U.S. patent application number 10/366445 was filed with the patent office on 2003-08-21 for human tissue inhibitor of metalloproteinase-4.
Invention is credited to Greene, John M., Rosen, Craig A..
Application Number | 20030157687 10/366445 |
Document ID | / |
Family ID | 46277851 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157687 |
Kind Code |
A1 |
Greene, John M. ; et
al. |
August 21, 2003 |
Human tissue inhibitor of metalloproteinase-4
Abstract
A human tissue inhibitor of metalloproteinases-4 polypeptide and
DNA (RNA) encoding such polypeptide and a procedure for producing
such polypeptide by recombinant techniques. Also disclosed are
methods for utilizing such polypeptide for the treatment of
diseases, including arthritis and cancer. Antagonists against such
polypeptides and their use as a therapeutic to resorb scar tissue
are also disclosed. Diagnostic assays for detecting levels of human
TIMP-4 protein and mutations in human TIMP-4 nucleic acid sequence
are also disclosed.
Inventors: |
Greene, John M.;
(Gaithersburg, MD) ; Rosen, Craig A.;
(Laytonsville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
9410 KEY WEST AVENUE
ROCKVILLE
MD
20850
|
Family ID: |
46277851 |
Appl. No.: |
10/366445 |
Filed: |
February 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10366445 |
Feb 14, 2003 |
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09901904 |
Jul 11, 2001 |
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6544761 |
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09901904 |
Jul 11, 2001 |
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09387525 |
Sep 1, 1999 |
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09387525 |
Sep 1, 1999 |
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08463261 |
Jun 5, 1995 |
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6448042 |
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08463261 |
Jun 5, 1995 |
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PCT/US94/14498 |
Dec 13, 1994 |
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60217419 |
Jul 11, 2000 |
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60220829 |
Jul 26, 2000 |
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Current U.S.
Class: |
435/226 ;
424/93.2; 424/94.65; 435/320.1; 435/325; 435/6.16; 435/69.1;
435/7.1; 536/23.2 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2799/026 20130101; C07K 14/8146 20130101 |
Class at
Publication: |
435/226 ;
435/69.1; 435/320.1; 435/325; 435/6; 435/7.1; 424/93.2; 424/94.65;
536/23.2 |
International
Class: |
C12Q 001/68; G01N
033/53; C07H 021/04; A61K 048/00; A61K 038/46; C12N 009/64 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide encoding the polypeptide
as set forth in SEQ ID NO:2; (b) a polynucleotide capable of
hybridizing to and which is at least 70% identical to the
polynucleotide of (a); and (c) a polynucleotide fragment of the
polynucleotide of (a) or (b).
2. The polynucleotide of claim 1 wherein the polynucleotide is
DNA.
3. The polynucleotide of claim 2 which encodes the polypeptide as
set forth in SEQ ID NO:2.
4. The polynucleotide of claim 2 which encodes the polypeptide
comprising amino acid 1 to 195 of of SEQ ID NO:2.
5. The polynucleotide of claim 2 comprising the nucleotide sequence
of SEQ ID NO:1 from nucleotide 1 to 675.
6. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide which encodes a mature
polypeptide encoded by the DNA contained in ATCC Deposit No. 75946;
(b) a polynucleotide which encodes a polypeptide expressed by the
DNA contained in ATCC Deposit No. 75946; (c) a polynucleotide
capable of hybridizing to and which is at least 70% identical to
the polynucleotide of (a) or (b); and (d) a polynucleotide fragment
of the polynucleotide of (a), (b) or (c).
7. A vector containing the DNA of claim 2.
8. A host cell genetically engineered with the vector of claim
7.
9. A process for producing a polypeptide comprising: expressing
from the host cell of claim 8 the polypeptide encoded by said
DNA.
10. A process for producing cells capable of expressing a
polypeptide comprising transforming or transfecting the cells with
the vector of claim 7.
11. A polypeptide comprising a member selected from the group
consisting of (i) a polypeptide having the deduced amino acid
sequence of SEQ ID NO:2 and fragments, analogs and derivatives
thereof; (ii) a polypeptide comprising amino acid 1 to amino acid
195 of SEQ ID NO:2; and (iii) a polypeptide encoded by the cDNA of
ATCC Deposit No. 75946 and fragments, analogs and derivatives of
said polypeptide.
12. A compound effective as an agonist for the polypeptide of claim
11.
13. A compound effective as an antagonist against the polypeptide
of claim 11.
14. A method for the treatment of a patient having need of TIMP-4
comprising: administering to the patient a therapeutically
effective amount of the polypeptide of claim 11.
15. The method of claim 14 wherein said therapeutically effective
amount of the polypeptide is administered by providing to the
patient DNA encoding said polypeptide and expressing said
polypeptide in vivo.
16. A method for the treatment of a patient having need of TIMP-4
comprising: administering to the patient a therapeutically
effective amount of the compound of claim 12.
17. A method for the treatment of a patient having need to inhibit
TIMP-4 comprising: administering to the patient a therapeutically
effective amount of the antagonist of claim 13.
18. A process for identifying compounds active as agonists or
antagonists to the polypeptide of claim 11 comprising: combining an
MMP, human TIMP-4, a compound to be screened and a reaction mixture
containing substrate capable of degradation by the MMP, wherein
said substrate is labeled; and determining the ability of the
compound to enhance or block the degradation of the substrate by
said MMP by measuring the label released from the substrate.
19. A process for diagnosing a disease or a susceptibility to a
disease related to a mutation in the polypeptide of claim 11
comprising: determining a mutation in the human TIMP-4 nucleic acid
sequence.
20. A diagnostic process comprising: analyzing for the presence of
the polypeptide of claim 11 in a sample derived from a host.
21. A method of treating restenosis in a patient, comprising
administering to the patient an isolated nucleic acid molecule
encoding a TIMP-4 polypeptide selected from the group consisting
of: (a) the amino acid sequence shown as residues -29 to 195 in SEQ
ID NO:2; (b) the amino acid sequence shown as residues -28 to 195
in SEQ ID NO:2; (c) the amino acid sequence shown as residues 1 to
195 in SEQ ID NO:2; and (d) a fragment of the sequence described in
(a) whereing a polypeptide consisting of the fragment retains
protease inhibiting activity; wherein said nucleic acid molecule is
operatively linked to a transcription control sequence; and wherein
the expression of said nucleic acid molecule results in an
increased amount of the TIMP-4 polypeptide in an amount effective
to inhibit metalloproteinase activity.
22. The method of claim 21, wherein said isolated nucleic
acidmolecule is administered to said patient in a viral vector
delivery vehicle.
23. The method of claim 22, wherein said viral vector delivery
vehicle is from adenovirus.
Description
[0001] This application is a Divisional of U.S. application Ser.
No. 09/901,904, filed Jul. 11, 2001, which is a
Continuation-in-Part of U.S. application Ser. No. 09/387,525, filed
Sep. 1, 1999, which is a Continuation of U.S. application Ser. No.
08/463,261, filed Jun. 5, 1995, which is a continuation-in-part of
PCT/US94/14498, filed Dec. 13, 1994 (filed in English), each of
which are hereby incorporated by reference in their entireties;
U.S. application Ser. No. 09/901,904 also claims benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/217,419,
filed Jul. 11, 2000, and No. 60/220,829, filed Jul. 26, 2000, each
of which are hereby incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides, polypeptides, and antibodies, as well as the
production of such polynucleotides and polypeptides. More
particularly, the polypeptides of the present invention are human
tissue inhibitor of metalloproteinase-4 polypeptides, hereinafter
referred to as "human TIMP-4". The invention also relates to
inhibiting the action of such polypeptides.
BACKGROUND OF THE INVENTION
[0003] The extracellular matrix is a complex structure that
contains collagen, proteoglycan, glycosaminoglycan, glycoproteins
(fibronectin, chondronectin, laminin) and in some tissues, elastin
(Hay, E. D., J. Cell Biol., 91:205-223 (1981)).
[0004] Matrix metalloproteinases (MMP's) constitute the major group
of zinc-binding endopeptidases that degrade extracellular matrix
proteins, for example connective tissue, collagen and gelatin,
during remodeling of connective tissue during normal physiological
and some pathological processes. The unrestrained activity of MMP's
may result in extensive tissue damage, and these enzymes have been
implicated in a variety of disease processes, including tumor cell
invasion, tumor angiogenesis and rheumatoid arthritis (Okada, Y.,
et al., J. Biol. Chem., 261:14245-14255 (1986)). The MMP's are
secreted from cells as inactive zymogens and their activity in the
extracellular environment is regulated by various activators and
inhibitors (Matrisian, L. M., Trends Genet., 6:121-125 (1990)).
[0005] Regulation of metalloproteinase-mediated proteolysis may
occur by naturally occurring inhibitor proteins, such as tissue
inhibitor of metalloproteinase (TIMP). The balance between the
production and activation of the MMP's, and their inhibition by
natural inhibitors such as TIMP, determines, in both physiological
and pathological conditions, whether connective tissue is
degraded.
[0006] MMP's include a number of proteases, exemplified by
interstitial (type I) collagenase itself, the stromelysins (also
known as proteoglycanases or transins), fibroblast and
polymorphonuclear leukocyte gelatinases (also known as
collagen-IV-ases), and pump-1 (putative metalloproteases 1, uterine
metalloproteases) [Goldberg et al, J. Biol. Chem. 2610:6600 (1986);
Whitham et al, Biochem. J. 240:913 (1986); Breathnach et al,
Nucleic Acids Res., 15:1139 (1987); Muller et al, Biochem. J.,
253:187 (1988); Collier et al, J. Biol. Chem., 263:6579 (1988);
Murphy et al, Biochem. J., 258:463 (1989); Quantin et al, Biochem.
(N.Y.), 28:5327 (1989); Birkedal-Hansen, J. Oral Pathol., 17:445
(1988)].
[0007] In general, the mammalian family of proteases has one or
more of the following properties: (a) optimal proteolytic activity
around neutral pH; (b) dependence of the enzyme's activity on the
presence of zinc, as evident by the loss of activity on treatment
with divalent metal ion chelators, such as 1.10 phenanthroline
(preferential chelation of zinc), or EDTA (less restricted
chelating properties; EDTA and EGTA also contribute to enzyme
inactivation via chelation of calcium ions required for enzyme
stability); (c) inhibition by TIMPs; (d) absence of significant
inhibition by known inhibitors of other families of neutral,
zinc-containing metalloproteases, such as thermolysis,
angiotensin-converting enzyme and `enkephalinases`; and (e)
biosynthesis and secretion as latent precursor forms (zymogens),
requiring extracellular activation. Activation has been achieved by
a number of endoproteases, organomercurials and chaotropic
agents.
[0008] In general, members of the family of neutral metalloprotease
enzymes have distinctive substrate specificities. Thus, collagenase
type I is unique in its ability to cleave a specific peptide bond
within the natural fibrils of the interstitial collagens (e.g.
types I, II and III). The gelatinases are only poorly active on
these collagens, but are able to degrade denatured interstitial
collagens, as well as the non-fibrillar collagens, e.g. type IV,
such as are found in the basement membrane. Pump 1 has been
reported to act preferentially on denatured collagens (gelatins),
though its profile differs from that of the stromelysins or the
collagenases type IV. Both the stromelysins and the gelatinases are
also capable of degrading non-collagenous structural proteins, such
as the core protein of proteoglycan and elastin. Macromolecules
involved in cell-to-substratum and cell-to-cell interactions, such
as laminin and fibronectin, are also susceptible to degradation by
several of these metalloproteases.
[0009] Enzymes of this family are produced by synovial and skin
fibroblasts, chondrocytes, peripheral mononuclear cells,
keratinocytes and gingival tissue, as well as existing within
granule storage vesicles in polymorphonuclear leukocytes
(PMNLs).
[0010] Current information suggests that there is a family of
metalloproteinase inhibitors which comprises TIMP-1 (tissue
inhibitor of metalloproteinases-l); TIMP-2; human TIMP-3 which has
been cloned, expressed and mapped to human chromosome 22; and
chicken tissue inhibitor of metalloproteinase (ChIMP-5). The
polypeptide of the present invention has been putatively identified
as a novel human TIMP polypeptide based on amino acid sequence
homology.
SUMMARY OF THE INVENTION
[0011] In accordance with one aspect of the present invention,
there is provided a novel mature polypeptide which is human TIMP-4,
as well as biologically active and diagnostically or
therapeutically useful fragments, analogs and derivatives
thereof.
[0012] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding human
TIMP-4, including mRNA's, DNA's, cDNA's, genomic DNA as well as
biologically active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof.
[0013] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptide by recombinant techniques which comprises culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
human TIMP-4 nucleic acid sequence under conditions promoting
expression of protein and subsequent recovery of said protein.
[0014] In accordance with yet a further aspect of the present
invention, there is provided a method for treating conditions which
are related to insufficient human TIMP-4 activity which comprises
administering to a patient in need thereof a pharmaceutical
composition containing the human TIMP-4 protein of the invention
which is effective to supplement a patient's endogenous human
TIMP-4 and thereby alleviate said conditions which include, for
example, arthritic diseases such as rheumatoid and osteoarthritis,
soft tissue rheumatism, polychondritis and tendonitis; bone
resorption diseases, such as osteoporosis, Paget's disease,
hyperparathyroidism and cholesteatoma; the enhanced collagen
destruction that occurs in association with diabetes; the recessive
classes of dystrophic epidermolysis bullosa; periodontal disease,
alveolitis and related consequences of gingival production of
collagenase; corneal ulceration; ulceration of the skin and
gastro-intestinal tract and abnormal wound healing; post-operative
conditions in which collagenase levels are raised; cancer by
blocking the destruction of tissue basement membranes leading to
cancer metastasis; demyelinating diseases of the central and
peripheral nervous systems; asthma; glomerulosclerosis; septic
shock and infection; and psoriasis.
[0015] In accordance with yet a further aspect of the present
invention, there is provided a method for treating or preventing
restenosis, which comprises administering to a patient in need
thereof a pharmaceutical composition containing the human TIMP-4
protein of the invention which is effective to treat or prevent
restenosis.
[0016] In accordance with yet a further aspect of the present
invention, there is provided an antibody against such
polypeptides.
[0017] In accordance with yet another aspect of the present
invention, there are provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to specifically
hybridize to human TIMP-4 sequences.
[0018] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides
which may be employed for therapeutic purposes, for example, for
remodeling and repairing tissue and for destruction of scar
tissue.
[0019] In accordance with another aspect of the present invention,
there are provided diagnostic assays for detecting diseases related
to mutations in human TIMP-4 sequences and over-expression of the
polypeptide.
[0020] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
[0021] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
BRIEF DESECRIPTION OF THE FIGURES
[0022] FIGS. 1A-B shows the cDNA sequence and corresponding deduced
amino acid sequence of the full-length human TIMP-4 polypeptide.
The standard one-letter abbreviations for amino acids are used.
Sequencing was performed using a 373 Automated DNA sequencer
(Applied Biosystems, Inc.). Sequencing accuracy is predicted to be
greater than 97% accurate.
[0023] FIGS. 2A-B is an amino acid sequence comparison between the
polypeptide of the present invention and other human TIMP
polypeptides.
[0024] FIGS. 3A-F shows the adenoviral plasmid maps used in the
gene therapy experiments described in Example 7.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] In accordance with an aspect of the present invention, there
is provided an isolated nucleic acid (polynucleotide) which encodes
for the mature polypeptide having the deduced amino acid sequence
of FIGS. 1A-B or for the mature polypeptide encoded by the cDNA of
the clone deposited as ATCC Deposit No. 75946 on Nov. 11, 1994.
[0026] A polynucleotide encoding a polypeptide of the present
invention may be obtained from an early stage human brain. This
contains an open reading frame and coding of protein of 224 amino
acid residues of which approximately the first 29 residues
represent the leader sequence such that the mature protein
comprises 195 amino acid residues. The polynucleotide of this
invention was discovered in a cDNA library derived from an early
stage human brain. The protein exhibits the highest degree of
homology to Human TIMP-2 with 48% identity and 72% similarity over
a 136 amino acid stretch. Human TIMP-4 has the signature 12
cysteine amino acids, which are conserved in all members of the
TIMP family. The 12 cysteine residues are all disulfide-linked in
TIMP-1 and TIMP-2. This evidence strongly suggests that the
polypeptide of the present invention is a novel member of the TIMP
family.
[0027] The polynucleotide of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown in FIGS. 1A-B or that of the deposited clone or may be a
different coding sequence which coding sequence, as a result of the
redundancy or degeneracy of the genetic code, encodes the same,
mature polypeptide as the DNA of FIGS. 1A-B or the deposited
cDNA.
[0028] The polynucleotide which encodes for the mature polypeptide
of FIGS. 1A-B or for the mature polypeptide encoded by the
deposited cDNA may include: only the coding sequence for the mature
polypeptide; the coding sequence for the mature polypeptide and
additional coding sequence such as a leader or secretory sequence
or a proprotein sequence; the coding sequence for the mature
polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequence 5'
and/or 3' of the coding sequence for the mature polypeptide.
[0029] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0030] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of FIGS. 1A-B or the polypeptide encoded by the cDNA
of the deposited clone. The variant of the polynucleotide may be a
naturally occurring allelic variant of the polynucleotide or a
non-naturally occurring variant of the polynucleotide.
[0031] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in FIGS. 1A-B or the
same mature polypeptide encoded by the cDNA of the deposited clone
as well as variants of such polynucleotides which variants encode
for a fragment, derivative or analog of the polypeptide of FIGS.
1A-B or the polypeptide encoded by the cDNA of the deposited clone.
Such nucleotide variants include deletion variants, substitution
variants and addition or insertion variants.
[0032] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence shown in FIGS. 1A-B or of the coding sequence
of the deposited clone. As known in the art, an allelic variant is
an alternate form of a polynucleotide sequence which may have a
substitution, deletion or addition of one or more nucleotides,
which does not substantially alter the function of the encoded
polypeptide.
[0033] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a leader sequence which functions as a secretory sequence
for controlling transport of a polypeptide from the cell. The
polypeptide having a leader sequence is a preprotein and may have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides may also encode for a
proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein remains. Thus, for
example, the polynucleotide of the present invention may encode for
a mature protein, or for a protein having a prosequence or for a
protein having both a prosequence and a presequence (leader
sequence).
[0034] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell, 37:767 (1984)).
[0035] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0036] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promotor regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0037] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 70%, preferably at least 90%, and more preferably at least
95% identity between the sequences. The present invention
particularly relates to polynucleotides which hybridize under
stringent conditions to the hereinabove-described polynucleotides.
As herein used, in one embodiment, the term "stringent conditions"
means hybridization will occur only if there is at least 95% and
preferably at least 97% identity between the sequences. The
polynucleotides which hybridize to the hereinabove described
polynucleotides in a preferred embodiment encode polypeptides which
either retain substantially the same biological function or
activity as the mature polypeptide encoded by the cDNAs of FIGS.
1A-B (SEQ ID NO:1) or the deposited cDNA(s). In an alternative
embodiment, by "stringent hybridization conditions" is intended
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0038] Alternatively, the polynucleotide may have at least 20
bases, preferably 30 bases, and more preferably at least 50 bases
which hybridize to a polynucleotide of the present invention and
which has an identity thereto, as hereinabove described, and which
may or may not retain activity. For example, such polynucleotides
may be employed as probes for the polynucleotide of SEQ ID NO:1,
for example, for recovery of the polynucleotide or as a diagnostic
probe or as a PCR primer.
[0039] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 80%, at least
85%, at least 90%, and more preferably at least a 95% identity, at
least a 96% identity, at least a 97% identity, at least a 98%
identity, or at least a 99% identity, to a polynucleotide which
encodes the polypeptide of SEQ ID NO:2 as well as fragments
thereof, which fragments have at least 30 bases and preferably at
least 50 bases and to polypeptides encoded by such polynucleotides.
The deposit(s) referred to herein will be maintained under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for purposes of Patent Procedure.
These deposits are provided merely as convenience to those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn.112. The sequence of the polynucleotides
contained in the deposited materials, as well as the amino acid
sequence of the polypeptides encoded thereby, are incorporated
herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such
license is hereby granted.
[0040] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" or "identity" to a reference
nucleotide sequence encoding a TIMP-4 polypeptide is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five mismatches per each 100 nucleotides of the
reference nucleotide sequence encoding the TIMP-4 polypeptide. In
other words, to obtain a polynucleotide having a nucleotide
sequence at least 95% identical to a reference nucleotide sequence,
up to 5% of the nucleotides in the reference sequence may be
deleted or substituted with another nucleotide, or a number of
nucleotides up to 5% of the total nucleotides in the reference
sequence may be inserted into the reference sequence. These
mutations of the reference sequence may occur at the 5' or 3'
terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. The reference
(query) sequence may be the entire nucleotide sequence encoding
TIMP-4, as shown in FIGS. 1A and 1B (SEQ ID NO:1) or any TIMP-4
polynucleotide sequence described herein.
[0041] As a practical matter, whether any particular nucleic acid
molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for instance, the nucleotide sequences shown in FIGS.
1A and 1B, or to the cDNA sequence of the deposited cDNA clone, or
fragments thereof, can be determined conventionally using known
computer programs such as the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711).
Bestfit uses the local homology algorithm of Smith and Waterman to
find the best segment of homology between two sequences (Advances
in Applied Mathematics 2:482-489 (1981)). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed.
[0042] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245
(1990)). In a sequence alignment the query and subject sequences
are both DNA sequences. An RNA sequence can be compared by
converting U's to T's. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB
alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject
nucleotide sequence, whichever is shorter. According to this
embodiment, if the subject sequence is shorter than the query
sequence because of 5' or 3' deletions, not because of internal
deletions, a manual correction is made to the results to take into
consideration the fact that the FASTDB program does not account for
5' and 3' truncations of the subject sequence when calculating
percent identity. For subject sequences truncated at the 5' or 3'
ends, relative to the query sequence, the percent identity is
corrected by calculating the number of bases of the query sequence
that are 5' and 3' of the subject sequence, which are not
matched/aligned, as a percent of the total bases of the query
sequence. A determination of whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this
embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score. For
example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the
5' end of the subject sequence and therefore, the FASTDB alignment
does not show a matched/alignment of the first 10 bases at 5' end.
The 10 unpaired bases represent 10% of the sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in
the query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90 bases
were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100
base query sequence. This time the deletions are internal deletions
so that there are no bases on the 5' or 3' of the subject sequence
which are not matched/aligned with the query. In this case the
percent identity calculated by FASTDB is not manually corrected.
Once again, only bases 5' and 3' of the subject sequence which are
not matched/aligned with the query sequence are manually corrected
for. No other manual corrections are made for the purposes of this
embodiment.
[0043] The present application is directed to nucleic acid
molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the nucleic acid sequences (i.e., polynucleotides)
disclosed herein (e.g., those disclosed in FIGS. 1A and 1B (SEQ ID
NO:1) or to the cDNA sequence of the deposited clone), irrespective
of whether they encode a polypeptide having TIMP-4 functional
activity (e.g., biological activity). This is because even where a
particular nucleic acid molecule does not encode a polypeptide
having TIMP-4 activity, one of skill in the art would still know
how to use the nucleic acid molecule, for instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer.
Uses of the nucleic acid molecules of the present invention that do
not encode a polypeptide having TIMP-4 activity include, inter
alia, (1) isolating the TIMP-4 gene or allelic variants thereof in
a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal spreads to provide precise chromosomal
location of the TIMP-4 gene, as described in Verma et al., Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
(1988); and Northern Blot analysis for detecting TIMP-4 mRNA
expression in specific tissues.
[0044] Preferred, however, are nucleic acid molecules having
sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to the nucleic acid sequences disclosed herein (e.g., the
nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) and the
cDNA sequence of the deposited clone, or fragments thereof), which
do, in fact, encode a polypeptide having TIMP-4 polypeptide
functional activity (e.g., biological activity).
[0045] By "a polypeptide having TIMP-4 functional activity" (e.g.,
biological activity) is intended polypeptides exhibiting activity
similar, but not necessarily identical, to an activity of TIMP-4
polypeptides of the invention, as measured in a particular
functional assay. TIMP-4 "functional activities include, but are
not limited to, biological activity (e.g., ability to inhibit
metalloproteinase activity, ability to inhibit the proliferation of
cardiac smooth muscles, ability to inhibit the formation of the
inner lining (neotima) of the carotid artery following balloon
angioplasty injury), antigenicity [ability to bind (or compete with
a TIMP-4 polypeptide for binding) to an anti-TIMP-4 antibody,],
immunogenicity (ability to generate antibody which binds to a
TIMP-4 polypeptide), and ability to bind to a TIMP-4
receptor/ligand. Techniques known in the art may be applied to
routinely determine if polypeptides of the invention exhibit TIMP-4
functional activities (e.g., biological activity (e.g., ability to
inhibit metalloproteinases (e.g., metalloproteinase 1, 2, 3, 7, and
9))).
[0046] In specific embodiments, the polynucleotides of the
invention comprise, or alternatively consist of, a polynucleotide
sequence that is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, contiguous
nucleotides of SEQ ID NO:1. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0047] In specific embodiments, the polynucleotides of the
invention comprise, or alternatively consist of, a polynucleotides
sequence encoding a polypeptide sequence that is at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200, contiguous amino acids of SEQ ID NO:2.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
[0048] In specific embodiments, the polynucleotides of the
invention comprise, or alternatively consist of, a nucleotide
sequence encoding a polypeptide sequence selected from the group:
(a) a polypeptide having the amino acid sequence of amino acids 22
to 28 of SEQ ID NO:2; and (b) a polypeptide having the amino acid
sequence of amino acids 34 to 40 of SEQ ID NO:2. Polypeptides
encoded by these polynucleotides are also encompassed by the
invention.
[0049] In specific embodiments, the polynucleotides of the
invention comprise, or alternatively consist of, a nucleotide
sequence encoding a polypeptide sequence selected from the group:
(a) a polypeptide having the amino acid sequence of amino acids 1
to 72 of SEQ ID NO:2; (b) a polypeptide having the amino acid
sequence of amino acids 73 to 127 of SEQ ID NO:2; (c) a polypeptide
having the amino acid sequence of amino acids 128 to 176 of SEQ ID
NO:2; and (d) a polypeptide having the amino acid sequence of amino
acids 1 to 176 of SEQ ID NO:2. Polypeptides encoded by these
polynucleotides are also encompassed by the invention.
[0050] In specific embodiments, the polynucleotides of the
invention comprise, or alternatively consist of, a nucleotide
sequence encoding a polypeptide sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide
sequence selected from the group: (a) a polypeptide having the
amino acid sequence of amino acids 1 to 72 of SEQ ID NO:2; (b) a
polypeptide having the amino acid sequence of amino acids 73 to 127
of SEQ ID NO:2; (c) a polypeptide having the amino acid sequence of
amino acids 128 to 176 of SEQ ID NO:2; and (d) a polypeptide having
the amino acid sequence of amino acids 1 to 176 of SEQ ID NO:2.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
[0051] The present invention further relates to a human TIMP-4
polypeptide which has the deduced amino acid sequence of FIGS. 1A-B
or which has the amino acid sequence encoded by the deposited cDNA,
as well as fragments, analogs and derivatives of such
polypeptide.
[0052] The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of FIGS. 1A-B or that encoded by the
deposited cDNA, means a polypeptide which retains essentially the
same biological function or activity as such polypeptide. Thus, an
analog includes a proprotein which can be activated by cleavage of
the proprotein portion to produce an active mature polypeptide.
[0053] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
[0054] The fragment, derivative or analog of the polypeptide of
FIGS. 1A-B or that encoded by the deposited cDNA may be (i) one in
which one or more of the amino acid residues are substituted with a
conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as a leader or secretory sequence or a
sequence which is employed for purification of the mature
polypeptide or a proprotein sequence. Such fragments, derivatives
and analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0055] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0056] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0057] In specific embodiments, the polypeptides of the invention
comprise, or alternatively consist of, an amino acid sequence that
is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, or 200, contiguous amino acids of SEQ
ID NO:2. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0058] In specific embodiments, the polypeptides of the invention
comprise, or alternatively consist of, a polypeptide selected from
the group: (a) a polypeptide having the amino acid sequence of
amino acids 1 to 72 of SEQ ID NO:2; (b) a polypeptide having the
amino acid sequence of amino acids 73 to 127 of SEQ ID NO:2; (c) a
polypeptide having the amino acid sequence of amino acids 128 to
176 of SEQ ID NO:2; and (d) a polypeptide having the amino acid
sequence of amino acids 1 to 176 of SEQ ID NO:2. Poynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0059] In specific embodiments, the polypeptides of the invention
comprise, or alternatively consist of, a polypeptide selected from
the group: (a) a polypeptide having the amino acid sequence of
amino acids 22 to 28 of SEQ ID NO:2; and (b) a polypeptide having
the amino acid sequence of amino acids 34 to 40 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0060] Preferred polypeptide fragments of the invention include the
secreted protein as well as the mature form. Further preferred
polypeptide fragments include the secreted protein or the mature
form having a continuous series of deleted residues from the amino
or the carboxy terminus, or both. Poynucleotides encoding these
polypeptides are also encompassed by the invention.
[0061] Accordingly, polypeptide fragments include the secreted
TIMP-4 protein as well as the mature form. Further preferred
polypeptide fragments include the secreted TIMP-4 protein or the
mature form having a continuous series of deleted residues from the
amino or the carboxy terminus, or both. For example, any number of
amino acids, ranging from 1-29 of the TIMP-4 sequence disclosed in
FIGS. 1A-B, can be deleted from the amino terminus of either the
secreted TIMP-4 polypeptide or the mature form. Similarly, any
number of amino acids, ranging from 1-30 of the TIMP-4 sequence
disclosed in FIGS. 1A-B, can be deleted from the carboxy terminus
of the secreted TIMP-4 protein or mature form. Furthermore, any
combination of the above amino and carboxy terminus deletions are
preferred. Similarly, polynucleotides encoding these polypeptide
fragments are also preferred. Particularly, N-terminal deletions of
the TIMP-4 polypeptide can be described by the general formula
m-224, where m is an integer from 2-218, where m corresponds to the
position of the amino acid residue identified in FIGS. 1A-B. More
in particular, the invention provides polypeptides comprising, or
alternatively consisting of, an amino acid sequence selected from:
P-2 to P-224; G-3 to P-224; S-4 to P-224; P-5 to P-224; R-6 to
P-224; P-7 to P-224; A-8 to P-224; P-9 to P-224; S-10 to P-224;
W-11 to P-224; V-12 to P-224; L-13 to P-224; L-14 to P-224; L-15 to
P-224; R-16 to P-224; L-17 to P-224; L-18 to P-224; A-19 to P-224;
L-20 to P-224; L-21 to P-224; R-22 to P-224; P-23 to P-224; P-24 to
P-224; G-25 to P-224; L-26 to P-224; G-27 to P-224; E-28 to P-224;
A-29 to P-224; C-30 to P-224; S-31 to P-224; C-32 to P-224; A-33 to
P-224; P-34 to P-224; A-35 to P-224; H-36 to P-224; P-37 to P-224;
Q-38 to P-224; Q-39 to P-224; H-40 to P-224; I-41 to P-224; C-42 to
P-224; H-43 to P-224; S-44 to P-224; A-45 to P-224; L-46 to P-224;
V-47 to P-224; I-48 to P-224; R-49 to P-224; A-50 to P-224; K-51 to
P-224; I-52 to P-224; S-53 to P-224; S-54 to P-224; E-55 to P-224;
K-56 to P-224; V-57 to P-224; V-58 to P-224; P-59 to P-224; A-60 to
P-224; S-61 to P-224; A-62 to P-224; D-63 to P-224; P-64 to P-224;
A-65 to P-224; D-66 to P-224; T-67 to P-224; E-68 to P-224; K-69 to
P-224; M-70 to P-224; L-71 to P-224; R-72 to P-224; Y-73 to P-224;
E-74 to P-224; I-75 to P-224; K-76 to P-224; Q-77 to P-224; I-78 to
P-224; K-79 to P-224; M-80 to P-224; F-81 to P-224; K-82 to P-224;
G-83 to P-224; F-84 to P-224; E-85 to P-224; K-86 to P-224; V-87 to
P-224; K-88 to P-224; D-89 to P-224; V-90 to P-224; Q-91 to P-224;
Y-92 to P-224; I-93 to P-224; Y-94 to P-224; T-95 to P-224; P-96 to
P-224; F-97 to P-224; D-98 to P-224; S-99 to P-224; S-100 to P-224;
L-101 to P-224; C-102 to P-224; G-103 to P-224; V-104 to P-224;
K-105 to P-224; L-106 to P-224; E-107 to P-224; A-108 to P-224;
N-109 to P-224; S-110 to P-224; Q-111 to P-224; K-112 to P-224;
Q-113 to P-224; Y-114 to P-224; L-115 to P-224; L-116 to P-224;
T-117 to P-224; G-118 to P-224; Q-119 to P-224; V-120 to P-224;
L-121 to P-224; S-122 to P-224; D-123 to P-224; G-124 to P-224;
K-125 to P-224; V-126 to P-224; F-127 to P-224; I-128 to P-224;
H-129 to P-224; L-130 to P-224; C-131 to P-224; N-132 to P-224;
Y-133 to P-224; I-134 to P-224; E-135 to P-224; P-136 to P-224;
W-137 to P-224; E-138 to P-224; D-139 to P-224; L-140 to P-224;
S-141 to P-224; L-142 to P-224; V-143 to P-224; Q-144 to P-224;
R-145 to P-224; E-146 to P-224; S-147 to P-224; L-148 to P-224;
N-149 to P-224; H-150 to P-224; H-151 to P-224; Y-152 to P-224;
H-153 to P-224; L-154 to P-224; N-155 to P-224; C-156 to P-224;
G-157 to P-224; C-158 to P-224; Q-159 to P-224; I-160 to P-224;
T-161 to P-224; T-162 to P-224; C-163 to P-224; Y-164 to P-224;
T-165 to P-224; V-166 to P-224; P-167 to P-224; C-168 to P-224;
T-169 to P-224; I-170 to P-224; S-171 to P-224; A-172 to P-224;
P-173 to P-224; N-174 to P-224; E-175 to P-224; C-176 to P-224;
L-177 to P-224; W-178 to P-224; T-179 to P-224; D-180 to P-224;
W-181 to P-224; L-182 to P-224; L-183 to P-224; E-184 to P-224;
R-185 to P-224; K-186 to P-224; L-187 to P-224; Y-188 to P-224;
G-189 to P-224; Y-190 to P-224; Q-191 to P-224; A-192 to P-224;
Q-193 to P-224; H-194 to P-224; Y-195 to P-224; V-196 to P-224;
C-197 to P-224; M-198 to P-224; K-199 to P-224; H-200 to P-224;
V-201 to P-224; D-202 to P-224; G-203 to P-224; T-204 to P-224;
C-205 to P-224; S-206 to P-224; W-207 to P-224; Y-208 to P-224;
R-209 to P-224; G-210 to P-224; H-211 to P-224; L-212 to P-224;
P-213 to P-224; L-214 to P-224; R-215 to P-224; K-216 to P-224;
E-217 to P-224; F-218 to P-224; and V-219 to P-224; of the amino
acid sequence in FIGS. 1A-B (the amino acid position in FIGS. 1A-B
correspond to that of the sequence in SEQ ID NO:2 plus 29). The
present application is also directed to polypeptides comprising, or
alternatively, consisting of, an amino acid sequence at least 90%,
92%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide
described above. The present invention also encompasses the above
polypeptide sequences fused to a heterologous polypeptide sequence.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0062] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other functional activities (e.g., biological activities,
ability to bind TIMP-4 ligand) may still be retained. For example,
the ability of the shortened TIMP-4 mutein to induce and/or bind to
antibodies which recognize the complete or mature forms of the
polypeptide generally will be retained when less than the majority
of the residues of the complete or mature polypeptide are removed
from the C-terminus. Whether a particular polypeptide lacking
C-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that an TIMP-4 mutein with a large number of deleted C-terminal
amino acid residues may retain some biological or immunogenic
activities. In fact, peptides composed of as few as six TIMP-4
amino acid residues may often evoke an immune response.
[0063] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the TIMP-4 polypeptide shown
in FIG. 1 (SEQ ID NO:2), as described by the general formula 1-n,
where n is an integer from 6-219, where n corresponds to the
position of amino acid residue identified in FIGS. 1A-B. More in
particular, the invention provides polypeptides comprising, or
alternatively consisting of, an amino acid sequence selected from:
M-1 to Q-223; M-1 to V-222; M-1 to 1-221; M-1 to D-220; M-1 to
V-219; M-1 to F-218; M-1 to E-217; M-1 to K-216; M-1 to R-215; M-1
to L-214; M-1 to P-213; M-1 to L-212; M-1 to H-211; M-1 to G-210;
M-1 to R-209; M-1 to Y-208; M-1 to W-207; M-1 to S-206; M-1 to
C-205; M-1 to T-204; M-1 to G-203; M-1 to D-202; M-1 to V-201; M-1
to H-200; M-1 to K-199; M-1 to M-198; M-1 to C-197; M-1 to V-196;
M-1 to Y-195; M-1 to H-194; M-1 to Q-193; M-1 to A-192; M-1 to
Q-191; M-1 to Y-190; M-1 to G-189; M-1 to Y-188; M-1 to L-187; M-1
to K-186; M-1 to R-185; M-1 to E-184; M-1 to L-183; M-1 to L-182;
M-1 to W-181; M-1 to D-180; M-1 to T-179; M-1 to W-178; M-1 to
L-177; M-1 to C-176; M-1 to E-175; M-1 to N-174; M-1 to P-173; M-1
to A-172; M-1 to S-171; M-1 to I-170; M-1 to T-169; M-1 to C-168;
M-1 to P-167; M-1 to V-166; M-1 to T-165; M-1 to Y-164; M-1 to
C-163; M-1 to T-162; M-1 to T-161; M-1 to 1-160; M-1 to Q-159; M-1
to C-158; M-1 to G-157; M-1 to C-156; M-1 to N-155; M-1 to L-154;
M-1 to H-153; M-1 to Y-152; M-1 to H-151; M-1 to H-150; M-1 to
N-149; M-1 to L-148; M-1 to S-147; M-1 to E-146; M-1 to R-145; M-1
to Q-144; M-1 to V-143; M-1 to L-142; M-1 to S-141; M-1 to L-140;
M-1 to D-139; M-1 to E-138; M-1 to W-137; M-1 to P-136; M-1 to
E-135; M-1 to 1-134; M-1 to Y-133; M-1 to N-132; M-1 to C-131; M-1
to L-130; M-1 to H-129; M-1 to I-128; M-1 to F-127; M-1 to V-126;
M-1 to K-125; M-1 to G-124; M-1 to D-123; M-1 to S-122; M-1 to
L-121; M-1 to V-120; M-1 to Q-119; M-1 to G-118; M-1 to T-117; M-1
to L-116; M-1 to L-115; M-1 to Y-114; M-1 to Q-113; M-1 to K-112;
M-1 to Q-111; M-1 to S-110; M-1 to N-109; M-1 to A-108; M-1 to
E-107; M-1 to L-106; M-1 to K-105; M-1 to V-104; M-1 to G-103; M-1
to C-102; M-1 to L-101; M-1 to S-100; M-1 to S-99; M-1 to D-98; M-1
to F-97; M-1 to P-96; M-1 to T-95; M-1 to Y-94; M-1 to 1-93; M-1 to
Y-92; M-1 to Q-91; M-1 to V-90; M-1 to D-89; M-1 to K-88; M-1 to
V-87; M-1 to K-86; M-1 to E-85; M-1 to F-84; M-1 to G-83; M-1 to
K-82; M-1 to F-81; M-1 to M-80; M-1 to K-79; M-1 to I-78; M-1 to
Q-77; M-1 to K-76; M-1 to 1-75; M-1 to E-74; M-1 to Y-73; M-1 to
R-72; M-1 to L-71; M-1 to M-70; M-1 to K-69; M-1 to E-68; M-1 to
T-67; M-1 to D-66; M-1 to A-65; M-1 to P-64; M-1 to D-63; M-1 to
A-62; M-1 to S-61; M-1 to A-60; M-1 to P-59; M-1 to V-58; M-1 to
V-57; M-1 to K-56; M-1 to E-55; M-1 to S-54; M-1 to S-53; M-1 to
1-52; M-1 to K-51; M-1 to A-50; M-1 to R-49; M-1 to 1-48; M-1 to
V-47; M-1 to L-46; M-1 to A-45; M-1 to S-44; M-1 to H-43; M-1 to
C-42; M-1 to 1-41; M-1 to H-40; M-1 to Q-39; M-1 to Q-38; M-1 to
P-37; M-1 to H-36; M-1 to A-35; M-1 to P-34; M-1 to A-33; M-1 to
C-32; M-1 to S-31; M-1 to C-30; M-1 to A-29; M-1 to E-28; M-1 to
G-27; M-1 to L-26; M-1 to G-25; M-1 to P-24; M-1 to P-23; M-1 to
R-22; M-1 to L-21; M-1 to L-20; M-1 to A-19; M-1 to L-18; M-1 to
L-17; M-1 to R-16; M-1 to L-15; M-1 to L-14; M-1 to L-13; M-1 to
V-12; M-1 to W-11; M-1 to S-10; M-1 to P-9; M-1 to A-8; and M-1 to
P-7; of FIGS. 1A-B (the amino acid position in FIGS. 1A-B
correspond to that of the sequence in SEQ ID NO:2 plus 29). The
present application is also directed to polypeptides comprising, or
alternatively, consisting of, an amino acid sequence at least 90%,
92%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide
described above. The present invention also encompasses the above
polypeptide sequences fused to a heterologous polypeptide sequence.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0064] In further embodiments, the present invention encompasses
polypeptides comprising, or alternatively consisting of, an epitope
of the polypeptide having an amino acid sequence of SEQ ID NO:2, or
an epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in deposited clone 75946 or encoded by a
polynucleotide that hybridizes to the complement of the sequence of
SEQ ID NO:1 or contained in deposited clone 75946 under stringent
hybridization conditions as defined supra. The present invention
further encompasses polynucleotide sequences encoding an epitope of
a polypeptide sequence of the invention (such as, for example, the
sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the
complementary strand of a polynucleotide sequence encoding an
epitope of the invention, and polynucleotide sequences which
hybridize to the complementary strand under stringent hybridization
conditions or lower stringency hybridization conditions defined
supra. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0065] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope," as used herein, is defined
as a portion of a protein to which an antibody can
immunospecifically bind its antigen as determined by any method
well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but
does not necessarily exclude cross-reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[0066] Fragments that function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0067] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
15, at least 20, at least 25, and, most preferably, between about
15 to about 30 amino acids. Preferred polypeptides comprising
immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino
acid residues in length. Antigenic epitopes are useful, for
example, to raise antibodies, including monoclonal antibodies, that
specifically bind the epitope. Antigenic epitopes can be used as
the target molecules in immunoassays. (See, for instance, Wilson et
al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666
(1983)).
[0068] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). A preferred immunogenic epitope
includes the secreted TIMP-4 protein. The polypeptides comprising
one or more immunogenic epitopes may be presented for eliciting an
antibody response together with a carrier protein, such as an
albumin, to an animal system (such as, for example, rabbit or
mouse), or, if the polypeptide is of sufficient length (at least
about 25 amino acids), the polypeptide may be presented without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10
amino acids have been shown to be sufficient to raise antibodies
capable of binding to, at the very least, linear epitopes in a
denatured polypeptide (e.g., in Western blotting).
[0069] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as, for example,
rabbits, rats, and mice are immunized with either free or
carrier-coupled peptides, for instance, by intraperitoneal and/or
intradermal injection of emulsions containing about 100 micrograms
of peptide or carrier protein and Freund's adjuvant or any other
adjuvant known for stimulating an immune response. Several booster
injections may be needed, for instance, at intervals of about two
weeks, to provide a useful titer of anti-peptide antibody that can
be detected, for example, by ELISA assay using free peptide
adsorbed to a solid surface. The titer of anti-peptide antibodies
in serum from an immunized animal may be increased by selection of
anti-peptide antibodies, for instance, by adsorption to the peptide
on a solid support and elution of the selected antibodies according
to methods well known in the art.
[0070] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides.
Such fusion proteins may facilitate purification and may increase
half-life in vivo. This has been shown for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins
that have a disulfide-linked dimeric structure due to the IgG
portion desulfide bonds have also been found to be more efficient
in binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid
in detection and purification of the expressed polypeptide. For
example, a system described by Janknecht et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag
consisting of six histidine residues. The tag serves as a
matrix-binding domain for the fusion protein. Extracts from cells
infected with the recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose column and histidine-tagged
proteins can be selectively eluted with imidazole-containing
buffers.
[0071] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to generate variation in the polynucleotide sequence.
In another embodiment, polynucleotides of the invention, or the
encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. In another embodiment, one or
more components, motifs, sections, parts, domains, fragments, etc.,
of a polynucleotide coding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0072] The polypeptides of the present invention include the
polypeptide of SEQ ID NO:2 (in particular the mature polypeptide)
as well as polypeptides which have at least 70% similarity
(preferably at least 70% identity) to the polypeptide of SEQ ID
NO:2 and more preferably at least 90% similarity (more preferably
at least 80% identity, or at least 85% identity) to the polypeptide
of SEQ ID NO:2 and still more preferably at least 95% similarity
(still more preferably at least 90% identity, at least 95%
identity, at least 96% identity, at least 97% identity, at least
98% identity, or at least 99% identity) to the polypeptide of SEQ
ID NO:2 and also include portions of such polypeptides with such
portion of the polypeptide generally containing at least 30 amino
acids and more preferably at least 50 amino acids.
[0073] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" or "identity" to a reference amino acid
sequence of a TIMP-4 polypeptide is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of the TIMP-4 polypeptide. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0074] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID
NO:2), the amino acid sequence encoded by the deposited cDNA clone,
or fragments thereof, can be determined conventionally using known
computer programs such the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711).
When using Bestfit or any other sequence alignment program to
determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5%
of the total number of amino acid residues in the reference
sequence are allowed.
[0075] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0076] In specific embodiments, the polypeptides of the invention
comprise, or alternatively consist of, a polypeptitde that is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
a polypeptide selected from the group: (a) a polypeptide having the
amino acid sequence of amino acids 1 to 72 of SEQ ID NO:2; (b) a
polypeptide having the amino acid sequence of amino acids 73 to 127
of SEQ ID NO:2; (c) a polypeptide having the amino acid sequence of
amino acids 128 to 176 of SEQ ID NO:2; and (d) a polypeptide having
the amino acid sequence of amino acids 1 to 176 of SEQ ID NO:2.
Poynucleotides encoding these polypeptides are also encompassed by
the invention.
[0077] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide.
[0078] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention. The present
invention also relates to vectors which include polynucleotides of
the present invention, host cells which are genetically engineered
with vectors of the invention and the production of polypeptides of
the invention by recombinant techniques.
[0079] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
human TIMP-4 genes. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to the ordinarily skilled
artisan.
[0080] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. Such vectors
include chromosomal, nonchromosomal and synthetic DNA sequences,
e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0081] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0082] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0083] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0084] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0085] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Sf9; animal cells such as CHO, COS or
Bowes melanoma; adenoviruses; plant cells, etc. The selection of an
appropriate host is deemed to be within the scope of those skilled
in the art from the teachings herein.
[0086] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10,
phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0087] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are PKK232-8 and PCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0088] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0089] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0090] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0091] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples including the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0092] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), alpha-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product.
[0093] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0094] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0095] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0096] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0097] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0098] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0099] The human TIMP-4 polypeptides can be recovered and purified
from recombinant cell cultures by methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0100] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
[0101] Antibodies
[0102] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, preferably an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying
specific antibody-antigen binding). Antibodies of the invention
include, but are not limited to, polyclonal, monoclonal,
multispecific, human, humanized or chimeric antibodies, single
chain antibodies, Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above.
The term "antibody," as used herein, refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that immunospecifically binds an antigen. The immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule. In specific
embodiments, the immunoglobulin molecule is IgG1. In other specific
embodiments, the immunoglobulin molecule is IgG4.
[0103] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, donkey, ship rabbit,
goat, guinea pig, camel, horse, or chicken. As used herein, "human"
antibodies include antibodies having the amino acid sequence of a
human immunoglobulin and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more
human immunoglobulin and that do not express endogenous
immunoglobulins, as described infra and, for example in, U.S. Pat.
No. 5,939,598 by Kucherlapati et al.
[0104] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0105] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention that they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies that specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0106] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less than 85%, less than 80%, less than 75%, less than
70%, less than 65%, less than 60%, less than 55%, and less than 50%
identity (as calculated using methods known in the art and
described herein) to a polypeptide of the present invention are
also included in the present invention. Further included in the
present invention are antibodies that bind polypeptides encoded by
polynucleotides which hybridize to a polynucleotide of the present
invention under stringent hybridization conditions (as described
herein). Antibodies of the present invention may also be described
or specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2M,
10.sup.-2M, 5.times.10.sup.-3M, 10.sup.-3M, 5.times.10.sup.4M,
10.sup.-4M, 5.times.10.sup.-5M, 10.sup.-5M, 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M,
10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0107] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 90%, at least 80%, at
least 70%, at least 60%, or at least 50%
[0108] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. In specific embodiments, the
antagonistic antibodies of the invention increase or enhance
metalloproteinase activity.). In specific embodiments, antibodies
are provided that increase or enhance metalloproteinase activity by
at least 90%, at least 80%, at least 70%, at least 60%, or at least
50% of the activity in absence of the antibody. In an alternative
example, the present invention includes antibodies which enhance
receptor/ligand interactions with the polypeptides of the invention
either partially or fully. In specific embodiments, the agonsitic
antibodies of the invention decrease or reduce metalloproteinase
activity. In specific embodiments, antibodies are provided that
decrease or reduce metalloproteinase activity by at least 90%, at
least 80%, at least 70%, at least 60%, or at least 50% of the
activity in absence of the antibody. The invention features both
receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. For example,
receptor activation can be determined by detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the
receptor or its substrate by immunoprecipitation followed by
western blot analysis (for example, as described supra). In
specific embodiments, antibodies are provided that enhance or
increase ligand or receptor activity by at least 90%, at least 80%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody. In specific embodiments, antibodies are
provided that inhibit ligand or receptor activity by at least 90%,
at least 80%, at least 70%, at least 60%, or at least 50% of the
activity in absence of the antibody.
[0109] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation. The antibodies may be
specified as agonists, antagonists or inverse agonists for
biological activities comprising the specific biological activities
of the peptides of the invention disclosed herein. The above
antibody agonists can be made using methods known in the art. See,
e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et
al., Blood 92(6):1981-1988 (1998); Chen, et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon, et al., J. Immunol. 160(7):3170-3179 (1998); Prat et
al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine
9(4):233-241 (1997); Carlson et al., J. Biol. Chem.
272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762
(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et
al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference herein in their entireties).
[0110] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0111] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs, or
toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO
89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
[0112] The antibodies of the invention include derivatives that are
modified, i.e, by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0113] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0114] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0115] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well-known in the art
and are discussed in detail in Example 6. Briefly, mice can be
immunized with a polypeptide of the invention or a cell expressing
such peptide. Once an immune response is detected, e.g., antibodies
specific for the antigen are detected in the mouse serum, the mouse
spleen is harvested and splenocytes isolated. The splenocytes are
then fused by well-known techniques to any suitable myeloma cells,
for example cells from cell line SP20 available from the ATCC.
Hybridomas are selected and cloned by limited dilution. The
hybridoma clones are then assayed by methods known in the art for
cells that secrete antibodies capable of binding a polypeptide of
the invention. Ascites fluid, which generally contains high levels
of antibodies, can be generated by immunizing mice with positive
hybridoma clones.
[0116] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0117] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0118] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0119] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0120] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397, which are incorporated herein by reference in their
entireties. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and framework regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties.)
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS
91:969-973 (1994)), and chain shuffling (U.S. Pat. No.
5,565,332).
[0121] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0122] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.
Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0123] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0124] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0125] Polynucleotides Encoding Antibodies
[0126] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:2.
[0127] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligation of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0128] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be obtained from a
suitable source (e.g., an antibody cDNA library, or a cDNA library
generated from, or nucleic acid, preferably poly A+ RNA, isolated
from, any tissue or cells expressing the antibody, such as
hybridoma cells selected to express an antibody of the invention)
by PCR amplification using synthetic primers hybridizable to the 3'
and 5' ends of the sequence or by cloning using an oligonucleotide
probe specific for the particular gene sequence to identify, e.g.,
a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into
replicable cloning vectors using any method well known in the
art.
[0129] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0130] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0131] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing 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. As described supra, 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, e.g.,
humanized antibodies.
[0132] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988,
Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be
adapted to produce single chain antibodies. 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. Techniques for the assembly of functional Fv fragments
in E. coli may also be used (Skerra et al., 1988, Science
242:1038-1041).
[0133] Methods of Producing Antibodies
[0134] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0135] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, e.g., a heavy or light
chain of an antibody of the invention, requires construction of an
expression vector containing a polynucleotide that encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody, or portion thereof (preferably
containing the heavy or light chain variable domain), of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
a protein by expressing a polynucleotide containing an antibody
encoding nucleotide sequence are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0136] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, operably linked to a heterologous promoter. In preferred
embodiments for the expression of double-chained antibodies,
vectors encoding both the heavy and light chains may be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
[0137] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,
NSO, 3T3 cells) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., 1986, Gene 45: 101; Cockett et al.,
1990, Bio/Technology 8:2).
[0138] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct 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., 1983, EMBO J. 2:1791), in which the antibody coding
sequence may be ligated individually into the vector in frame with
the lac Z coding region so that a fusion protein is produced; pIN
vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509); 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 and
binding to a matrix 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.
[0139] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence 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).
[0140] 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 antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., 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 (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. 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., 1987, Methods in Enzymol.
153:51-544).
[0141] 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 (e.g., glycosylation) and processing (e.g., 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. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0142] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule 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 (e.g., 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 are 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 may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0143] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthineguanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:817) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl.
Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991,
Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and
Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215); and hygro, which confers resistance to hygromycin
(Santerre et al., 1984, Gene 30:147). Methods commonly known in the
art of recombinant DNA technology which can be used are described
in Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer
and Expression, A Laboratory Manual, Stockton Press, NY; and in
Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols
in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et
al., 1981, J. Mol. Biol. 150:1, which are incorporated by reference
herein in their entireties.
[0144] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning,
Vol.3. (Academic Press, New York, 1987)). When a marker in the
vector system expressing antibody is amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody gene, production of the antibody
will also increase (Crouse et al., 1983, Mol. Cell. Biol.
3:257).
[0145] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0146] Once an antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
[0147] Antibody Conjugates
[0148] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20 or 50 amino acids of the polypeptide) of
the present invention to generate fusion proteins. The fusion does
not necessarily need to be direct, but may occur through linker
sequences. The antibodies may be specific for antigens other than
polypeptides (or portion thereof, preferably at least 10, 20 or 50
amino acids of the polypeptide) of the present invention. For
example, antibodies may be used to target the polypeptides of the
present invention to particular cell types, either in vitro or in
vivo, by fusing or conjugating the polypeptides of the present
invention to antibodies specific for particular cell surface
receptors. Antibodies fused or conjugated to the polypeptides of
the present invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452(1991), which are incorporated by
reference in their entireties.
[0149] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341(1992) (said references incorporated by
reference in their entireties).
[0150] As discussed, supra, the polypeptides of the present
invention may be fused or conjugated to the above antibody portions
to increase the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to facilitate purification. One
reported example describes chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86
(1988). The polypeptides of the present invention fused or
conjugated to an antibody having disulfide-linked dimeric
structures (due to the IgG) may also be more efficient in binding
and neutralizing other molecules, than the monomeric secreted
protein or protein fragment alone. (Fountoulakis et al., J.
Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a
fusion protein is beneficial in therapy and diagnosis, and thus can
result in, for example, improved pharmacokinetic properties. (EP A
232,262). Alternatively, deleting the Fc part after the fusion
protein has been expressed, detected, and purified, would be
desired. For example, the Fc portion may hinder therapy and
diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
(See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995);
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)0.
[0151] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitates their purification. In preferred embodiments, the
marker amino acid sequence is a hexa-histidine peptide, such as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. Other
peptide tags useful for purification include, but are not limited
to, the "HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))
and the "flag" tag.
[0152] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal
ions which can be conjugated to antibodies for use as diagnostics
according to the present invention. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc.
[0153] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion. A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0154] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, a thrombotic agent or an anti-angiogenic agent, e.g.,
angiostatin or endostatin; or, biological response modifiers such
as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0155] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0156] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0157] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0158] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[0159] Assays for Antibody Binding
[0160] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0161] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0162] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0163] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0164] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest is conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
[0165] Therapeutic Uses
[0166] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the described disorders.
Therapeutic compounds of the invention include, but are not limited
to, antibodies of the invention (including fragments, analogs and
derivatives thereof as described herein) and nucleic acids encoding
antibodies of the invention (including fragments, analogs and
derivatives thereof as described herein). The antibodies of the
invention can be used to treat, inhibit or prevent diseases and
disorders associated with aberrant expression and/or activity of a
polypeptide of the invention. The treatment and/or prevention of
diseases and disorders associated with aberrant expression and/or
activity of a polypeptide of the invention includes, but is not
limited to, alleviating symptoms associated with those diseases and
disorders. Antibodies of the invention may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0167] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0168] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0169] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0170] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides, including fragments thereof. Preferred binding
affinities include those with a dissociation constant or Kd less
than 5.times.10-6 M, 10-6 M, 5.times.10-7 M, 10-7 M, 5.times.10-8
M, 10-8 M, 5.times.10-9 M, 10-9 M, 5.times.10-10 M, 10-10 M,
5.times.10-11 M, 10-11 M, 5.times.10-12 M, 10-12 M, 5.times.10-13
M, 10-13 M, 5.times.10-14 M, 10-14 M, 5.times.10-15 M, and 10-15
M.
[0171] Gene Therapy
[0172] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0173] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0174] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0175] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody nucleic acids (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature 342:435-438). In specific embodiments, the expressed
antibody molecule is a single chain antibody; alternatively, the
nucleic acid sequences include sequences encoding both the heavy
and light chains, or fragments thereof, of the antibody.
[0176] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0177] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec.
23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis
et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO
93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0178] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors
have been to delete retroviral sequences that are not necessary for
packaging of the viral genome and integration into host cell DNA.
The nucleic acid sequences encoding the antibody to be used in gene
therapy are cloned into one or more vectors, which facilitates
delivery of the gene into a patient. More detail about retroviral
vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302,
which describes the use of a retroviral vector to deliver the mdr1
gene to hematopoietic stem cells in order to make the stem cells
more resistant to chemotherapy. Other references illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., 1994,
J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0179] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang, et al., 1995, Gene Therapy
2:775-783.
[0180] In cases where an adenovirus is used as an expression
vector, the antibody coding sequence of interest may be ligated to
an adenovirus transcription/translation control complex, e.g., 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 (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the TIMP-4 molecule
in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.
Acad. Sci. USA 81:355-359). Specific initiation signals may also be
required for efficient translation of inserted antibody coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. 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., 1987, Methods in Enzymol. 153:51-544).
[0181] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0182] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0183] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0184] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0185] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0186] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0187] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598, dated Apr. 28, 1994; Stemple and
Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.
21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc.
61:771).
[0188] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0189] Demonstration of Therapeutic or Prophylactic Activity
[0190] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[0191] Therapeutic/Prophylactic Administration and Composition
[0192] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0193] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0194] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other-biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0195] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0196] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer, 1990,
Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0197] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek
et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem.
23:61; see also Levy et al., 1985, Science 228:190; During et al.,
1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg.
71:105). In yet another embodiment, a controlled release system can
be placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol.
2, pp. 115-138 (1984)).
[0198] Other controlled release systems are discussed in the review
by Langer (1990, Science 249:1527-1533).
[0199] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0200] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0201] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0202] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0203] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0204] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0205] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0206] Diagnosis and Imaging
[0207] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or disorders associated with the aberrant expression and/or
activity of a polypeptide of the invention. The invention provides
for the detection of aberrant expression of a polypeptide of
interest, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0208] The invention provides a diagnostic assay for diagnosising a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0209] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, M.,
et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J.
Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods
useful for detecting protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99Tc); luminescent labels, such
as luminol; and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0210] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of the interest in an animal, preferably a mammal and
most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0211] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[0212] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0213] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0214] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0215] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI). Kits
[0216] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0217] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0218] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0219] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0220] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0221] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0222] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
[0223] Therapeutic and Diagnostic Uses
[0224] The present invention is also directed, in part, to human
TIMP-4 which has, as a defining characteristic, the ability to
inhibit the action of MMP's. The human TIMP-4 polypeptide may be
employed as a metalloproteinase inhibitor to prevent tumor invasion
and angiogeneses and subsequent metastases. The human TIMP-4
polypeptide may also be employed to treat arthritic diseases, such
as rheumatoid arthritis and osteoarthritis, soft tissue rheumatism,
polychondritis and tendonitis; and bone resorption diseases, such
as osteoporosis, Paget's disease, hyperparathyroidism and
cholesteatoma. Human TIMP-4 may also be employed to prevent
enhanced collagen destruction that occurs in association with
diabetes, the recessive classes of dystrophic epidermolysis
bullosa, periodontal disease and related consequences of gingival
production of collagenase. human TIMP-4 may also be employed to
inhibit PMNL collagenase release following cellular infiltration to
inflamed gingiva, including combatting the greater susceptibility
of diabetes patients to periodontal disease.
[0225] Human TIMP-4 may also be employed to treat corneal
ulceration, for example, that induced by alkali or other burns, by
radiation, by Vitamin E or retinoid deficiency; ulceration of the
skin and gastro-intestinal tract, and abnormal wound healing, and
post-operative conditions including colonic anastomosis, in which
collagenase levels are raised.
[0226] MMP's mediate tumor growth in situ. Accordingly, human
TIMP-4 may be used to block the destruction of cellular basement
membranes, which is the mechanism by which cancer cells
metastasize. MMP's have been implicated in neovascularization
required to support tumor growth and survival, in the tissue
remodeling required to accommodate the growing primary and
secondary tumors, and in the penetration of tumor cells through the
basement membranes of the vascular walls during metastasis.
[0227] Thus, in specific embodiments, the invention provides for
treatment or prevention of various diseases and disorders involving
cell proliferation, tumor cell invasion, and tumor
angiogenesis.
[0228] TIMP-4 polynucleotides, polypeptides, antibodies (e.g.,
agonistic anti-TIMP-4 antibodies) and/or agonists of the invention,
may be used for therapeutic/prophylactic purposes alone or in
combination with other therapeutics useful in the treatment of
cancer and hyperproliferative or dysproliferative disorders.
Diseases and disorders involving cell overproliferation are treated
or prevented by administration of a TIMP-4 polynucleotide,
polypeptide, antibody and/or agonist of the invention that promotes
(i.e., increases or supplies) TIMP-4 function.
[0229] Diseases and disorders involving cell overproliferation that
can be treated or prevented using the polynucleotides,
polypeptides, antibodies, and/or agonists of the invention include,
but are not limited to, malignancies, premalignant conditions
(e.g., hyperplasia, metaplasia, dysplasia), benign tumors,
hyperproliferative disorders, benign dysproliferative disorders,
etc.
[0230] Malignancies and related disorders that can be treated or
prevented by administration of a TIMP-4 polynucleotide,
polypeptide, antibody and/or agonist of the invention, include but
are not limited to, leukemia, acute leukemia, acute lymphocytic
leukemia, acute myelocytic leukemia, myeloblastic leukemia,
promyelocytic leukemia, myelomonocytic leukemia, monocytic
leukemia, erythroleukemia, chronic leukemia, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, Polycythemia
vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple
myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease,
solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
colorectal carcinoma, pancreatic cancer, breast cancer, ovarian
cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical cancer, uterine cancer, testicular tumor, lung carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, nasopharyngeal carcinoma, and esophageal
carcinoma.(for a review of such disorders, see Fishman et al.,
1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).
[0231] In a specific embodiment, digestive tract tumors are treated
or prevented, including but not limited to, esophageal, stomach,
colon, and colorectal cancers. In another specific embodiment,
airway cancers such as lung cancers (e.g., small cell lung
carcinoma) and nasopharyngeal carcinoma are treated or prevented.
In yet other specific embodiments, malignancy or dysproliferative
changes (such as metaplasias and dysplasias), or hyperproliferative
disorders, are treated or prevented in the head, neck, cervix,
kidney, stomach, skin, ovary, bladder, breast, colon, lung, or
uterus. In other specific embodiments, sarcoma, or leukemia is
treated or prevented. In another particular embodiment,
osteosarcoma or renal cell carcinoma is treated or prevented.
[0232] TIMP-4 polynucleotides, polypeptides, antibodies and/or
agonists of the invention can also be administered to treat
premalignant conditions and to prevent progression to a neoplastic
or malignant state, including but not limited to, those disorders
listed above. Such prophylactic or therapeutic use is indicated in
conditions known or suspected of preceding progression to neoplasia
or cancer, in particular, where non-neoplastic cell growth
consisting of hyperplasia, metaplasia, or most particularly,
dysplasia has occurred (for review of such abnormal growth
conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed.,
W. B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a form
of controlled cell proliferation involving an increase in cell
number in a tissue or organ, without significant alteration in
structure or function. As but one example, endometrial hyperplasia
often precedes endometrial cancer. Metaplasia is a form of
controlled cell growth in which one type of adult or fully
differentiated cell substitutes for another type of adult cell.
Metaplasia can occur in epithelial or connective tissue cells.
Atypical metaplasia involves a somewhat disorderly metaplastic
epithelium. Dysplasia is frequently a forerunner of cancer, and is
found mainly in the epithelia; it is the most disorderly form of
non-neoplastic cell growth, involving a loss in individual cell
uniformity and in the architectural orientation of cells.
Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0233] In a specific embodiment, a TIMP-4 polypeptide,
polynucleotide, antibody, and/or agonist of the invention is
administered to a human patient to prevent progression to breast,
colon, lung, stomach or uterine cancer, or melanoma or sarcoma.
[0234] In another embodiment of the invention, a polynucleotide,
polypeptide, antibody and/or agonist of the invention is used to
treat or prevent hyperproliferative or benign dysproliferative
disorders. Specific embodiments are directed to treatment or
prevention of benign tumors, fibrocystic conditions, and tissue
hypertrophy (e.g., prostatic hyperplasia).
[0235] In a specific embodiment TIMP-4 polynucleotides,
polypeptides, antibodies, and/or agonists of the invention are
administered to treat or prevent an immune system related disease,
disorder or condition. In a preferred embodiment, the immune system
disease, disorder, or condition is an autoimmune diesease,
disorder, or condition. In a most preferred embodiment, the immune
system diesease, disorder, or condition, is rheumatoid
arthritis.
[0236] MMP's are responsible for localized degradation of the
follicular wall during ovulation and localized degradation of the
uterine wall for blastocyte implantation. Accordingly, human TIMP-4
may be employed as a contraceptive.
[0237] Human TIMP-4 may also be employed as a general growth factor
to treat restenosis and similar diseases. Human TIMP-4 may be
employed particularly as a growth factor for erythroid cell
lineages.
[0238] TIMP-4 is a strong inhibitor of metalloproteinases which
degrade structural components of tissues and are involved in the
remodeling of tissues in normal and certain pathological states.
Accordingly, potential therapeutic applications of the TIMP-4
polynucleotides, polypeptides, antibodies, and/or agonists of the
invention include, but are not limited to, the treatment and/or
prevention of restenosis, or obstruction of blood vessels, such as
coronary arteries, and heart failure. Restenosis is a medical
condition characterized by the constriction of coronary arteries.
Restenosis usually occurs following treatment to open coronary
arteries clogged by plaque accumulation. Balloon angioplasty,
insertion of a catheter into the clogged artery followed by
expansion of a balloon at the site of blockage, compresses the
plaque and opens the arteries. This procedure can damage the wall
of the artery. The damaged vessel often responds to the balloon
angioplasty injury by overgrowth, which can lead to reconstriction
of the artery. While not intending to be bound by theory, it is
believed that TIMP-4 acts to treat or prevent restenosis by
blocking metalloproteinases, a family of genes that become active
after injury to the artery and are thought to play a major role in
restenosis.
[0239] TIMP-4 polynucleotides or polypeptides, or agonists or
antagonists of TIMP-4, encoding TIMP-4 may be used to treat,
prevent, and/or diagnose cardiovascular diseases, disorders, and/or
conditions, including peripheral artery disease, such as limb
ischemia.
[0240] Cardiovascular diseases, disorders, and/or conditions that
may be treated, prevented and/or diagnosed with the
polynucleotides, polypeptides (including antibodies), agonists
and/or antagonists of the invention include, but are not limited
to, cardiovascular abnormalities, such as arterio-arterial fistula,
arteriovenous fistula, cerebral arteriovenous malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
Congenital heart defects include aortic coarctation, cor
triatriatum, coronary vessel anomalies, crisscross heart,
dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of fallot, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[0241] Cardiovascular diseases, disorders, and/or conditions that
may be treated, prevented and/or diagnosed with the
polynucleotides, polypeptides (including antibodies), agonists
and/or antagonists of the invention also include, but are not
limited to, heart disease, such as arrhythmias, carcinoid heart
disease, high cardiac output, low cardiac output, cardiac
tamponade, endocarditis (including bacterial), heart aneurysm,
cardiac arrest, congestive heart failure, congestive
cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy, right ventricular hypertrophy, post-infarction heart
rupture, ventricular septal rupture, heart valve diseases,
myocardial diseases, myocardial ischemia, pericardial effusion,
pericarditis (including constrictive and tuberculous),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart
disease, rheumatic heart disease, ventricular dysfunction,
hyperemia, cardiovascular pregnancy complications, Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[0242] Arrhythmias that may be treated, prevented and/or diagnosed
with the polynucleotides, polypeptides (including antibodies),
agonists and/or antagonists of the invention include, but are not
lmited to, sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch
block, sinoatrial block, long QT syndrome, parasystole,
Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,
Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias,
and ventricular fibrillation. Tachycardias include paroxysmal
tachycardia, supraventricular tachycardia, accelerated
idioventricular rhythm, atrioventricular nodal reentry tachycardia,
ectopic atrial tachycardia, ectopic junctional tachycardia,
sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de Pointes, and ventricular tachycardia.
[0243] Heart valve disease that may be treated, prevented and/or
diagnosed with the polynucleotides, polypeptides (including
antibodies), agonists and/or antagonists of the invention include,
but are not limited to, aortic valve insufficiency, aortic valve
stenosis, hear murmurs, aortic valve prolapse, mitral valve
prolapse, tricuspid valve prolapse, mitral valve insufficiency,
mitral valve stenosis, pulmonary atresia, pulmonary valve
insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[0244] Myocardial diseases that may be treated, prevented and/or
diagnosed with the polynucleotides, polypeptides (including
antibodies), agonists and/or antagonists of the invention include,
but are not limited to, alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular
stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[0245] Myocardial ischemias that may be treated, prevented and/or
diagnosed with the polynucleotides, polypeptides (including
antibodies), agonists and/or antagonists of the invention include,
but are not limited to, coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,
coronary vasospasm, myocardial infarction and myocardial
stunning.
[0246] Cardiovascular diseases that may be treated, prevented
and/or diagnosed with the polynucleotides, polypeptides (including
antibodies), agonists and/or antagonists of the invention also
include, but are not limited to, vascular diseases such as
aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular diseases, disorders, and/or conditions, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,
hypertension, hypotension, ischemia, peripheral vascular diseases,
phlebitis, pulmonary veno-occlusive disease, Raynaud's disease,
CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena cava syndrome, telangiectasia, atacia telangiectasia,
hereditary hemorrhagic telangiectasia, varicocele, varicose veins,
varicose ulcer, vasculitis, and venous insufficiency.
[0247] Aneurysms that may be treated, prevented and/or diagnosed
with the polynucleotides, polypeptides (including antibodies),
agonists and/or antagonists of the invention include, but are not
limited to, dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0248] Arterial occlusive diseases that may be treated, prevented
and/or diagnosed with the polynucleotides, polypeptides (including
antibodies), agonists and/or antagonists of the invention include,
but are not limited to, arteriosclerosis, intermittent
claudication, carotid stenosis, fibromuscular dysplasias,
mesenteric vascular occlusion, Moyamoya disease, renal artery
obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[0249] Cerebrovascular diseases, disorders, and/or conditions that
may be treated, prevented and/or diagnosed with the
polynucleotides, polypeptides (including antibodies), agonists
and/or antagonists of the invention include, but are not limited
to, carotid artery diseases, cerebral amyloid angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral
arteriovenous malformation, cerebral artery diseases, cerebral
embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[0250] Embolisms that may be treated, prevented and/or diagnosed
with the polynucleotides, polypeptides (including antibodies),
agonists and/or antagonists of the invention include, but are not
limited to, air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms,
and thromoboembolisms. Thrombosis include coronary thrombosis,
hepatic vein thrombosis, retinal vein occlusion, carotid artery
thrombosis, sinus thrombosis, Wallenberg's syndrome, and
thrombophlebitis.
[0251] Ischemia that may be treated, prevented and/or diagnosed
with the polynucleotides, polypeptides (including antibodies),
agonists and/or antagonists of the invention includes, but are not
limited to, cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia,
reperfusion injuries, and peripheral limb ischemia. Vasculitis
includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss
Syndrome, mucocutaneous lymph node syndrome, thromboangiitis
obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
[0252] TIMP-4 polypeptides may be administered using any method
known in the art, including, but not limited to, gene therapy
(e.g., via techniques known in the art utilizing adenovirus
vectors), gene gun, direct needle injection at the delivery site,
intravenous injection, topical administration, catheter infusion,
biolistic injectors, particle accelerators, gelfoam sponge depots,
other commercially available depot materials, osmotic pumps, oral
or suppositorial solid pharmaceutical formulations, decanting or
topical applications during surgery, aerosol delivery. Such methods
are known in the art. TIMP-4 polypeptides may be administered as
part of a Therapeutic, described in more detail below. Methods of
delivering TIMP-4 polynucleotides are described in more detail
herein.
[0253] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. Rastinejad et al., Cell
56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulatory controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al.,
Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein
and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science
221:719-725 (1983). In a number of pathological conditions, the
process of angiogenesis contributes to the disease state. For
example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis. Folkman and
Klagsbrun, Science 235:442-447 (1987).
[0254] The present invention provides for treatment or prevention
of diseases or disorders associated with neovascularization by
administration of the polynucleotides, polypeptides, antibodies,
and/or agonists of the invention. Malignant and metastatic
conditions which can be treated with the polynucleotides,
polypeptides, antibodies, and/or agonists of the invention include,
but are not limited to, malignancies, solid tumors, and cancers
described herein and otherwise known in the art (for a review of
such disorders, see Fishman et al., Medicine, 2d Ed., J. B.
Lippincott Co., Philadelphia (1985)).
[0255] Ocular disorders associated with neovascularization which
can be treated or prevented with the TIMP-4 polynucleotides,
polypeptides, antibodies, and/or agonists of the present invention
include, but are not limited to: neovascular glaucoma, diabetic
retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,
retinopathy of prematurity macular degeneration, corneal graft
neovascularization, as well as other eye inflammatory diseases,
ocular tumors and diseases associated with choroidal or iris
neovascularization. See, e.g., reviews by Waltman et al., Am. J.
Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal.
22:291-312 (1978).
[0256] In another embodiment, TIMP-4 polypeptides, polynucleotides,
antibodies and/or agonists or antagonists of the invention are used
to stimulate differentiation and/or survival of photoreceptor cells
and/or to treat or prevent diseases, disorders, or conditions
associated with decreased number, differentiation and/or survival
of photoreceptor cells.
[0257] Additionally, disorders which can be treated with the TIMP-4
polynucleotides, polypeptides, antibodies, agonists and/or
antagonist of the present invention include, but are not limited
to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic
plaques, delayed wound healing, granulations, hemophilic joints,
hypertrophic scars, nonunion fractures, Osler-Weber syndrome,
pyogenic granuloma, scleroderma, trachoma, and vascular
adhesions.
[0258] In further embodiments, the TIMP-4 polynucleotides,
polypeptides, antibodies, agonists, and/or antagonists of the
inveniton, are used to promote wound healing.
[0259] In alternative embodiments, TIMP-4 polynucleotides,
polypeptides, antibodies (e.g., antagonistic anti-TIMP-4
antibodies) and/or antagonists of the invention, are useful in the
treatment of disorders in which stimulation of new blood vessel
development would ameliorate the disorder. Such disorders include,
but are not limited to, heart failure, angina, blood vessel (e.g.
coronary artery) blockage and ischemia, including critical limb
ischemia and refractory myocardial ischemia. Antagonistic TIMP-4
polynucleotides of the invention can be delivered to individuals to
using gene therapy techniques and materials described herein or
otherwise known in the art.
[0260] As a result of the ability to stimulate vascular endothelial
cell growth, TIMP-4 antagonists of the invention, such as for
example, antagonistic anti-TIMP-4 antibodies, may be employed in
treatment for stimulating re-vascularization of ischemic tissues
due to various disease conditions such as thrombosis,
arteriosclerosis, and other cardiovascular conditions. The
polypeptides, polynucleotides, antibodies, agonists and/or
antagonists of the present invention may also be employed to
stimulate angiogenesis and limb regeneration, as discussed
herein.
[0261] TIMP-4 polynucleotides, polypeptides, antibodies (e.g.,
agonistic anti-TIMP-4 antibodies), and/or agonists can be used to
inhibit MMP mediated extracellular matrix degradation or
alternatively differentiate, proliferate, and attract cells, and
thereby lead to the regeneration of tissues. (See, Science
276:59-87 (1997).) The regeneration of tissues could be used to
repair, replace, or protect tissue damaged by congenital defects,
trauma (wounds, burns, incisions, or ulcers), age, disease (e.g.
osteoporosis, osteocarthritis, periodontal disease, liver failure),
surgery, including cosmetic plastic surgery, fibrosis, reperfusion
injury, or systemic cytokine damage.
[0262] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, heart, intestine,
kidney, skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[0263] Moreover, TIMP-4 polynucleotides, polypeptides, antibodies,
and agonists or antagonists of the invention may increase
regeneration of tissues difficult to heal. For example, increased
tendon/ligament regeneration would quicken recovery time after
damage. TIMP-4 polynucleotides, polypeptides, antibodies, and
agonists or antagonists of the present invention could also be used
prophylactically in an effort to avoid damage. Specific diseases
that could be treated or prevented include of tendinitis, carpal
tunnel syndrome, and other tendon or ligament defects. A further
example of tissue regeneration of non-healing wounds includes
pressure ulcers, ulcers associated with vascular insufficiency,
surgical, and traumatic wounds.
[0264] Similarly, nerve and brain tissue could also be regenerated
according to the present invention by using TIMP-4 polynucleotides,
polypeptides, antibodies, agonists and/or antagonists to, for
example, proliferate and differentiate nerve cells. Diseases that
could be treated or prevented using this method include, but are
not limited to, central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the TIMP-4 polynucleotides,
polypeptides, antibodies, and agonists or antagonists of the
invention.
[0265] Among the other diseases which TIMP-4 may be employed to
treat includes alveolitis, asthma, psoriasis, glomerulosclerosis,
and septic shock since MMP's are involved in the tissue
invasiveness of some parasites.
[0266] An effective amount of the TIMP-4 polynucleotides,
polypeptides, antibodies and/or agonists or antagonists can be
administered in vitro, ex vivo, or in vivo using techniques and
compositions described herein described herein (e.g., in the
section entitled Therapeutic/Prophylactic Administration and
Composition) or otherwise known in the art. By administration of an
"effective amount" of TIMP-4 polynucleotides, polypeptides,
antibodies and/or agonists or antagonists is intended an amount of
the compound that is sufficient to enhance or inhibit a cellular
response to one or more metalloproteinases. In particular, by
administration of an "effective amount" of an agonist or
antagonists is intended an amount effective to enhance or inhibit
TIMP-4 polynucleotides, polypeptides, antibodies and/or agonists or
antagonists mediated metalloproteinase actiivity. One of ordinary
skill will appreciate that effective amounts of an agonist or
antagonist can be determined empirically and may be employed in
pure form or in pharmaceutically acceptable salt, ester or pro-drug
form. The agonist or antagonist may be administered in compositions
in combination with one or more pharmaceutically acceptable
excipients.
[0267] It will be understood that, when administered to a human
patient, the total daily usage of the compounds and compositions of
the present invention will be decided by the attending physician
within the scope of sound medical judgement. The specific
therapeutically effective dose level for any particular patient
will depend upon factors well known in the medical arts.
[0268] As a general proposition, the total pharmaceutically
effective amount of a TIMP-4 polypeptide administered parenterally
per dose will be in the range of about 1 .mu.g/kg/day to 10
mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the TIMP-4 polypeptides are typically administered at
a dose rate of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed.
[0269] Pharmaceutical compositions containing the TIMP-4
polypeptides of the invention may be administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, drops or transdermal patch),
bucally, or as an oral or nasal spray. By "pharmaceutically
acceptable carrier" is meant a non-toxic solid, semisolid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
[0270] The pharmaceutical compositions may be administered in a
convenient manner such as by the topical, intravenous,
intra-articular, intra-tumor, intraperitoneal, intramuscular,
subcutaneous, intranasal or intradermal routes. The pharmaceutical
compositions are administered in an amount which is effective for
treating and/or prophylaxis of the specific indication. In general,
the pharmaceutical compositions are administered in an amount of at
least about 10 micrograms/kg body weight and in most cases they
will be administered in an amount not in excess of about 8 mg/Kg
body weight per day and preferably the dosage is from about 10
micrograms/kg to about 1 mg/kg body weight daily, taking into
account the routes of administration, symptoms, etc.
[0271] The compositions of the invention may be administered alone
or in combination with other therapeutic agents, including but not
limited to, chemotherapeutic agents, anti-angiogenic agents,
angiogenic agents, anti-opportunistic infection agents, antivirals,
antibiotics, steroidal and non-steroidal anti-inflammatories,
immunosuppressants, conventional immunotherapeutic agents and
cytokines. Combinations may be administered either concomitantly,
e.g., as an admixture, separately but simultaneously or
concurrently; or sequentially. This includes presentations in which
the combined agents are administered together as a therapeutic
mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through
separate intravenous lines into the same individual. Administration
"in combination" further includes the separate administration of
one of the compounds or agents given first, followed by the
second.
[0272] In one embodiment, the compositions of the invention are
administered in combination with a member of the TNF family. TNF,
TNF-related or TNF-like molecules that may be administered with the
compositions of the invention include, but are not limited to,
soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known
as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L,
TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), AIM-II (International
Publication No. WO 97/34911), APRIL, endokine-alpha (International
Publication No. WO 98/07880), TR6 (International Publication No. WO
98/30694), OPG, and neutrokine-alpha (International application
publication number WO 98/18921), TWEAK, OX40, and nerve growth
factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB,
TR2 (International application publication number WO 96/34095), DR3
(International Publication No. WO 97/33904), DR4 (International
application publication number WO 98/32856), TR5 (International
application publication number WO 98/30693), TR7 (International
application publication number WO 98/41629), TRANK, TR9
(International application publication number WO 98/56892), TRIO
(International application publication number WO 98/54202),312C2
(International application publication number WO 98/06842), and
TR12.
[0273] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, cyclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents that act by suppressing the function of
responding T cells.
[0274] In specific embodiments, compositions of the invention are
administered in combination with immunosuppressants.
Immunosuppressants preparations that may be administered with the
compositions of the invention include, but are not limited to,
ORTHOCLONE.TM. (OKT3), SANDIMMUNE.TM./NEORAL.TM./SANGDYA.TM.
(cyclosporin), PROGRAF.TM. (tacrolimus), CELLCEPT.TM.
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE.TM.
(sirolimus). In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow
transplantation.
[0275] In certain embodiments, compositions of the invention are
administered in combination with antiretroviral agents, nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors. Nucleoside
reverse transcriptase inhibitors that may be administered in
combination with the compositions of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the
compositions of the invention, include, but are not limited to,
VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM. (delavirdine), and
SUSTVA.TM. (efavirenz). Protease inhibitors that may be
administered in combination with the compositions of the invention,
include, but are not limited to, CRIXIVAN.TM. (indinavir),
NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and VIRACEPT.TM.
(nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors may be used in
any combination with compositions of the invention to treat AIDS
and/or to prevent or treat HIV infection.
[0276] In other embodiments, compositions of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the compositions of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHO- XAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat or prevent an
opportunistic Pneumocystis carinii pneumonia infection. In another
specific embodiment, compositions of the invention are used in any
combination with ISONIAZID.TM., RIFAMPIN.TM., PYRAZINAMIDE.TM.,
and/or ETHAMBUTOL.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium avium complex infection. In another
specific embodiment, compositions of the invention are used in any
combination with RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or
AZITHROMYCIN.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium tuberculosis infection. In another
specific embodiment, compositions of the invention are used in any
combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment,
compositions of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat or prevent an opportunistic fungal
infection. In another specific embodiment, compositions of the
invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection.
In another specific embodiment, compositions of the invention are
used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplasma gondii infection. In another specific
embodiment, compositions of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat or prevent an opportunistic bacterial
infection.
[0277] In a further embodiment, the compositions of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the compositions of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine
[0278] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole, penicillins, quinolones, rifampin, streptomycin,
sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
[0279] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0280] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0281] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or any combination of the
components of CHOP. In another embodiment, compositions of the
invention are administered in combination with Rituximab. In a
further embodiment, compositions of the invention are administered
with Rituxmab and CHOP, or Rituxmab and any combination of the
components of CHOP.
[0282] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5,
IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha,
IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another
embodiment, compositions of the invention may be administered with
any interleukin, including, but not limited to, IL-1alpha,
IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, and IL-21. In a preferred embodiment, the compositions of
the invention are administered in combination with TNF-alpha. In
another preferred embodiment, the compositions of the invention are
administered in combination with IFN-alpha.
[0283] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-angiogenic agent. Anti-angiogenic agents that may be
administered with the compositions of the invention include, but
are not limited to, Angiostatin (Entremed, Rockville, Md.),
Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol),
Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor
of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1,
Plasminogen Activator Inhibitor-2, and various forms of the lighter
"d group" transition metals.
[0284] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[0285] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[0286] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[0287] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include, but are not limited to, platelet
factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from queen crab shells), (Murata et al., Cancer Res.
51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine; modulators of matrix metabolism, including for
example, proline analogs, cishydroxyproline,
d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,
aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, 1992); and
metalloproteinase inhibitors such as BB94.
[0288] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and
J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v
beta 3 antagonist (C. Storgard et al., J Clin. Invest. 103:47-54
(1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI)
(National Cancer Institute, Bethesda, Md.); Conbretastatin A-4
(CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin
Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap
Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London,
UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251
(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;
Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide
(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88;
Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen
(Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine);
and 5-Fluorouracil.
[0289] Anti-angiogenic agents that may be administed in combination
with the compounds of the invention may work through a variety of
mechanisms including, but not limited to, inhibiting proteolysis of
the extracellular matrix, blocking the function of endothelial
cell-extracellular matrix adhesion molecules, by antagonizing the
function of angiogenesis inducers such as growth factors, and
inhibiting integrin receptors expressed on proliferating
endothelial cells. Examples of anti-angiogenic inhibitors that
interfere with extracellular matrix proteolysis and which may be
administered in combination with the compositons of the invention
include, but are not lmited to, AG-3340 (Agouron, La Jolla,
Calif.), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol
Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover,
N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat
(Aeterna, St-Foy, Quebec). Examples of anti-angiogenic inhibitors
that act by blocking the function of endothelial cell-extracellular
matrix adhesion molecules and which may be administered in
combination with the compositons of the invention include, but are
not Imited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and
Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.).
Examples of anti-angiogenic agents that act by directly
antagonizing or inhibiting angiogenesis inducers and which may be
administered in combination with the compositons of the invention
include, but are not Imited to, Angiozyme (Ribozyme, Boulder,
Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),
PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S.
San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn,
Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic
agents act to indirectly inhibit angiogenesis. Examples of indirect
inhibitors of angiogenesis which may be administered in combination
with the compositons of the invention include, but are not lmited
to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12
(Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown
University, Washington, D.C.).
[0290] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
an autoimmune disease, such as for example, an autoimmune disease
described herein.
[0291] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use of compositions
of the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
rheumatoid arthritis.
[0292] In an additional embodiment, the compositions of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the compositions
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-6821 10; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PIGF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PIGF-2), as disclosed in Hauser et al., Gorwth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B186), as
disclosed in International Publication Number WO 96/26736; Vascular
Endothelial Growth Factor-D (VEGF-D), as disclosed in International
Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D), as disclosed in International Publication Number
WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as
disclosed in German Patent Number DE19639601. The above mentioned
references are incorporated herein by reference herein.
[0293] In an additional embodiment, the compositions of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0294] In additional embodiments, the compositions of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
[0295] This invention is also related to the use of the human
TIMP-4 gene as part of a diagnostic assay for detecting diseases or
susceptibility to diseases related to the presence of mutated human
TIMP-4.
[0296] Individuals carrying mutations in the human TIMP-4 gene may
be detected at the DNA level by a variety of techniques. Nucleic
acids for diagnosis may be obtained from a patient's cells, such as
from blood, urine, saliva, tissue biopsy and autopsy material. The
genomic DNA may be used directly for detection or may be amplified
enzymatically by using PCR (Saiki et al., Nature, 324:163-166
(1986)) prior to analysis. RNA or cDNA may also be used for the
same purpose. As an example, PCR primers complementary to the
nucleic acid encoding human TIMP-4 can be used to identify and
analyze human TIMP-4 mutations. For example, deletions and
insertions can be detected by a change in size of the amplified
product in comparison to the normal genotype. Point mutations can
be identified by hybridizing amplified DNA to radiolabeled human
TIMP-4 RNA or alternatively, radiolabeled human TIMP-4 antisense
DNA sequences. Perfectly matched sequences can be distinguished
from mismatched duplexes by RNase A digestion or by differences in
melting temperatures.
[0297] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230:1242 (1985)).
[0298] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., PNAS, USA,
85:4397-4401 (1985)).
[0299] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting of genomic DNA.
[0300] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0301] The present invention also relates to a diagnostic assay for
detecting altered levels of human TIMP-4 protein in various tissues
since an over-expression of the proteins compared to normal control
tissue samples may detect the presence of a disease or
susceptibility to a disease regulated by human TIMP-4. Assays used
to detect levels of human TIMP-4 protein in a sample derived from a
host are well-known to those of skill in the art and include
radioimmunoassays, competitive-binding assays, Western Blot
analysis, ELISA assays and "sandwich" assay. An ELISA assay
(Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6,
(1991)) initially comprises preparing an antibody specific to the
human TIMP-4 antigen, preferably a monoclonal antibody. In addition
a reporter antibody is prepared against the monoclonal antibody. To
the reporter antibody is attached a detectable reagent such as
radioactivity, fluorescence or, in this example, a horseradish
peroxidase enzyme. A sample is removed from a host and incubated on
a solid support, e.g. a polystyrene dish, that binds the proteins
in the sample. Any free protein binding sites on the dish are then
covered by incubating with a non-specific protein like BSA. Next,
the monoclonal antibody is incubated in the dish during which time
the monoclonal antibodies attach to any human TIMP-4 proteins
attached to the polystyrene dish. All unbound monoclonal antibody
is washed out with buffer. The reporter antibody linked to
horseradish peroxidase is now placed in the dish resulting in
binding of the reporter antibody to any monoclonal antibody bound
to human TIMP-4. Unattached reporter antibody is then washed out.
Peroxidase substrates are then added to the dish and the amount of
color developed in a given time period is a measurement of the
amount of human TIMP-4 protein present in a given volume of patient
sample when compared against a standard curve.
[0302] A competition assay may be employed wherein antibodies
specific to human TIMP-4 are attached to a solid support and
labeled human TIMP-4 and a sample derived from the host are passed
over the solid support and the amount of label detected, for
example by liquid scintillation chromatography, can be correlated
to a quantity of human TIMP-4 in the sample.
[0303] A "sandwich" assay is similar to an ELISA assay. In a
"sandwich" assay human TIMP-4 is passed over a solid support and
binds to antibody attached to a solid support. A second antibody is
then bound to the human TIMP-4. A third antibody which is labeled
and specific to the second antibody is then passed over the solid
support and binds to the second antibody and an amount can then be
quantitated.
[0304] This invention also provides a method of screening compounds
to identify those which are agonists or antagonists to be human
TIMP-4 polypeptide. An example of such a method comprises obtaining
mammalian tissue comprising an extra-cellular matrix, for example,
bovine radiocarpal joints. The articular cartilage is cut into
smaller disks and labeled with .sup.35S-sodium sulfate (10 micro
Ci/ml) in DMEM for a sufficient time for the cartilage to
incorporate the labeled Sodium sulfate. An MMP, for example,
stromelysin, or IL1 or TNF is then added to the cartilage disks
under appropriate conditions such that tissue breakdown would
normally occur. Human TIMP-4 and the compounds to be screened are
then added to the reaction mixture for a sufficient time for the
MMP to normally break down the cartilage disks. The supernatant,
which is the media outside the cartilage disks, is then collected
and radioactivity is counted by a liquid scintillation counter. The
percentage of 35S released into the media is then calculated. This
release of .sup.35S-GAG is representative of the proteoglycan pool
in the extracellular matrix of cartilage, and reflects proteoglycan
degradation by the MMP. The amount of .sup.35S-GAG, as determined
by liquid scintillation chromatography, is then compared to a
control assay done in the absence of the compound to be screened
and the ability of the compound to agonize or antagonize the action
of human TIMP-4 may then be determined.
[0305] Examples of potential human TIMP-4 antagonists, in addition
to those identified above, include an antibody, or in some cases,
an oligonucleotide, which binds to the polypeptide. Alternatively,
a potential antagonist may be a mutated form of human TIMP-4, which
recognizes natural substrates, but is inactive, and thereby prevent
the action of human TIMP-4.
[0306] Potential human TIMP-4 antagonists also include antisense
constructs prepared using antisense technology. Antisense
technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or RNA. For
example, the 5' coding portion of the polynucleotide sequence,
which encodes for the mature polypeptides of the present invention,
is used to design an antisense RNA oligonucleotide of from about 10
to 40 base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
(triple helix--see Lee et al., Nucl. Acids Res., 6:3073 (1979);
Cooney et al, Science, 241:456 (1988); and Dervan et al., Science,
251: 1360 (1991)), thereby preventing transcription and the
production of human TIMP-4. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA
molecule into the human TIMP-4 (antisense--Okano, J. Neurochem.,
56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of human TIMP-4.
[0307] Another potential human TIMP-4 antagonist is a small
molecule which binds to and occupies the active site of the human
TIMP-4 thereby preventing human TIMP-4 from interacting with MMP's
such that normal biological activity is prevented. Examples of
small molecules include but are not limited to small peptides or
peptide-like molecules, for example a peptide-bonded molecule.
[0308] The human TIMP-4 antagonists may be employed for tissue
repair and remodeling, for example, where destruction of scar
tissue is desired. In some situations, enhanced connective tissue
turnover or remodeling may be desirable, e.g. in resorption of scar
tissue; in uterine involution post-partum; in remodeling of
fibrotic deposits in the lung, liver or joints. To appropriately
control turnover of extra-cellular matrix proteins in these
situations would require a balance between the MMP's and human
TIMP-4 to appropriately control degradation.
[0309] The polypeptides and agonists or antagonists that are also
polypeptides may be employed in accordance with the present
invention by expression of such polypeptides in vivo, which is
often referred to as "gene therapy."
[0310] Gene Therapy
[0311] The invention also encompasses gene therapy methods for
treating or preventing disorders, diseases and conditions, such as,
for example restenosis. Vectors and techniques described herein
(e.g, below or in the Antibody section of the application) or known
in the art may be routinely applied or modified for such therapy.
Gene therapy methods relate to the introduction of nucleic acid
(DNA, RNA and antisense DNA or RNA) sequence of the invention into
an animal to achieve expression of the TIMP-4 polypeptide of the
present invention. This method requires a polynucleotide which
codes for a TIMP-4 polypeptide operatively linked to a promoter and
any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery
techniques are known in the art, see, for example, WO90/11092,
which is herein incorporated by reference.
[0312] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a TIMP-4 polynucleotide ex vivo, with the engineered
cells then being provided to a patient to be treated with the
polypeptide. Such methods are well-known in the art. For example,
see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216
(1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112
(1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994);
Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et
al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene
Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene
Therapy 3: 31-38 (1996)), which are herein incorporated by
reference. In one embodiment, the cells which are engineered are
arterial cells. The arterial cells may be reintroduced into the
patient through direct injection to the artery, the tissues
surrounding the artery, or through catheter injection.
[0313] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang, et al., 1995, Gene Therapy
2:775-783.
[0314] As discussed in more detail herein (e.g., below and in the
antibody section of the application), the TIMP-4 polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The TIMP-4 polynucleotide constructs may be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0315] In one embodiment, the TIMP-4 polynucleotide is delivered as
a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA
refers to sequences that are free from any delivery vehicle that
acts to assist, promote or facilitate entry into the cell,
including viral sequences, viral particles, liposome formulations,
lipofectin or precipitating agents and the like. However, the
TIMP-4 polynucleotides can also be delivered in liposome
formulations and lipofectin formulations and the like can be
prepared by methods well known to those skilled in the art. Such
methods are described, for example, in U.S. Pat. Nos. 5,593,972,
5,589,466, and 5,580,859, which are herein incorporated by
reference.
[0316] The TIMP-4 polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2
available from Invitrogen. Other suitable vectors will be readily
apparent to the skilled artisan.
[0317] Any strong promoter known to those skilled in the art can be
used for driving the expression of TIMP-4 polynucleotide sequence.
Suitable promoters include adenoviral promoters, such as the
adenoviral major late promoter; or heterologous promoters, such as
the cytomegalovirus (CMV) promoter; the respiratory syncytial virus
(RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for TIMP-4.
[0318] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0319] The TIMP-4 polynucleotide construct can be delivered to the
interstitial space of tissues within the an animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular, fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0320] For the naked nucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0321] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
TIMP-4 DNA constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[0322] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0323] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0324] In certain embodiments, the TIMP-4 polynucleotide constructs
are complexed in a liposome preparation. Liposomal preparations for
use in the instant invention include cationic (positively charged),
anionic (negatively charged) and neutral preparations. However,
cationic liposomes are particularly preferred because a tight
charge complex can be formed between the cationic liposome and the
polyanionic nucleic acid. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad.
Sci. USA (1989) 86:6077-6081, which is herein incorporated by
reference); and purified transcription factors (Debs et al., J.
Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by
reference), in functional form.
[0325] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner
et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-7416, which is
herein incorporated by reference). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0326] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimet- hylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0327] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0328] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0329] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983),
101:512-527, which is herein incorporated by reference. For
example, MLVs containing nucleic acid can be prepared by depositing
a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating with a solution of the material to be
encapsulated. SUVs are prepared by extended sonication of MLVs to
produce a homogeneous population of unilamellar liposomes. The
material to be entrapped is added to a suspension of preformed MLVs
and then sonicated. When using liposomes containing cationic
lipids, the dried lipid film is resuspended in an appropriate
solution such as sterile water or an isotonic buffer solution such
as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are
mixed directly with the DNA. The liposome and DNA form a very
stable complex due to binding of the positively charged liposomes
to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in
the art. Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell (1979) 17:77); ether injection (Deamer, D. and
Bangham, A., Biochim. Biophys. Acta (1976) 443:629; Ostro et al.,
Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc.
Natl. Acad. Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76:145);
and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
(1980) 255:10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl.
Acad. Sci. USA (1978) 75:145; Schaefer-Ridder et al., Science
(1982) 215:166), which are herein incorporated by reference.
[0330] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0331] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication no. WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0332] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding TIMP-4. Retroviruses from which the retroviral
plasmid vectors may be derived include, but are not limited to,
Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape
leukemia virus, human immunodeficiency virus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
[0333] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, .PSI.-2, .PSI.-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0334] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding TIMP-4. Such
retroviral vector particles then may be employed, to transduce
eukaryotic cells, either in vitro or in vivo. The transduced
eukaryotic cells will express TIMP-4.
[0335] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with TIMP-4 polynucleotide contained in an adenovirus
vector. Adenovirus can be manipulated such that it encodes and
expresses TIMP-4, and at the same time is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle.
Adenovirus expression is achieved without integration of the viral
DNA into the host cell chromosome, thereby alleviating concerns
about insertional mutagenesis. Furthermore, adenoviruses have been
used as live enteric vaccines for many years with an excellent
safety profile (Schwartz, A. R. et al. (1974) Am. Rev. Respir.
Dis.109:233-238). Finally, adenovirus mediated gene transfer has
been demonstrated in a number of instances including transfer of
alpha-1-antitrypsin and CFTR to the lungs of cotton rats
(Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et
al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.
Acad. Sci. USA 76:6606).
[0336] In cases where an adenovirus is used as an expression
vector, the TIMP-4 coding sequence of interest may be ligated to an
adenovirus transcription/translation control complex, e.g., 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 (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the TIMP-4 molecule
in infected hosts. (e.g., see Logan & Shenk, 1984, Proc. Natl.
Acad. Sci. USA 81:355-359). Specific initiation signals may also be
required for efficient translation of inserted antibody coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. 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., 1987, Methods in Enzymol. 153:51-544).
[0337] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0338] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); U.S. Pat. No. 6,040,174, U.S. Pat. No.
6,013,638, and U.S. Pat. No. 5,652,224, each of which are herein
incorporated by reference in its entirety. For example, the
adenovirus vector Ad2 is useful and can be grown in human 293
cells. These cells contain the E1 region of adenovirus and
constitutively express E1a and E1b, which complement the defective
adenoviruses by providing the products of the genes deleted from
the vector. In addition to Ad2, other varieties of adenovirus
(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
(See e.g., U.S. Pat. Nos. 6,040,174 and 6,013,638, the contents of
each of which are incorporated by reference in its entirety).
[0339] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0340] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377, the contents of
each of which are herein incorporated by reference in its
entirety.
[0341] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The TIMP-4
polynucleotide construct is inserted into the AAV vector using
standard cloning methods, such as those found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). The recombinant AAV vector is then transfected into
packaging cells which are infected with a helper virus, using any
standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
TIMP-4 polynucleotide construct. These viral particles are then
used to transduce eukaryotic cells, either ex vivo or in vivo. The
transduced cells will contain the TIMP-4 polynucleotide construct
integrated into its genome, and will express TIMP-4.
[0342] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding TIMP-4) via homologous recombination (see,
e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International
Publication No. WO 96/29411, published Sep. 26, 1996; International
Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et
al., Nature 342:435-438 (1989). This method involves the activation
of a gene which is present in the target cells, but which is not
normally expressed in the cells, or is expressed at a lower level
than desired.
[0343] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the TIMP-4 desired endogenous polynucleotide
sequence so the promoter will be operably linked to the endogenous
sequence upon homologous recombination.
[0344] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0345] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0346] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous TIMP-4
sequence is placed under the control of the promoter. The promoter
then drives the expression of the endogenous TIMP-4 sequence.
[0347] The polynucleotides encoding TIMP-4 may be administered
along with other polynucleotides encoding an angiogenic protein.
Examples of angiogenic proteins include, but are not limited to,
acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3,
epidermal growth factor alpha and beta, platelet-derived
endothelial cell growth factor, platelet-derived growth factor,
tumor necrosis factor alpha, hepatocyte growth factor, insulin like
growth factor, colony stimulating factor, macrophage colony
stimulating factor, granulocyte/macrophage colony stimulating
factor, and nitric oxide synthase.
[0348] Preferably, the polynucleotide encoding TIMP-4 contains a
secretory signal sequence that facilitates secretion of the
protein. Typically, the signal sequence is positioned in the coding
region of the polynucleotide to be expressed towards or at the 5'
end of the coding region. The signal sequence may be homologous or
heterologous to the polynucleotide of interest and may be
homologous or heterologous to the cells to be transfected.
Additionally, the signal sequence may be chemically synthesized
using methods known in the art.
[0349] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0350] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0351] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0352] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0353] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0354] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0355] Therapeutic compositions of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
[0356] Kits
[0357] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the pharmaceutical compositions
may be employed in conjunction with other therapeutic
compounds.
[0358] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0359] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region is used to rapidly select primers
that do not span more than one exon in the genomic DNA, thus
complicating the amplification process. These primers are then used
for PCR screening of somatic cell hybrids containing individual
human chromosomes. Only those hybrids containing the human gene
corresponding to the primer will yield an amplified fragment.
[0360] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0361] Fluorescence in situ hybridization (FISH) of a cDNA clones
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bases. For a review of this technique,
see Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
[0362] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0363] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0364] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0365] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0366] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0367] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0368] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0369] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0370] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0371] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0372] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 microgram of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 microliters of buffer solution. For the purpose of
isolating DNA fragments for plasmid construction, typically 5 to 50
micrograms of DNA are digested with 20 to 250 units of enzyme in a
larger volume. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the manufacturer.
Incubation times of about 1 hour at 37.degree. C. are ordinarily
used, but may vary in accordance with the supplier's instructions.
After digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0373] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8:4057 (1980).
[0374] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0375] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units to
T4 DNA ligase ("ligase") per 0.5 micrograms of approximately
equimolar amounts of the DNA fragments to be ligated.
[0376] Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
[0377] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, Detailed Description, and Examples of this
specification is hereby incorporated by reference in its
entirety.
[0378] In addition, the entire disclosure, including the
specifications and sequence listings, of related U.S. application
Ser. No. 09/901,904, filed Jul. 11, 2001; Ser. No. 09/387,525,
filed Sep. 1, 1999; Ser. No. 08/463,261, filed Jun. 5, 1995; No.
60/217,419, filed Jul. 11, 2000; No. 60/220,829, filed Jul. 26,
2000; and International Application No. PCT/US94/14498, filed Dec.
13, 1994 (in English), are each hereby incorporated by reference in
their entireties.
[0379] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
[0380] Bacterial Expression and Purification of Human TIMP-4
[0381] The DNA sequence encoding for human TIMP-4, ATCC #75946, is
initially amplified using PCR oligonucleotide primers corresponding
to the 5' and sequences of the processed human TIMP-4 protein
(minus the signal peptide sequence) and the vector sequences 3' to
the TIMP-4 gene. Additional nucleotides corresponding to human
TIMP-4 were added to the 5' and 3' sequences respectively. The 5'
oligonucleotide primer has the sequence 5'
GCCAGAGGATCCTGCAGCTGCGCCCCGGCGCAC 3' (SEQ ID NO:3) contains a BamH1
restriction enzyme site followed by 21 nucleotides of human TIMP-4
coding sequence starting from the presumed terminal amino acid of
the processed protein codon. The 3' sequence
5'CGGCTTCTAGAACTAGGGCTGAACGATGTC- AAC 3' (SEQ ID NO:4) contains an
XbaI site and is followed by 18 nucleotides of human TIMP-4. The
restriction enzyme sites correspond to the restriction enzyme sites
on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, Calif., 91311). pQE-9 encodes antibiotic
resistance (Amp.sup.r), a bacterial origin of replication (ori), an
IPTG-regulatable promoter operator (P/O), a ribosome binding site
(RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then
digested with BamHI and XbaI. The amplified sequences were ligated
into pQE-9 and were inserted in frame with the sequence encoding
for the histidine tag and the RBS. The ligation mixture was then
used to transform E. coli strain m15/pREP4 available from Qiagen by
the procedure described in Sambrook, J. et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Laboratory Press, (1989).
m15/pREP4 contains multiple copies of the plasmid pREP4, which
expresses the lacI repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies were
selected. Plasmid DNA was isolated and confirmed by restriction
analysis.
[0382] Clones containing the desired constructs were grown
overnight (O/N) in liquid culture in LB media supplemented with
both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to
inoculate a large culture at a ratio of 1:100 to 1:250. The cells
were grown to an optical density 600 (O.D..sup.600) of between 0.4
and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then
added to a final concentration of 1 mM. IPTG induces by
inactivating the lacI repressor, clearing the P/O leading to
increased gene expression. Cells were grown an extra 3 to 4 hours.
Cells were then harvested by centrifugation. The cell pellet was
solubilized in the chaotropic agent 6 Molar Guanidine HCl. After
clarification, solubilized human TIMP-4 was purified from this
solution by chromatography on a Nickel-Chelate column under
conditions that allow for tight binding by proteins containing the
6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184
(1984). Human TIMP-4 (90% pure) was eluted from the column in 6
molar guanidine HCl pH 5.0 and for the purpose of renaturation
adjusted to 3 molar guanidine HCl, 100 nM sodium phosphate, 10
mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized).
After incubation in this solution for 12 hours the protein was
dialyzed to 10 mmolar sodium phosphate.
EXAMPLE 2
[0383] Expression of Recombinant Human TIMP-4 in COS Cells
[0384] The expression of human TIMP-4 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication,
2) ampicillin resistance gene, 3) E. coli replication origin, 4)
CMV promoter followed by a polylinker region, a SV40 intron and
polyadenylation site. A DNA fragment encoding the entire human
TIMP-4 precursor and a HA tag fused in frame to its 3' end was
cloned into the polylinker region of the vector, therefore, the
recombinant protein expression is directed under the CMV promoter.
The HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (I. Wilson, H. Niman,
R. Heighten, A Cherenson, M. Connolly, and R. Lemer, 1984, Cell 37,
767). The fusion of HA tag to the target protein allows easy
detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0385] The plasmid construction strategy is described as
follows:
[0386] The DNA sequence ATCC #75946, encoding for human TIMP-4 was
constructed by PCR using two primers: the 5' primer 5'
GCCAGAGGATCCGC CACCATGCCTGGGAGCCCTCGGCCC 3' (SEQ ID NO:5) contains
a BamHI site followed by 21 nucleotides of human TIMP-4 coding
sequence starting from the initiation codon; the 3' sequence
5'CGGCTTCTAGAATCAAGCGTAGTCTGGGACGTCG TATGGGTAGGGCTGAACGATGTCAAC 3'
(SEQ ID NO:6) contains complementary sequences to an XbaI site,
translation stop codon, HA tag and the last 18 nucleotides of the
human TIMP-4 coding sequence (not including the stop codon).
Therefore, the PCR product contains a BamHI site, human TIMP-4
coding sequence followed by HA tag fused in frame, a translation
termination stop codon next to the HA tag, and an XbaI site. The
PCR amplified DNA fragment and the vector, pcDNAI/Amp, were
digested with BamHI and XbaI restriction enzyme and ligated. The
ligation mixture was transformed into E. coli strain SURE
(available from Stratagene Cloning Systems, 11099 North Torrey
Pines Road, La Jolla, Calif. 92037) the transformed culture was
plated on ampicillin media plates and resistant colonies were
selected. Plasmid DNA was isolated from transformants and examined
by restriction analysis for the presence of the correct fragment.
For expression of the recombinant human TIMP-4, COS cells were
transfected with the expression vector by DEAE-DEXTRAN method (J.
Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory Press, (1989)). The expression of
the human TIMP-4 HA protein was detected by radiolabelling and
immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)).
Cells were labelled for 8 hours with .sup.35S-cysteine two days
post transfection. Culture media were then collected and cells were
lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS,
1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id.
37:767 (1984)). Both cell lysate and culture media were
precipitated with a HA specific monoclonal antibody. Proteins
precipitated were analyzed on 15% SDS-PAGE gels.
EXAMPLE 3
[0387] Cloning and Expression of TIMP-4 Using the Baculovirus
Expression System
[0388] The DNA sequence encoding the full length TIMP-4 protein,
ATCC #75946, was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
[0389] The 5' primer has the sequence 5' GCCAGAGGATCCATGCCTGG
GAGCCCTCGGCCC 3' (SEQ ID NO:7) and contains a BamHI restriction
enzyme site (in bold) just behind the first 21 nucleotides of the
TIMP-4 gene (the initiation codon for translation "ATG" is
underlined).
[0390] The 3' primer has the sequence 5'CGGCTTCTAGAACTAGGGCTG
AACGATGTCAAC 3' (SEQ ID NO:8) and contains the cleavage site for
the restriction endonuclease XbaI and 18 nucleotides complementary
to the 3' non-translated sequence of the TIMP-4 gene. The amplified
sequences were isolated from a 1% agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Calif.). The
fragment was then digested with the endonucleases BamHI and XbaI
and then purified again on a 1% agarose gel. This fragment is
designated F2.
[0391] The vector pA2 (modification of pVL941 vector, discussed
below) is used for the expression of the TIMP-4 protein using the
baculovirus expression system (for review see: Summers, M. D. and
Smith, G. E. 1987, A manual of methods for baculovirus vectors and
insect cell culture procedures, Texas Agricultural Experimental
Station Bulletin No. 1555). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhidrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamHI and XbaI. The polyadenylation
site of the simian virus (SV)40 is used for efficient
polyadenylation. For an easy selection of recombinant viruses the
beta-galactosidase gene from E. coli is inserted in the same
orientation as the polyhedrin promoter followed by the
polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the
cell-mediated homologous recombination of co-transfected wild-type
viral DNA. Many other baculovirus vectors could be used in place of
pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers,
M. D., Virology, 170:31-39).
[0392] The plasmid was digested with the restriction enzymes BamHI
and XbaI. The DNA was then isolated from a 1% agarose gel using the
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla,
Calif.). This vector DNA is designated V2.
[0393] Fragment F2 and the plasmid V2 were ligated with T4 DNA
ligase. E. coli HB101 cells were then transformed and bacteria
identified that contained the plasmid (pBacTIMP-4) with the TIMP-4
gene using the enzymes BamHI and XbaI. The sequence of the cloned
fragment was confirmed by DNA sequencing.
[0394] 5 micrograms of the plasmid pBacTIMP-4 was co-transfected
with 1.0 microgram of a commercially available linearized
baculovirus ("BaculoGold.TM. baculovirus DNA", Pharmingen, San
Diego, Calif.) using the lipofection method (Felgner et al. Proc.
Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
[0395] One microgram of BaculoGold.TM. virus DNA and 5 micrograsm
of the plasmid pBacTIMP-4 were mixed in a sterile well of a
microtiter plate containing 50 microliters of serum free Grace's
medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards 10
microliters Lipofectin plus 90 microliters Grace's medium were
added, mixed and incubated for 15 minutes at room temperature. Then
the transfection mixture was added drop-wise to the Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate was rocked back and
forth to mix the newly added solution. The plate was then incubated
for 5 hours at 27 degreec. After 5 hours the transfection solution
was removed from the plate and 1 ml of Grace's insect medium
supplemented with 10% fetal calf serum was added. The plate was put
back into an incubator and cultivation continued at 27 degree C.
for four days.
[0396] After four days the supernatant was collected and a plaque
assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies
Inc., Gaithersburg) was used which allows an easy isolation of blue
stained plaques. (A detailed description of a "plaque assay" can
also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0397] Four days after the serial dilution, the viruses were added
to the cells and blue stained plaques were picked with the tip of
an Eppendorf pipette. The agar containing the recombinant viruses
was then resuspended in an Eppendorf tube containing 200
microliters of Grace's medium. The agar was removed by a brief
centrifugation and the supernatant containing the recombinant
baculovirus was used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes were
harvested and then stored at 4 degree C.
[0398] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-TIMP-4 at a multiplicity of infection (MOI) of 2. Six
hours later the medium was removed and replaced with SF900 II
medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 microCi of .sup.35S-methionine and
5 microCi 35S cysteine (Amersham) were added. The cells were
further incubated for 16 hours before they were harvested by
centrifugation and the labelled proteins visualized by SDS-PAGE and
autoradiography.
EXAMPLE 4
[0399] Expression Pattern of Human TIMP-4 in Human Tissues
[0400] 20 micrograms of total RNA from each of the above tissues
was denatured and run on a 1.2% formaldehyde agarose gel and
capillary blotted onto a nylon filter overnight. RNA was
immobilized on the filter by UV cross-linking. A random primer
probe was prepared from the EcoRI-Xhol insert of the partial TIMP-4
nucleic acid sequence and used to probe the blot by overnight
hybridization in Church buffer with 100 .mu.g/ml denatured herring
sperm DNA as a blocking agent. Washing was performed sequentially
with 2.times.SSC/0.1% SDA and 0.2.times.SSC/0.1% SDS at 65 degrees
Celsius.
EXAMPLE 5
[0401] Expression via Gene Therapy
[0402] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin, is added. This is
then incubated at 37 degreeC. for approximately one week. At this
time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of
fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
[0403] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0404] The cDNA encoding a polypeptide of the present invention is
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively. The 5' primer contains an EcoRI site and
the 3' primer further includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is used to transform bacteria HB101, which are then plated onto
agar-containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
[0405] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles
containing the gene (the packaging cells are now referred to as
producer cells).
[0406] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his.
[0407] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product.
EXAMPLE 6
[0408] Production of an Antibody
[0409] a) Hybridoma Technology
[0410] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing TIMP-4 are administered
to an animal to induce the production of sera containing polyclonal
antibodies. In a preferred method, a preparation of TIMP-4 protein
is prepared and purified to render it substantially free of natural
contaminants. Such a preparation is then introduced into an animal
in order to produce polyclonal antisera of greater specific
activity.
[0411] Monoclonal antibodies specific for protein TIMP-4 are
prepared using hybridoma technology. (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., in:
Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.
563-681 (1981)). In general, an animal (preferably a mouse) is
immunized with TIMP-4 polypeptide or, more preferably, with a
secreted TIMP-4 polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 g/l of nonessential
amino acids, about 1,000 U/ml of penicillin, and about 100 .mu.g/ml
of streptomycin.
[0412] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP20), available
from the ATCC. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981). The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the TIMP-4 polypeptide.
[0413] Alternatively, additional antibodies capable of binding to
TIMP-4 polypeptide can be produced in a two-step procedure using
anti-idiotypic antibodies. Such a method makes use of the fact that
antibodies are themselves antigens, and therefore, it is possible
to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the TIMP-4 protein-specific
antibody can be blocked by TIMP-4. Such antibodies comprise
anti-idiotypic antibodies to the TIMP-4 protein-specific antibody
and are used to immunize an animal to induce formation of further
TIMP-4 protein-specific antibodies.
[0414] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed infra.
(See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neubergeret al., Nature
314:268 (1985).)
[0415] b) Isolation of Antibody Fragments Directed Against TIMP-4
From a Library of scFvs
[0416] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against TIMP-4 to which the donor may or may not have
been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein
by reference in its entirety).
[0417] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2xTY containing 1% glucose and 100 .mu.g/ml of
ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.
Five ml of this culture is used to innoculate 50 ml of
2xTY-AMP-GLU, 2.times.10.sup.8 TU of delta gene 3 helper (M 13
delta gene III, see PCT publication WO 92/01047) are added and the
culture incubated at 37.degree. C. for 45 minutes without shaking
and then at 37.degree. C. for 45 minutes with shaking. The culture
is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2xTY containing 100 .mu.g/ml ampicillin
and 50 ug/ml kanamycin and grown overnight. Phage are prepared as
described in PCT publication WO 92/01047.
[0418] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2xTY broth containing 100 .mu.g ampicillin/ml
and 25 .mu.g kanamycin/ml (2xTY-AMP-KAN) and grown overnight,
shaking at 37.degree. C. Phage particles are purified and
concentrated from the culture medium by two PEG-precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through
a 0.45 .mu.m filter (Minisart NML; Sartorius) to give a final
concentration of approximately 1013 transducing units/ml
(ampicillin-resistant clones).
[0419] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0420] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing.
EXAMPLE 7
[0421] Adenoviral Mediated Gene Therapy
[0422] Adenoviral expression constructs were used to express human
and rat TIMP-4 polypeptides in rat thoracic aorta smooth muscle
cells and human and porcine coronary artery smooth muscle cells in
vitro, as well as in an in vivo rat model of carotid artery balloon
injury.
[0423] Adenoviral Constructions:
[0424] The human cDNA sequence encoding the full length TIMP-4
protein, contained in ATCC Deposit No. 75946, was isolated by PCR
from clone HGFAM58 using oligonucleotide primers corresponding to
the 5' and 3' sequence of the gene. The 5' primer has the sequence
5'CCGGAATTCCACCATGCCTGGGAGCCCTCG 3' (SEQ ID NO:9) and contains an
EcoRI restriction enzyme site behind the first 17 nucleotides of
the TIMP-4 gene. The 3' primer has the sequence 5'
ATCTTTGGTACCTTTCTAGAACTAGGGCTG 3' (SEQ ID NO: 10) and contains a
XbaI restriction enzyme site and 14 nucleotides complementary to
the 3' non-translated sequence of the TIMP-4 gene.
[0425] The rat cDNA sequence encoding the full length TIMP-4
protein was isolated by RT-PCR using RNA extracted from rat aortic
smooth muscle cells. Briefly, 2 .mu.g of total RNA was reverse
transcribed in 20 .mu.l final reaction volume in the presence of
the four dNTPs, 100 pmol of pdN6 (Boehringer Mannheim, Ingelheim,
Germany) and 200IU of reverse transcriptase (Superscript, Promega,
France). The reaction was performed at 37.degree. C. for 75 min.
Then, the enzyme was denatured at 90.degree. C. for 5 min. 2 .mu.l
of the reaction was used as a template for PCR using specific
oligonucleotide promoters for the rat TIMP-4. The sense primer has
the sequence 5'CCGGAATTCCACCATGCCCTGGAGTCCC3' (SEQ ID NO:11) and
contains an EcoRI restriction enzyme site behind the first 17
nucleotides of the TIMP-4 gene. The reverse primer has the sequence
5' CTAGTCTAGACTAGGGCTGGACGATGTCAA 3' (SEQ ID NO:12) and contains a
XbaI restriction enzyme site and 24 nucleotides complementary to
the 3' non-translated sequence of the TIMP-4 gene.
[0426] The amplified sequences were isolated from a 1% agarose gel
using a commercially available kit ("Qiaquick", Qiagen,
Courtaboeuf, France), and then was EcoRI/Xba I subcloned in a
transfer vector containing the CMV promoter. This transfer vector
contains the Ad5 1-458 region followed by the CMV enhancer/promoter
and a chimeric intron generated by combining the splice donor from
the human beta-globin intron 1 and the splice acceptor from the IgG
intervening sequence obtained from pCI plasmid (Promega,
Charbonnieres, France). A poly-linker containing, among others, the
recognition sites for the restriction endonucleases XbaI and EcoRI,
was inserted upstream to the bovine growth hormone polyadenylation
site followed by the Ad5 3511-5788 region. This vector contains the
ampicillin resistance gene.
[0427] The E1/E3-deleted adenoviral vector containing the gene
encoding TIMP-4 (named AdTG14854) was obtained by homologous
recombination in Escherichia coli BJ (Chartier et al., J. Virol.
70(7): 4805-10 (1996)), between the TIMP-4 transfer vector (named
pTG14846 for human and pTG14847 for rat; see FIGS. 3A and 3B) and
the adenoviral DNA plasmid (named pTG6624; see FIG. 3F) linearized
by ClaI. The adenoviral vector containing human TIMP-4 (pTG14854;
FIG. 3C) was deposited at the Collection Nationale de Cultures de
Microorganismes, Institute Pasteur (25 Rue du Docteur Roux, F-74724
Paris Cedex 15, France), on Jul. 9, 2001, and received deposit
registration number CNCM 1-2696.
[0428] Virus propagation, purification and titration of infectious
units (iu) by indirect immunofluorescence of the viral DNA binding
protein were carried out as described previously (Lusky et al., J.
Virol. 72(3):2022-32 (1998)). Purified virus was stored in viral
storage buffer (1 M sucrose, 10 mM Tris-HCl [pH=8.5], 1 mM
MgCl.sub.2, 150 mM NaCl, 0.005% [vol/vol] Tween 80). Bacteria
comprising DNA plasmid containing human TIMP-4.
[0429] Cells and Culture Conditions:
[0430] Rat thoracic aorta smooth muscle cells were isolated from
normal rats (ratAoSMCs) and from injured rats 15 days after balloon
catheter deendothelialization (ratIT15) by enzymatic digestion as
previously described (Orlandi at al., Arterioscler. Thromb.
14(6):982-9 (1994)). Porcine coronary artery SMCs were isolated
from normal pigs (pigCoSMCs) and from injured animals 15 days after
stent placement (pigIT15) by enzymatic digestion as previously
described (Christen et al., Circ. Res. 85(1):99-107 (1999)). The
human coronary artery SMCs were purchased from Clonetics
(Walkersville, Md., USA). Rat and pig cells were cultured in DMEM
containing 10% FCS (Life Technologies, Cergy-Pontoise, France).
Human cells were cultured in SMGM2 medium containing 5% FCS
(Bioproducts, Gagny, France).
[0431] Adenoviral Cell Infection:
[0432] SMCs were infected in suspension at an MOI corresponding to
80% of infected cells. Briefly, cells were trypsinized, centrifuged
and then resuspended in 2% FCS cell culture medium
(5.times.10.sup.6 cells in 500 .mu.l). The virus was added for a 30
min. incubation time at 37.degree. C., 5% CO2. Cells were rinsed in
fresh medium and finally resuspended and plated in 10% or 5% FCS
corresponding medium.
[0433] Gelatin Zymography:
[0434] Recombinant human MMP2 was purchased from R&D System
(Oxon, UK) and used at 2.5 ng in the gel. Briefly, cell lysates
(from 2.times.10.sup.5 cells) were mixed with Novex tris-Glycine
Sample Buffer and let stand 10 minutes at room temperature. Samples
(20 .mu.l) were then subjected to electrophoresis on 10%
Tris-Glycine gel with 0.1% gelatine incorporated as a substrate.
Gels were washed in Novex Renaturing Buffer with gentle agitation
for 30 min. at room temperature. Renaturing Buffer is then decanted
and replaced with Developing Buffer for a 4 hour incubation at
37.degree. C. Gels were stained with Coomassie Blue R-250 for 30
min. Metalloproteinase produced clear areas of lysis in the
gel.
[0435] Rat Carotid Artery Balloon Injury Model:
[0436] Adult male Wistar rats (body weight >400 g) were used for
experiments (Iffa-Credo). Anesthesia was induced with
intraperitoneal injection of Ketamine (Imalgene, Rhne-Mrieux, Lyon,
France) and Acepromazin (Vetranquil 0.5%, Sanofi, Libourne, France)
in doses of 23.1 and 3.84 mg/kg respectively. Animals were
anticoagulated with intravenous injection of 200U/kg of human
heparin (Choay, Sanofi Winthrop, Gentilly, France). The left common
carotid artery was surgically exposed and an arteriotomy was made
on the left external carotid artery. Deendothelialization was
achieved by three passages of a 2F Fogarty balloon catheter
(Baxter, Maurepas, France) filled with 0.2 ml air. A 1 cm length
segment of the carotid was isolated with microsurgical clamps and a
24-gauge catheter was introduced through the arteriotomy. The
segment was flushed with 0.2 ml NaCl 0.9% and 50 .mu.l of
adenoviral solution (2.times.10.sup.9 iu) was infused. The solution
was allowed to dwell in the carotid for 5 minutes during which the
carotid segment remained distended. The solution was withdrawn, the
external carotid artery was ligated and blood flow was
reestablished through the common and the internal carotid arteries.
Rats were sacrificed at D14 post injury. After lethal pentobarbital
injection and cannulation of the heart, vessels were perfused with
1.times. PBS solution and perfusion-fixed either with 2% or 4%
formaldehyde in PBS at normal blood pressure. Then, carotids were
excised and treated for histological analyses.
[0437] For morphometric analysis, carotids were fixed in 4%
formaldehyde and embedded in paraffin. Five .mu.m sections were
stained either with hematoxylin or with hematoxylin and eosin and
the media and intimal area, as well as medial and neointimal cell
number were evaluated by image analysis on 3 cross sections for
each vessel (NIH Image software).
[0438] TIMP-4 mRNA Expression
[0439] Total RNA was extracted (RNA Now reagent, Ozyme, Montigny,
France) at 24 and 48 hours after AdTG14854 (CMV-human TIMP-4; see
FIG. 3C) infection of human and porcine CaSMCs, and AdTG14855
(CMV-rat TIMP-4; see FIG. 3D) infection of rat AoSMCs. The presence
of the mRNA was detected by Northern blot. Briefly, 10 .mu.g of
total RNA extracted from each of the above cell populations were
denatured and run on a 1% formaldehyde agarose gel and capillary
blotted onto a Hybond nylon membrane overnight. RNA was fixed on
the membrane by heating at 80.degree. C. for 2 hours. A random
primer probe (Amersham Multi Prime Kit) was prepared from the
SmaI-KpnI insert of the partial TIMP-4 nucleic acid sequence and
used to probe the blot by 3 hour hybridization in Amersham
hybridization buffer containing 200 .mu.g/ml denatured herring
sperm DNA as a blocking agent. Washing was performed sequentially
with 1.times.SSC-0.1% SDS (2.times.15 min.) and 0.1.times.SSC-0.1%
SDS (1.times.10 min.). Results indicate that the exogeneous TIMP-4
mRNA is present in infected SMC of human and porcine coronary
arteries and rat aorta. In all cell types, two major bands were
observed in agreement with published data (Gomez, European Journal
of Cell Biology, 74:111 (1997)).
[0440] TIMP-4 Protein Expression
[0441] Total proteins were extracted from cells and culture
supernatants 2 days, 3 days, 5 days and 7 days after AdTG14854
infection of human and porcine CaSMCs, and AdTG14855 infection of
rat AoSMCs. The presence of TIMP-4 protein was detected by Western
blot. Briefly, 150 .mu.g of extracted proteins were run on a 10%
Nupage gel (Novex, Invitrogen, Groningen, the Netherlands), and
then blotted on a nitrocellulose membrane (Novex). The membrane was
incubated in a blocking solution (PBS-2% milk) overnight before
detection of the blotted antigen, using an anti-TIMP-4 antibody
(clone S720, Abcam, Cambridge, UK). Western blot detection of the
TIMP-4 protein indicates that TIMP-4 is present in large amounts in
human coronary cell supernatants and in lower amounts in pig cell
supernatants. In rat SMC supernatants, no protein was detected in
IT15 and only a faint signal in ratAo. These results indicate
either that the protein is not equally expressed by all cell types
or that the antibody does not recognize the rat TIMP-4. By
infecting human cells with the rat TIMP-4 adenovirus and the rat
cells with the human TIMP-4 adenovirus, it was observed by western
blot that the production was equivalent in the different cell types
for a defined vector suggesting that the antibody is specific for
the human protein.
[0442] TIMP-4 Activity
[0443] Staining of gelatin zymogram gels revealed a gelatin lysis
activity (MMP activity) with cell lysates corresponding to the
Ad-null infection whereas, no areas of gel lysis were observed with
cell lysate from Ad-TIMP-4 infected cells. Taken together with
Western blot results showing an equal amount of MMP2 in cells
infected either with Ad-null or Ad-TIMP-4 (data not shown), the
zymogram data indicated that the decrease of MMP activity was due
to the TIMP-4 inhibitory effect.
[0444] Effects on Cell Proliferation
[0445] SMC proliferation was studied in SMCs infected in suspension
at an MOI corresponding to 80% of infected cells. The results
indicate that on rat and pig cells, TIMP-4 is not able to
significantly inhibit cell growth. On human cells, an inhibitory
effect was observed which could be due to adenovirus toxicity. To
confirm this hypothesis, lower MOIs of AdTIMP-4 were used to infect
human cells. The results are summarized in Table 1:
1TABLE 1 MOI Adenovirus MOI 0 MOI 1 MOI 10 MOI 50 MOI 100 300
AdTG6401 100% 75% 75% 67% 44% 32.7% AdTG14854 100% 123% 84% 49% 30%
21.4%
[0446] An antiproliferative effect was observed at MOIs starting
from MOI 50. At this and higher MOIs a toxic effect of the Adnull
itself and a slight additional toxicity of the TIMP-4 was observed.
In conclusion, these data suggest that TIMP-4 has no growth
inhibitory effect on rat, pig and human SMCs except at very high
adenoviral load. These results are in agreement with published
reports on the effects of other members of the TIMP family.
[0447] Effects on Cell Migration
[0448] The assay of migration in matrigel drops was used. TIMP-4
infected Human CaSMCs were incorporated into 50 .mu.l of matrigel.
A non-selective MMP inhibitor (doxycycline) was added at different
concentrations in the aim to inhibit most of the non-gelatinase
(collagenase) activity. In addition, the medium was changed after
24 hours to remove soluble MMPs. Four days after seeding, an
anti-migratory activity of TIMP-4 was observed. This effect is
gelatinase-dependent since TIMP-4 and doxycycline have cummulative
effects. This experiment was repeated twice with the same
result.
[0449] TIMP-4 Inhibition of Neointimal Thickening in the Rat
Injured Carotid Model
[0450] To examine the effect of Ad-TIMP-4 infection on neointimal
formation, 12 rats were infected after injury with 2.times.10.sup.9
IU of AdTG14855 or AdTG6401. The carotids of these animals were
collected at 14 days after injury and both neointimal and medial
areas were measured (see Table 2). There was a significant 74%
reduction in neointimal area in Ad-TIMP-4 infected vessels
(1.04+/-0.32 mm.sup.2; p=0.00018; n=6) compared with Ad-null
infected vessels (5.03+/-1.66 mm.sup.2; n=6). No significant
difference was seen in medial area (5.86+/-0.32 versus 6.14+/-0.65
for AdTIMP-4 and Ad-Null infected vessels, respectively). As
expected, the ratio of neointima to media showed a significant
difference between Ad-TIMP-4 and Ad-Null infected vessels
(0.18+/-0.05 versus 0.80+/-0.16; p=0.01). These results show that
adenovirus-mediated gene transfer of rat TIMP-4 to the rat carotid
artery immediately after injury, causes a significant decrease in
neointima development.
2 TABLE 2 AdTG6401 AdTG14855 (2 .times. 10.sup.9 iu) (2 .times.
10.sup.9 iu) Lumen area 13.81 .+-. 2.10 15.77 .+-. 1.84 (p = 0.11)
Media area 6.14 .+-. 0.65 5.86 .+-. 0.32 (p = 0.37) Intima area
5.03 .+-. 1.66 1.04 .+-. 0.32 (p = 0.00018) Neointima/media 0.80
.+-. 0.16 0.18 .+-. 0.05 (p = 0.000012) Lumen perimeter 13.88 .+-.
0.88 15.25 .+-. 0.59 (p = 0.01)
[0451]
Sequence CWU 1
1
8 1 675 DNA Homo sapiens CDS (1)..(672) sig_peptide (1)..(87)
mat_peptide (88)..() 1 atg cct ggg agc cct cgg ccc gcg cca agc tgg
gtg ctg ttg ctg cgg 48 Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Trp
Val Leu Leu Leu Arg -25 -20 -15 ctg ctg gcg ttg ctg cgg ccc ccg ggg
ctg ggt gag gca tgc agc tgc 96 Leu Leu Ala Leu Leu Arg Pro Pro Gly
Leu Gly Glu Ala Cys Ser Cys -10 -5 -1 1 gcc ccg gcg cac cct cag cag
cac atc tgc cac tcg gca ctt gtg att 144 Ala Pro Ala His Pro Gln Gln
His Ile Cys His Ser Ala Leu Val Ile 5 10 15 cgg gcc aaa atc tcc agt
gag aag gta gtt ccg gcc agt gca gac cct 192 Arg Ala Lys Ile Ser Ser
Glu Lys Val Val Pro Ala Ser Ala Asp Pro 20 25 30 35 gct gac act gaa
aaa atg ctc cgg tat gaa atc aaa cag ata aag atg 240 Ala Asp Thr Glu
Lys Met Leu Arg Tyr Glu Ile Lys Gln Ile Lys Met 40 45 50 ttc aaa
ggg ttt gag aaa gtc aag gat gtt cag tat atc tat acg cct 288 Phe Lys
Gly Phe Glu Lys Val Lys Asp Val Gln Tyr Ile Tyr Thr Pro 55 60 65
ttt gac tct tcc ctc tgt ggt gtg aaa cta gaa gcc aac agc cag aag 336
Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala Asn Ser Gln Lys 70
75 80 cag tat ctc ttg act ggt cag gtc ctc agt gat gga aaa gtc ttc
atc 384 Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys Val Phe
Ile 85 90 95 cat ctg tgc aac tac atc gag ccc tgg gag gac ctg tcc
ttg gtg cag 432 His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser
Leu Val Gln 100 105 110 115 agg gaa agt ctg aat cat cac tac cat ctg
aac tgt ggc tgc caa atc 480 Arg Glu Ser Leu Asn His His Tyr His Leu
Asn Cys Gly Cys Gln Ile 120 125 130 acc acc tgc tac aca gta ccc tgt
acc atc tcg gcc cct aac gag tgc 528 Thr Thr Cys Tyr Thr Val Pro Cys
Thr Ile Ser Ala Pro Asn Glu Cys 135 140 145 ctc tgg aca gac tgg ctg
ttg gaa cga aag ctc tat ggt tac cag gct 576 Leu Trp Thr Asp Trp Leu
Leu Glu Arg Lys Leu Tyr Gly Tyr Gln Ala 150 155 160 cag cat tat gtc
tgt atg aag cat gtt gac ggc acc tgc agc tgg tac 624 Gln His Tyr Val
Cys Met Lys His Val Asp Gly Thr Cys Ser Trp Tyr 165 170 175 cgg ggc
cac ctg cct ctc agg aag gag ttt gtt gac atc gtt cag ccc 672 Arg Gly
His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile Val Gln Pro 180 185 190
195 tag 675 2 224 PRT Homo sapiens 2 Met Pro Gly Ser Pro Arg Pro
Ala Pro Ser Trp Val Leu Leu Leu Arg -25 -20 -15 Leu Leu Ala Leu Leu
Arg Pro Pro Gly Leu Gly Glu Ala Cys Ser Cys -10 -5 -1 1 Ala Pro Ala
His Pro Gln Gln His Ile Cys His Ser Ala Leu Val Ile 5 10 15 Arg Ala
Lys Ile Ser Ser Glu Lys Val Val Pro Ala Ser Ala Asp Pro 20 25 30 35
Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Ile Lys Gln Ile Lys Met 40
45 50 Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gln Tyr Ile Tyr Thr
Pro 55 60 65 Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala Asn
Ser Gln Lys 70 75 80 Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp
Gly Lys Val Phe Ile 85 90 95 His Leu Cys Asn Tyr Ile Glu Pro Trp
Glu Asp Leu Ser Leu Val Gln 100 105 110 115 Arg Glu Ser Leu Asn His
His Tyr His Leu Asn Cys Gly Cys Gln Ile 120 125 130 Thr Thr Cys Tyr
Thr Val Pro Cys Thr Ile Ser Ala Pro Asn Glu Cys 135 140 145 Leu Trp
Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr Gly Tyr Gln Ala 150 155 160
Gln His Tyr Val Cys Met Lys His Val Asp Gly Thr Cys Ser Trp Tyr 165
170 175 Arg Gly His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile Val Gln
Pro 180 185 190 195 3 33 DNA Artificial Sequence 5' TIMP-4 primer
with BamH1site 3 gccagaggat cctgcagctg cgccccggcg cac 33 4 33 DNA
Artificial Sequence 3' TIMP-4 primer with XbaI site 4 cggcttctag
aactagggct gaacgatgtc aac 33 5 39 DNA Artificial Sequence 5' TIMP-4
primer with BamHI site 5 gccagaggat ccgccaccat gcctgggagc cctcggccc
39 6 60 DNA Artificial Sequence 3' TIMP-4 primer with XbaI site 6
cggcttctag aatcaagcgt agtctgggac gtcgtatggg tagggctgaa cgatgtcaac
60 7 33 DNA Artificial Sequence 5' TIMP-4 primer with BamHI site 7
gccagaggat ccatgcctgg gagccctcgg ccc 33 8 33 DNA Artificial
Sequence 3' TIMP-4 primer with XbaI site 8 cggcttctag aactagggct
gaacgatgtc aac 33
* * * * *