep, horse, pig, rabbit, monkey, dog, or cat.
Combinations or modifications of the nucleotides within these types
of nucleic acids are also encompassed.
[0057] In addition, the isolated nucleic acid molecules of the
invention encompass fragments that are not found as such in the
natural state. Thus, the invention encompasses recombinant
molecules, such as those in which a nucleic acid molecule (for
example, an isolated nucleic acid molecule encoding Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83) is incorporated into a
vector (for example, a plasmid or viral vector) or into the genome
of a heterologous cell (or the genome of a homologous cell, at a
position other than the natural chromosomal location). Recombinant
nucleic acid molecules and uses therefor are discussed further
below.
[0058] In the event the nucleic acid molecules of the invention
encode or act as antisense molecules, they can be used for example,
to regulate translation of Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 mRNA. Techniques associated with detection or
regulation of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
expression are well known to skilled artisans and can be used to
diagnose and/or treat disorders associated with aberrant Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 expression.
[0059] The invention also encompasses nucleic acid molecules that
hybridize under stringent conditions to a nucleic acid molecule
encoding a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
polypeptide (e.g., nucleic acid molecules having the sequence of
the protein coding portion of SEQ ID NO:1, 3, 5, 7, or 9). The cDNA
sequences described herein can be used to identify these
hybridizing nucleic acids, which include, for example, nucleic
acids that encode homologous polypeptides in other species and
splice variants of the Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 gene in humans or other mammals. Accordingly, the
invention features methods of detecting and isolating these nucleic
acid molecules. Using these methods, a sample (for example, a
nucleic acid library, such as a cDNA or genomic library) is
contacted (or "screened") with a Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83-specific probe (for example, a fragment of
SEQ ID NO:1, 3, 5, 7, or 9 that is at least 25 or 50 or 100
nucleotides long) The probe will selectively hybridize to nucleic
acids encoding related polypeptides (or to complementary sequences
thereof). The probe, which can contain at least 25 (for example,
25, 50, 100, or 200 nucleotides) can be produced using any of
several standard methods (see, for example, Ausubel et al.,
"Current Protocols in Molecular Biology, Vol. I," Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., NY, 1989). For
example, the probe can be generated using PCR amplification methods
in which oligonucleotide primers are used to amplify a Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83-specific nucleic acid
sequence that can be used as a probe to screen a nucleic acid
library and thereby detect nucleic acid molecules (within the
library) that hybridize to the probe. one single-stranded nucleic
acid is said to hybridize to another if a duplex forms between
them. This occurs when one nucleic acid contains a sequence that is
the reverse and complement of the other (this same arrangement
gives rise to the natural interaction between the sense and
antisense strands of DNA in the genome and underlies the
configuration of the "double helix"). Complete complementarity
between the hybridizing regions is not required in order for a
duplex to form; it is only necessary that the number of paired
bases is sufficient to maintain the duplex under the hybridization
conditions used.
[0060] Typically, hybridization conditions are of low to moderate
stringency. These conditions favor specific interactions between
completely complementary sequences, but allow some non-specific
interaction between less than perfectly matched sequences to occur
as well. After hybridization, the nucleic acids can be "washed"
under moderate or high conditions of stringency to dissociate
duplexes that are bound together by some non-specific interaction
(the nucleic acids that form these duplexes are thus not completely
complementary).
[0061] As is known in the art, the optimal conditions for washing
are determined empirically, often by gradually increasing the
stringency. The parameters that can be changed to affect stringency
include, primarily, temperature and salt concentration. In general,
the lower the salt concentration and the higher the temperature,
the higher the stringency. Washing can be initiated at a low
temperature (for example, room temperature) using a solution
containing a salt concentration that is equivalent to or lower than
that of the hybridization solution. Subsequent washing can be
carried out using progressively warmer solutions having the same
salt concentration. As alternatives, the salt concentration can be
lowered and the temperature maintained in the washing step, or the
salt concentration can be lowered and the temperature increased.
Additional parameters can also be altered. For example, use of a
destabilizing agent, such as formamide, alters the stringency
conditions.
[0062] In reactions where nucleic acids are hybridized, the
conditions used to achieve a given level of stringency will vary.
There is not one set of conditions, for example, that will allow
duplexes to form between all nucleic acids that are 85% identical
to one another; hybridization also depends on unique features of
each nucleic acid. The length of the sequence, the composition of
the sequence (for example, the content of purine-like nucleotides
versus the content of pyrimidine-like nucleotides) and the type of
nucleic acid (for example, DNA or RNA) affect hybridization. An
additional consideration is whether one of the nucleic acids is
immobilized (for example, on a filter).
[0063] An example of a progression from lower to higher stringency
conditions is the following, where the salt content is given as the
relative abundance of SSC (a salt solution containing sodium
chloride and sodium citrate; 2.times.SSC is 10-fold more
concentrated than 0.2.times.SSC). Nucleic acids are hybridized at
42.degree. C. in 2.times.SSC/0.1% SDS (sodium dodecylsulfate; a
detergent) and then washed in 0.2.times.SSC/0.1% SDS at room
temperature (for conditions of low stringency); 0.2.times.SSC/0.1%
SDS at 42.degree. C. (for conditions of moderate stringency); and
0.1.times.SSC at 68.degree. C. (for conditions of high stringency).
Washing can be carried out using only one of the conditions given,
or each of the conditions can be used (for example, washing for
10-15 minutes each in the order listed above). Any or all of the
washes can be repeated. As mentioned above, optimal conditions will
vary and can be determined empirically.
[0064] A second set of conditions that are considered "stringent
conditions" are those in which hybridization is carried out at
50.degree. C. in Church buffer (7% SDS, 0.5% NaHPO.sub.4, 1 M EDTA,
1% BSA) and washing is carried out at 50.degree. C. in
2.times.SSC.
[0065] Once detected, the nucleic acid molecules can be isolated by
any of a number of standard techniques (see, for example, Sambrook
et al., "Molecular Cloning, A Laboratory Manual," 2nd Ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
[0066] The invention also encompasses: (a) expression vectors that
contain any of the foregoing Tango-71, Tango-73, Tango-74,
Tango-76, and Tango-83-related coding sequences and/or their
complements (that is, "antisense" sequence); (b) expression vectors
that contain any of the foregoing Tango-71, Tango-73, Tango-74,
Tango-76, and Tango-83-related coding sequences operatively
associated with a regulatory element (examples of which are given
below) that directs the expression of the coding sequences; (c)
expression vectors containing, in addition to sequences encoding a
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide,
nucleic acid sequences that are unrelated to nucleic acid sequences
encoding Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83, such
as molecules encoding a reporter or marker; and (d) genetically
engineered host cells that contain any of the foregoing expression
vectors and thereby express the nucleic acid molecules of the
invention in the host cell.
[0067] Recombinant nucleic acid molecules can contain a sequence
encoding a soluble Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 polypeptide; mature Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83; or Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 having a signal sequence. A full length Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide; a domain of
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; or a fragment
thereof may be fused to additional polypeptides, as described
below. Similarly, the nucleic acid molecules of the invention can
encode the mature form of Tango-71, Tango-73, Tango-74, Tango-76,
and Tango-83 or a form that encodes a polypeptide which facilitates
secretion. In the latter instance, the polypeptide is typically
referred to as a proprotein, which can be converted into an active
form by removal of the signal sequence, for example, within the
host cell. Proproteins can be converted into the active form of the
protein by removal of the inactivating sequence.
[0068] The regulatory elements referred to above include, but are
not limited to, inducible and non-inducible promoters, enhancers,
operators and other elements, which are known to those skilled in
the art, and which drive or otherwise regulate gene expression.
Such regulatory elements include but are not limited to the
cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, the lac system, the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage A, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase, the promoters of acid phosphatase,
and the promoters of the yeast .alpha.-mating factors.
[0069] Similarly, the nucleic acid can form part of a hybrid gene
encoding additional polypeptide sequences, for example, sequences
that function as a marker or reporter. Examples of marker or
reporter genes include .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (neo.sup.r, G418.sup.r) dihydrofolate reductase
(DHFR), hygromycin-B-phosphotransfera- se (HPH), thymidine kinase
(TK), lacZ (encoding .beta.-galactosidase), and xanthine guanine
phosphoribosyltransferase (XGPRT). As with many of the standard
procedures associated with the practice of the invention, skilled
artisans will be aware of additional useful reagents, for example,
of additional sequences that can serve the function of a marker or
reporter. Generally, the hybrid polypeptide will include a first
portion and a second portion; the first portion being a Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 polypeptide and the
second portion being, for example, the reporter described above or
an immunoglobulin constant region.
[0070] The expression systems that may be used for purposes of the
invention include, but are not limited to, microorganisms such as
bacteria (for example, E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA
expression vectors containing the nucleic acid molecules of the
invention; yeast (for example, Saccharomyces and Pichia)
transformed with recombinant yeast expression vectors containing
the nucleic acid molecules of the invention (preferably containing
the nucleic acid sequence encoding Tango-71, Tango-73, Tango-74,
Tango-76, and Tango-83); insect cell systems infected with
recombinant virus expression vectors (for example, baculovirus)
containing the nucleic acid molecules of the invention; plant cell
systems infected with recombinant virus expression vectors (for
example, cauliflower mosaic virus (CaMV) and tobacco mosaic virus
(TMV)) or transformed with recombinant plasmid expression vectors
(for example, Ti plasmid) containing Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 nucleotide sequences; or mammalian cell
systems (for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38,
and NIH 3T3 cells) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells
(for example, the metallothionein promoter) or from mammalian
viruses (for example, the adenovirus late promoter and the vaccinia
virus 7.5K promoter).
[0071] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
gene product being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions containing Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 polypeptides or for raising
antibodies to those polypeptides, vectors that are capable of
directing the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited to, the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791, 1983), in which the coding sequence
of the insert may be ligated individually into the vector in frame
with the lacZ coding region so that a fusion protein is produced;
pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109,
1985; Van Heeke and Schuster, J. Biol. Chem. 264:5503-5509, 1989);
and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0072] In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) can be used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda cells. The
coding sequence of the insert may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of the coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed. (for example, see
Smith et al., J. Virol. 46:584, 1983; Smith, U.S. Pat. No.
4,215,051).
[0073] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the nucleic acid molecule of the invention may
be ligated to an adenovirus transcription/translation control
complex, for example, the late promoter and tripartite leader
sequence. This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (for example, region E1 or
E3) will result in a recombinant virus that is viable and capable
of expressing a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
gene product in infected hosts (for example, see Logan and Shenk,
Proc. Natl. Acad. Sci. USA 81:3655-3659, 1984). Specific initiation
signals may also be required for efficient translation of inserted
nucleic acid molecules. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire gene or
cDNA, including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of the coding sequence is inserted, exogenous
translational control signals, including, perhaps, the ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:516-544, 1987).
[0074] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (for example, glycosylation) and processing (for
example, cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic
and specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines
or host systems can be chosen to ensure the correct modification
and processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. The mammalian cell
types listed above are among those that could serve as suitable
host cells.
[0075] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express a Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 sequences described above may be engineered. Rather than
using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (for example, promoter,
enhancer sequences, transcription terminators, polyadenylation
sites, etc.), and a selectable marker. Following the introduction
of the foreign DNA, engineered cells may be allowed to grow for 1-2
days in an enriched media, and then switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance
to the selection and allows cells to stably integrate the plasmid
into their chromosomes and grow to form foci which in turn can be
cloned and expanded into cell lines. This method can advantageously
be used to engineer cell lines which express Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds that
affect the endogenous activity of the gene product.
[0076] A number of selection systems can be used. For example, the
herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223,
1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and
adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980)
genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, anti-metabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci.
USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci. USA
78:1527, 1981); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,
which confers resistance to hygromycin (Santerre et al., Gene
30:147, 1984).
[0077] Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83 nucleic
acid molecules are useful in genetic mapping and chromosome
identification.
Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83
Polypeptides
[0078] The Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83
polypeptides described herein are those encoded by any of the
nucleic acid molecules described above and include Tango-71,
Tango-73, Tango-74, Tango-76, and Tango-83 fragments, mutants,
truncated forms, and fusion proteins. These polypeptides can be
prepared for a variety of uses, including but not limited to the
generation of antibodies, as reagents in diagnostic assays, for the
identification of other cellular gene products or compounds that
can modulate the activity or expression of Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83, and as pharmaceutical reagents
useful for the treatment of disorders associated with aberrant
expression or activity of Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83.
[0079] Preferred polypeptides are substantially pure Tango-71,
Tango-73, Tango-74, Tango-76, and Tango-83 polypeptides, including
those that correspond to the polypeptide with an intact signal
sequence, the secreted form of a Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 polypeptide. Especially preferred are
polypeptides that are soluble under normal physiological
conditions.
[0080] The invention also encompasses polypeptides that are
functionally equivalent to Tango-71, Tango-73, Tango-74, Tango-76,
and Tango-83. These polypeptides are equivalent to Tango-71,
Tango-73, Tango-74, Tango-76, and Tango-83 in that they are capable
of carrying out one or more of the functions of Tango-71, Tango-73,
Tango-74, Tango-76, and Tango-83 in a biological system. Preferred
Tango-71, Tango-73, Tango-.sup.74, Tango-76, and Tango-83
polypeptides have 20%, 40%, 50%, 75%, 80%, or even 90% of one or
more of the biological activities of the full-length, mature human
form of Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83. Such
comparisons are generally based on an assay of biological activity
in which equal concentrations of the polypeptides are used and
compared. The comparison can also be based on the amount of the
polypeptide required to reach 50% of the maximal stimulation
obtainable.
[0081] Functionally equivalent proteins can be those, for example,
that contain additional or substituted amino acid residues.
Substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues involved. Amino acids that are
typically considered to provide a conservative substitution for one
another are specified in the summary of the invention.
[0082] Polypeptides that are functionally equivalent to Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 can be made using random
mutagenesis techniques well known to those skilled in the art. It
is more likely, however, that such polypeptides will be generated
by site-directed mutagenesis (again using techniques well known to
those skilled in the art). These polypeptides may have increased
functionality or decreased functionality.
[0083] To design functionally equivalent polypeptides, it is useful
to distinguish between conserved positions and variable positions.
This can be done by aligning the amino acid sequence of Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 with the amino acid
sequence of the homologons protein from another species. Skilled
artisans will recognize that conserved amino acid residues are more
likely to be necessary for preservation of function. Thus, it is
preferable that conserved residues are not altered.
[0084] Mutations within the Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 coding sequence can be made to generate variant
Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83 genes that are
better suited for expression in a selected host cell. For example,
N-linked glycosylation sites can be altered or eliminated to
achieve, for example, expression of a homogeneous product that is
more easily recovered and purified from yeast hosts which are known
to hyperglycosylate N-linked sites. To this end, a variety of amino
acid substitutions at one or both of the first or third amino acid
positions of any one or more of the glycosylation recognition
sequences which occur, and/or an amino acid deletion at the second
position of any one or more of such recognition sequences, will
prevent glycosylation at the modified tripeptide sequence (see, for
example, Miyajima et al., EMBO J. 5:1193, 1986).
[0085] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Proc. Natl. Acad. Sci. USA 88:
8972-8976, 1991). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the gene's open
reading frame is translationally fused to an amino-terminal tag
consisting of six histidine residues. Extracts from cells infected
with recombinant vaccinia virus are loaded onto Ni.sup.2+
nitriloacetic acid-agarose columns and histidine-tagged proteins
are selectively eluted with imidazole-containing buffers.
[0086] The polypeptides of the invention can be expressed fused to
another polypeptide, for example, a marker polypeptide or fusion
partner. For example, the polypeptide can be fused to a
hexa-histidine tag to facilitate purification of bacterially
expressed protein or a hemagglutinin tag to facilitate purification
of protein expressed in eukaryotic cells.
[0087] The polypeptides of the invention can be chemically
synthesized (for example, see Creighton, "Proteins: Structures and
Molecular Principles," W. H. Freeman & Co., NY, 1983), or,
perhaps more advantageously, produced by recombinant DNA technology
as described herein. For additional guidance, skilled artisans may
consult Ausubel et al. (supra), Sambrook et al. ("Molecular
Cloning, A Laboratory Manual," Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 1989), and, particularly for examples of
chemical synthesis Gait, M. J. Ed. ("Oligonucleotide Synthesis,"
IRL Press, Oxford, 1984).
[0088] The invention also features polypeptides that interact with
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (and the genes
that encode them) and thereby alter the function of Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83. Interacting polypeptides
can be identified using methods known to those skilled in the art.
One suitable method is the "two-hybrid system," which detects
protein interactions in vivo (Chien et al., Proc. Natl. Acad. Sci.
USA, 88:9578, 1991). A kit for practicing this method is available
from Clontech (Palo Alto, Calif.).
[0089] Transgenic Animals
[0090] Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83
polypeptides can also be expressed in transgenic animals. These
animals represent a model system for the study of disorders that
are caused by or exacerbated by overexpression or underexpression
of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83, and for the
development of therapeutic agents that modulate the expression or
activity of Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83.
[0091] Transgenic animals can be farm animals (pigs, goats, sheep,
cows, horses, rabbits, and the like) rodents (such as rats, guinea
pigs, and mice), non-human primates (for example, baboons, monkeys,
and chimpanzees), and domestic animals (for example, dogs and
cats). Transgenic mice are especially preferred.
[0092] Any technique known in the art can be used to introduce a
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 transgene into
animals to produce the founder lines of transgenic animals. Such
techniques include, but are not limited to, pronuclear
microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene
transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.
Sci., USA 82:6148, 1985); gene targeting into embryonic stem cells
(Thompson et al., Cell 56:313, 1989); and electroporation of
embryos (Lo, Mol. Cell. Biol. 3:1803, 1983).
[0093] The present invention provides for transgenic animals that
carry a the Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
transgene in all their cells, as well as animals that carry a
transgene in some, but not all of their cells. That is, the
invention provides for mosaic animals. The transgene can be
integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene can
also be selectively introduced into and activated in a particular
cell type (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232, 1992).
The regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art.
[0094] When it is desired that the Tango-71, Tango-73, Tango-74,
Tango-76, and Tango-83 transgene be integrated into the chromosomal
site of the endogenous gene, gene targeting is preferred. Briefly,
when such a technique is to be used, vectors containing some
nucleotide sequences homologous to an endogenous Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 gene are designed for the
purpose of integrating, via homologous recombination with
chromosomal sequences, into and disrupting the function of the
nucleotide sequence of the endogenous gene. The transgene also can
be selectively introduced into a particular cell type, thus
inactivating the endogenous Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 gene in only that cell type (Gu et al., Science
265:103, 1984). The regulatory sequences required for such a
cell-type specific inactivation will depend upon the particular
cell type of interest, and will be apparent to those of skill in
the art.
[0095] Once transgenic animals have been generated, expression of
the recombinant Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
gene can be assayed utilizing standard techniques. Initial
screening may be accomplished by Southern blot analysis or PCR
techniques to determine whether integration of the transgene has
taken place. The level of mRNA expression of the transgene in the
tissues of the transgenic animals may also be assessed using
techniques which include, but are not limited to, Northern blot
analysis of tissue samples obtained from the animal, in situ
hybridization analysis, and RT-PCR. Samples of Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 gene-expressing tissue can also be
evaluated immunocytochemically using antibodies specific for the
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 transgene
product.
[0096] For a review of techniques that can be used to generate and
assess transgenic animals, skilled artisans can consult Gordon
(Intl. Rev. Cytol. 115:171-229, 1989), and may obtain additional
guidance from, for example: Hogan et al. "Manipulating the Mouse
Embryo" (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1986;
Krimpenfort et al., Bio/Technology 9:86, 1991; Palmiter et al.,
Cell 41:343, 1985; Kraemer et al., "Genetic Manipulation of the
Early Mammalian Embryo," Cold Spring Harbor Press, Cold Spring
Harbor, N.Y., 1985; Hammer et al., Nature 315:680, 1985; Purcel et
al., Science, 244:1281, 1986; Wagner et al., U.S. Pat. No.
5,175,385; and Krimpenfort et al., U.S. Pat. No. 5,175,384 (the
latter two publications are hereby incorporated by reference).
Anti-Tango-71 Tango-73, Tango-74, Tango-76, and Tango-83
Antibodies
[0097] Human Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83
polypeptides (or immunogenic fragments or analogs) can be used to
raise antibodies useful in the invention; such polypeptides can be
produced by recombinant techniques or synthesized (see, for
example, "Solid Phase Peptide Synthesis," supra; Ausubel et al.,
supra). In general, the peptides can be coupled to a carrier
protein, such as KLH, as described in Ausubel et al., supra, mixed
with an adjuvant, and injected into a host mammal. Antibodies can
be purified by peptide antigen affinity chromatography.
[0098] In particular, various host animals can be immunized by
injection with a Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 protein or polypeptide. Host animals include rabbits,
mice, guinea pigs, and rats. Various adjuvants that can be used to
increase the immunological response depend on the host species and
include Freund's adjuvant (complete and incomplete), mineral gels
such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hem ocyanin, and dinitrophenol.
Potentially useful human adjuvants include BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies
are heterogeneous populations of antibody molecules that are
contained in the sera of the immunized animals.
[0099] Antibodies within the invention therefore include polyclonal
antibodies and, in addition, monoclonal antibodies, humanized or
chimeric antibodies, single chain antibodies, Fab fragments,
F(ab').sub.2 fragments, and molecules produced using a Fab
expression library.
[0100] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be prepared using the
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 polypeptides
described above and standard hybridoma technology (see, for
example, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur.
J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292,
1976; Hammerling et al., "Monoclonal Antibodies and T Cell
Hybridomas," Elsevier, N.Y., 1981; Ausubel et al., supra).
[0101] In particular, monoclonal antibodies can be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture such as described in Kohler et
al., Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human
B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72,
1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and
the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies
and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96, 1983). Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo. The
ability to produce high titers of mAbs in vivo makes this a
particularly useful method of production.
[0102] Once produced, polyclonal or monoclonal antibodies are
tested for specific Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 recognition by Western blot or immunoprecipitation
analysis by standard methods, e.g., as described in Ausubel et al.,
supra. Antibodies that specifically recognize and bind to Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 are useful. For example,
such antibodies can be used in an immunoassay to monitor the level
of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 produced by
a mammal (for example, to determine the amount or subcellular
location of Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83).
[0103] Preferably, antibodies of the invention are produced using
fragments of the Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 protein which lie outside highly conserved regions and
appear likely to be antigenic, by criteria such as high frequency
of charged residues. In one specific example, such fragments are
generated by standard techniques of PCR, and are then cloned into
the pGEX expression vector (Ausubel et al., supra). Fusion proteins
are expressed in E. coli and purified using a glutathione agarose
affinity matrix as described in Ausubel, et al., supra.
[0104] In some cases it may be desirable to minimize the potential
problems of low affinity or specificity of antisera. In such
circumstances, two or three fusions can be generated for each
protein, and each fusion can be injected into at least two rabbits.
Antisera can be raised by injections in a series, preferably
including at least three booster injections.
[0105] Antisera is also checked for its ability to
immunoprecipitate recombinant Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 protein or control proteins, such as
glucocorticoid receptor, CAT, or luciferase.
[0106] The antibodies can be used, for example, in the detection of
the Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 in a
biological sample as part of a diagnostic assay. Antibodies also
can be used in a screening assay to measure the effect of a
candidate compound on expression or localization of Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83. Additionally, antibodies
can be used in conjunction with the gene therapy techniques
described to, for example, evaluate the normal and/or engineered
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83-expressing
cells prior to their introduction into the patient. Such antibodies
additionally can be used in a method for inhibiting abnormal
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 activity.
[0107] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et
al., Nature, 314:452, 1984) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. 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.
[0108] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and
4,704,692) can be adapted to produce single chain antibodies
against a Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
polypeptide. Single chain antibodies are formed by linking the
heavy and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0109] Antibody fragments that recognize and bind to specific
epitopes can be generated by known techniques. For example, such
fragments include but are not limited to F(ab').sub.2 fragments
that can be produced by pepsin digestion of the antibody molecule,
and Fab fragments that can be generated by reducing the disulfide
bridges of F(ab').sub.2 fragments. Alternatively, Fab expression
libraries can be constructed (Huse et al., Science, 246:1275, 1989)
to allow rapid and easy identification of monoclonal Fab fragments
with the desired specificity.
[0110] Antibodies to Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 can, in turn, be used to generate anti-idiotype antibodies
that resemble a portion of Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 using techniques well known to those skilled in the art
(see, e.g., Greenspan et al., FASEB J. 7:437, 1993; Nissinoff, J.
Immunol. 147:2429, 1991). For example, antibodies that bind to
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 and
competitively inhibit the binding of a binding partner of the
protein can be used to generate anti-idiotypes that resemble a
binding partner binding domain of the protein and, therefore, bind
and neutralize a binding partner of the protein. Such neutralizing
anti-idiotypic antibodies or Fab fragments of such anti-idiotypic
antibodies can be used in therapeutic regimens.
[0111] Antibodies can be humanized by methods known in the art. For
example, monoclonal antibodies with a desired binding specificity
can be commercially humanized (Scotgene, Scotland; Oxford
Molecular, Palo Alto, Calif.). Fully human antibodies, such as
those expressed in transgenic animals are also features of the
invention (Green et al., Nature Genetics 7:13-21, 1994; see also
U.S. Pat. Nos. 5,545,806 and 5,569,825, both of which are hereby
incorporated by reference).
[0112] The methods described herein in which anti-Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 antibodies are employed
may be performed, for example, by utilizing pre-packaged diagnostic
kits comprising at least one specific Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 antibody reagent described herein, which may
be conveniently used, for example, in clinical settings, to
diagnose patients exhibiting symptoms of the disorders described
below.
[0113] Antisense Nucleic Acids
[0114] Treatment regimes based on an "antisense" approach involve
the design of oligonucleotides (either DNA or RNA) that are
complementary to Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 mRNA. These oligonucleotides bind to the complementary
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 mRNA
transcripts and prevent translation. Absolute complementarity,
although preferred, is not required. A sequence "complementary" to
a portion of an RNA, as referred to herein, means a sequence having
sufficient complementarily to be able to hybridize with the RNA,
forming a stable duplex; in the case of double-stranded antisense
nucleic acids, a single strand of the duplex DNA may be tested, or
triplex formation may be assayed. The ability to hybridize will
depend on both the degree of complementarily and the length of the
antisense nucleic acid. Generally, the longer the hybridizing
nucleic acid, the more base mismatches with an RNA it may contain
and still form a stable duplex (or triplex, as the case may be) One
skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.
[0115] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs recently have been shown to be
effective at inhibiting translation of mRNAs as well (Wagner,
Nature 372:333, 1984). Thus, oligonucleotides complementary to
either the 5' or 3' non-translated, non-coding regions of the mRNA,
could be used in an antisense approach to inhibit translation of
endogenous Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83
mRNA. Oligonucleotides complementary to the 5' untranslated region
of the mRNA should include the complement of the AUG start
codon.
[0116] Antisense oligonucleotides complementary to mRNA coding
regions are less efficient inhibitors of translation but could be
used in accordance with the invention. Whether designed to
hybridize to the 5', 3', or coding region of mRNA, antisense
nucleic acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides, or
at least 50 nucleotides.
[0117] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0118] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (as described,
e.g., in Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553,
1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648, 1987; PCT
Publication No. WO 88/09810) or the blood-brain barrier (see, for
example, PCT Publication No. WO 89/10134), or
hybridization-triggered cleavage agents (see, for example, Krol et
al., BioTechniques 6:958, 1988), or intercalating agents (see, for
example, Zon, Pharm. Res. 5:539, 1988). To this end, the
oligonucleotide can be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, or hybridization-triggered cleavage agent.
[0119] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethyl-aminomethylurac- il, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
2-(3-amino-3-N-2-carboxypropl)uracil, (acp3)w, and
2,6-diaminopurine.
[0120] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0121] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal, or
an analog of any of these backbones.
[0122] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids.
Res. 15:6625, 1987). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,
1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett.
215:327, 1987).
[0123] Antisense oligonucleotides of the invention can be
synthesized by standard methods known in the art, e.g., by use of
an automated DNA synthesizer (such as are commercially available
from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides can be synthesized by the method
of Stein et al. (Nucl. Acids Res. 16:3209, 1988), and
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al., Proc. Natl.
Acad. Sci. USA 85:7448, 1988).
[0124] The antisense molecules should be delivered to cells that
express the protein of interest in vivo. A number of methods have
been developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systemically.
[0125] However, it is often difficult to achieve intracellular
concentrations of the antisense molecule sufficient to suppress
translation of endogenous mRNAs. Therefore, a preferred approach
uses a recombinant DNA construct in which the antisense
oligonucleotide is placed under the control of a strong pol III or
pol II promoter. The use of such a construct to transfect target
cells in the patient will result in the transcription of sufficient
amounts of single stranded RNAs that will form complementary base
pairs with the endogenous Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 transcripts and thereby prevent translation. For
example, a vector can be introduced in vivo such that it 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.
[0126] 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. 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 et al., Nature 290:304,
1981); the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., Cell 22:787-797, 1988); the
herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad.
Sci. USA 78:1441, 1981); or the regulatory sequences of the
metallothionein gene (Brinster et al., Nature 296:39, 1988).
[0127] Ribozymes
[0128] Ribozyme molecules designed to catalytically cleave
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 mRNA
transcripts also can be used to prevent translation and expression
of Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 (see, e.g.,
PCT Publication WO 90/11364; Saraver et al., Science 247:1222,
1990). While various ribozymes that cleave mRNA at site-specific
recognition sequences can be used to destroy Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 mRNAs, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at
locations dictated by flanking regions that form complementary base
pairs with the target mRNA. The sole requirement is that the target
mRNA have the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well known
in the art (Haseloff et al., Nature 334:585, 1988). There are
numerous examples of potential hammerhead ribozyme cleavage sites
within the nucleotide sequence of human Tango-71, Tango-73,
Tango-74, Tango-76, and Tango-83 cDNA. Preferably, the ribozyme is
engineered so that the cleavage recognition site is located near
the 5' end of the mRNA, i.e., to increase efficiency and minimize
the intracellular accumulation of non-functional mRNA
transcripts.
[0129] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one that occurs naturally in Tetrahymena Thermophila (known as the
IVS or L-19 IVS RNA), and which has been extensively described by
Cech and his collaborators (Zaug et al., Science 224:574, 1984;
Zaug et al., Science, 231:470, 1986; Zug et al., Nature 324:429,
1986; PCT Application No. WO 88/04300; and Been et al., Cell
47:207, 1986). The Cech-type ribozymes have an eight base-pair
sequence that hybridizes to a target RNA sequence, whereafter
cleavage of the target RNA takes place. The invention encompasses
those Cech-type ribozymes that target eight base-pair active site
sequences present in Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83.
[0130] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.), and should be delivered to cells which express
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 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 destroy endogenous
messages and inhibit translation. Because ribozymes, unlike
antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
Other Methods for Reducing Tango-71, Tango-73, Tango-74, Tango-76,
and Tango-83 Expression
[0131] Endogenous Tango-71, Tango-73, Tango-74, Tango-76, and
Tango-83 gene expression can also be reduced by inactivating the
endogenous gene or its promoter using targeted homologous
recombination (see, e.g., U.S. Pat. No. 5,464,764). For example, a
mutant, non-functional Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous gene (either the coding regions or
regulatory regions) can be used, with or without a selectable
marker and/or a negative selectable marker, to transfect cells that
express the endogenous gene in vivo. Insertion of the DNA
construct, via targeted homologous recombination, results in
inactivation of the Tango-71, Tango-73, Tango-74, Tango-76, or
Tango-83 gene. Such approaches are particularly suited for use in
the agricultural field where modifications to ES (embryonic stem)
cells can be used to generate animal offspring with an inactive
Tango-71, Tango-73, Tango-74, Tango-76, and Tango-83. However, this
approach can be adapted for use in humans, provided the recombinant
DNA constructs are directly administered or targeted to the
required site in vivo using appropriate viral vectors.
[0132] Alternatively, endogenous Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 gene expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory
region of the gene (i.e., the promoter and/or enhancers) to form
triple helical structures that prevent transcription of the gene in
target cells in the body (Helene Anticancer Drug Res. 6:569, 1981;
Helene et al., Ann. N.Y. Acad. Sci. 660:27, 1992; and Maher,
Bioassays 14:807, 1992).
Detecting Proteins Associated with Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83
[0133] The invention also features polypeptides which interact with
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. Any method
suitable for detecting protein-protein interactions may be employed
for identifying transmembrane proteins, intracellular, or
extracellular proteins that interact with Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83. Among the traditional methods
which may be employed are co-immunoprecipitation, crosslinking and
co-purification through gradients or chromatographic columns of
cell lysates or proteins obtained from cell lysates and the use of
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 to identify
proteins in the lysate that interact with Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83. For these assays, the Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 polypetide can be: a full
length Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83; a
soluble extracellular domain of Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83; or some other suitable Tango-71, Tango-73,
Tango-74, Tango-76, or Tango-83 polypeptide. Once isolated, such an
interacting protein can be identified and cloned and then used, in
conjunction with standard techniques, to identify proteins with
which it interacts. For example, at least a portion of the amino
acid sequence of a protein which interacts with the Tango-71,
Tango-73, Tango-74, Tango-76, or Tango-83 can be ascertained using
techniques well known to those of skill in the art, such as via the
Edman degradation technique. The amino acid sequence obtained may
be used as a guide for the generation of oligonucleotide mixtures
that can be used to screen for gene sequences encoding the
interacting protein. Screening may be accomplished, for example, by
standard hybridization or PCR techniques. Techniques for the
generation of oligonucleotide mixtures and the screening are
well-known (Ausubel, supra; and "PCR Protocols: A Guide to Methods
and Applications," Innis et al., eds. Academic Press, Inc., NY,
1990).
[0134] Additionally, methods may be employed which result directly
in the identification of genes which encode proteins which interact
with Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83. These
methods include, for example, screening expression libraries, in a
manner similar to the well known technique of antibody probing of
.lambda.gt11 libraries, using labeled Tango-71, Tango-73, Tango-74,
Tango-76, or Tango-83 polypeptide or a Tango-71, Tango-73,
Tango-74, Tango-76, and Tango-83 fusion protein, e.g., a Tango-71,
Tango-73, Tango-74, Tango-76, and Tango-83 polypeptide or domain
fused to a marker such as an enzyme, fluorescent dye, a luminescent
protein, or to an IgFc domain.
[0135] There are also methods which are capable of detecting
protein interaction. A method which detects protein interactions in
vivo is the two-hybrid system (Chien et al., Proc. Natl. Acad. Sci.
USA, 88:9578, 1991). A kit for practicing this method is available
from Clontech (Palo Alto, Calif.).
Compounds which bind Tango-71, Tango-73, Tango-74, Tango-76, and
Tango-83
[0136] Compounds which bind Tango-71, Tango-73, Tango-74, Tango-76,
or Tango-83 can be identified using any standard binding assay. For
example, candidate compounds can be bound to a solid support.
Tango-71, Tango-73, Tango-74, Tango-76, or Tango-83 is then exposed
to the immobilized compound and binding is measured (European
Patent Application 84/03564).
EXAMPLES
[0137] Tango-71 cDNA (FIG. 1; SEQ ID NO:1) was isolated from human
melanocytes as follows.
[0138] Human melanocytes (Clonetics Corporation; San Diego, Calif.)
were expanded in culture with Melanocyte Growth Media (MGM;
Clonetics) according to the recommendations of the supplier. When
the cells reached .about.80-90% confluence, they were starved in
MGM without growth factors for 46 hours. The starved cells were
then stimulated with complete MGM supplemented with 20 ng/ml TNF
(Gibco BRL; Gaithersburg, Md.) and cycloheximide (CHI;40
micrograms/ml) for 4 hours. Total RNA was isolated using the RNeasy
Midi Kit (Qiagen; Chatsworth, Calif.), and the poly A+ fraction was
further purified using Oligotex beads (Qiagen).
[0139] Three micrograms of poly A+ RNA were used to synthesize a
cDNA library using the Superscript cDNA Synthesis kit (Gibco BRL).
Complementary DNA was directionally cloned into the expression
plasmid pMET7 using the SalI and NotI sites in the polylinker to
construct a plasmid library. Transformants were picked and grown up
for single-pass sequencing. Additionally, astrocyte cDNA was
ligated into the SalI/NotI sites of the ZipLox vector (Gibco BRL)
for construction of a lambda phage cDNA library.
[0140] Northern blot analysis of Tango-71 expression was performed
using Tango-71 labeled with .sup.32P-dCTP using the Prime-It kit
(Stratagene, LaJolla, Calif.). Human mRNA blots (MTNI and MTNII;
Clonetech; Palo Alto, Calif.) were probed and washed at high
stringency as recommended by the manufacturer. Tango-71 is
expressed as an approximately 6.0 kb transcript in all tissues:
heart brain, placenta, lung, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testes, ovary, small intestine,
colon, PBLs.
[0141] The amino acid sequence of a portion of Tango-71 is 90%
identical to the amino acid sequence of murine ADAMTS-1 (FIG. 9), a
cellular disintegrin and metalloprotease that is thought to be
involved in inflammation and development of cancer cachexia (Kuno
et al., J. Biol. Chem. 272:556, 1997). Based on sequence comparison
to ADAMTS-1, Tango-71, using the amino acid numbering in FIG. 9,
has the following domains: amino acids 1-160 (metalloproteinase
domain, partial); amino acids 170-242 (disintegrin domain); amino
acids 257-307 (thrombospondin domain). A less apparent
thrombospondin domain is present at amino acid 558-608. Portions of
Tango-71 shown in FIG. 1, but not in FIG. 9, may also be homologous
ADAMTS-1. Tango-71 may represent the human homolog of ADAMTS-1 or a
splice variant thereof.
[0142] Tango-71 expression may be androgen regulated. Tango-71
expression in LNCaP cells, an androgen-dependent prostate cancer
cell line, is induced by R1881, a testosterone analog. Tango-71
expression is downregulated in LNCaP cells treated with casodex, an
anti-androgen.
[0143] Tango-73 cDNA (FIG. 2; SEQ ID NO:2) was isolated from human
prostate epithelial cells as follows.
[0144] Human prostate epithelial cells (Clonetics) were expanded in
culture with Prostate Epithelial Growth Medium (PEGM) (Clonetics).
When cells reached confluence cells were grown in Prostate Basal
Media (Clonetics) for 24 hours. They were stimulated with PEGM
(prostate epithelial growth medium; Clonetics) and 40 ug/ml
cycloheximide for 3 hours.
[0145] Total RNA was isolated using the RNeasy Midi Kit (Qiagen).
Poly (A)+ was isolated using the Oligotex beads (Qiagen). Next,
cDNA was constructed using the Superscript cDNA Synthesis Kit
(Gibco BRL). The cDNA was cloned into the expression vector pMET7
using the SalI and NotI sites in the polylinker. Transformants were
picked and sequenced.
[0146] Northern blot analysis of Tango-73 expression was carried
out as described above. This analysis revealed the presence of 4.0
kb and 3.0 kb transcripts in the placenta and liver. A 4.0 kb
transcript was present in lung, kidney, thymus, prostate, spleen,
testes, and colon, with the highest expression in lung, pancreas,
prostate, and testes.
[0147] The amino acid sequence of Tango-73 is 48% identical to rat
RVP.1 (Briehl et al., Mol. Endocrinol. 5:1381, 1991) and 46.1%
identical to TMVCF (Sirotkin et al., Genomics 42:245, 1997).
[0148] RVP.1 is up-regulated during apoptosis (Briehl et al.,
supra). TMVCF, a 219 amino acid protein with two putative membrane
spanning domains, is deleted in velo-cardio-facial syndrome
(Sirotkin et al., supra).
[0149] Tango-83 (FIGS. 7 and 8) and Tango-74 cDNAs (FIG. 5) were
isolated from human astrocytes as follows.
[0150] Human astrocytes (Clonetics) were expanded in culture with
Astocyte Growth Media (AGM; Clonetics) according to the
recommendations of the supplier. When the cells reached -80-90%
confluence, they were stimulated with 200 units/ml Interleukin
1-Beta (Boehringer Mannheim) and cycloheximide (CHI: 40
micrograms/ml) for 4 hours. Total RNA was isolated using the RNeasy
Midi Kit (Qiagen), and the poly A+ fraction was further purified
using Oligotex beads (Qiagen).
[0151] Three micrograms of poly A+ RNA were used to synthesize a
cDNA library using the Superscript cDNA Synthesis kit (Gibco BRL).
Complementary DNA was directionally cloned into the expression
plasmid pMET7 using the SalI and NotI sites in the polylinker to
construct a plasmid library. Transformants were picked and grown up
for single-pass sequencing. Additionally, astrocyte cDNA was
ligated into the SalI/NotI sites of the ZipLox vector (Gibco BERL)
for construction of a lambda phage cDNA library.
[0152] Northern blot analysis of Tango-83 expression, performed as
described above, revealed that Tango-83 is expressed as an
approximately 9.0 kb transcript in brain (FIG. 13).
[0153] Northern blot analysis, performed as described above,
revealed that Tango-74 is expressed as an approximately 4.0 kb
transcript in heart, brain, lung, liver, kidney, pancreas, spleen,
prostate, testes, ovary, small intestine, colon and peripheral
blood lymphocytes. Higher expression was seen in lung, liver,
skeletal muscle, spleen, testes, colon and peripheral blood
lymphocytes.
[0154] The amino acid sequence of Tango-74 is homologous to the
amino acid sequence of the TRAIL receptor (Pan et al., Science
276:111, 1997) (FIG. 5).
[0155] Tango-76 cDNA (SEQ ID NO:7) was isolated form an adult rat
frontal cortex library. The amino acid sequence of Tango-76 is
homologous to the amino acid sequence of ADAMTS-1 (FIG. 14).
[0156] Northern blot analysis of human mRNA probed with a Tango-76
probe revealed a 4.2 kb band in lung. Analysis of rat mRNA revealed
a weak 3.8 kb transcript in heart, brain, spleen, liver, skeletal
muscle, and kidney and a weak 1.8 kb transcript in spleen and
liver.
[0157] Effective Dose
[0158] Toxicity and therapeutic efficacy of the polypeptides of the
invention and the compounds that modulate their expression or
activity can be determined by standard pharmaceutical procedures,
using either cells in culture or experimental animals to determine
the LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50. Polypeptides or other compounds that exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0159] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (that is, the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0160] Formulations and Use
[0161] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0162] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0163] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (for example, pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(for example, lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (for example, magnesium stearate,
talc or silica); disintegrants (for example, potato starch or
sodium starch glycolate); or wetting agents (for example, sodium
lauryl sulphate). The tablets may be coated by methods well known
in the art. Liquid preparations for oral administration may take
the form of, for example, solutions, syrups or suspensions, or they
may be presented as a dry product for constitution with water or
other suitable vehicle before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable
additives such as suspending agents (for example, sorbitol syrup,
cellulose derivatives or hydrogenated edible fats); emulsifying
agents (for example, lecithin or acacia); non-aqueous vehicles (for
example, almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (for example, methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated to give controlled release of the active
compound.
[0164] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0165] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, for example,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, for example, gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0166] The compounds may be formulated for parenteral
administration by injection, for example, by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, for example, in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, for example, sterile pyrogen-free water, before
use.
[0167] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, for example, containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0168] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0169] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0170] The therapeutic compositions of the invention can also
contain a carrier or excipient, many of which are known to skilled
artisans. Excipients which can be used include buffers (for
example, citrate buffer, phosphate buffer, acetate buffer, and
bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, proteins (for example, serum albumin), EDTA, sodium
chloride, liposomes, mannitol, sorbitol, and glycerol. The nucleic
acids, polypeptides, antibodies, or modulatory compounds of the
invention can be administered by any standard route of
administration. For example, administration can be parenteral,
intravenous, subcutaneous, intramuscular, intracranial,
intraorbital, opthalmic, intraventricular, intracapsular,
intraspinal, intracisternal, intraperitoneal, transmucosal, or
oral. The modulatory compound can be formulated in various ways,
according to the corresponding route of administration. For
example, liquid solutions can be made for ingestion or injection;
gels or powders can be made for ingestion, inhalation, or topical
application. Methods for making such formulations are well known
and can be found in, for example, "Remington's Pharmaceutical
Sciences." It is expected that the preferred route of
administration will be intravenous.
* * * * *
References