Tissue inhibitor of metalloproteinases

The present invention provides a nucleic acid sequence which encodes a novel tissue inhibitor of metalloproteinases (TIMP-4) which was isolated from cells of human uterus. The invention provides for genetically engineered expression vectors and host cells comprising nucleic acid sequence encoding TIMP-4. The invention also provides for purified TIMP-4.

FIELD OF THE INVENTION 
The present invention relates to nucleic acid and amino acid sequences of a 
novel metalloproteinase inhibitor from human uterus and to the use of 
these sequences in the diagnosis, study, prevention and treatment of 
disease. 
BACKGROUND OF THE INVENTION 
Tissue inhibitors of metalloproteinases (TIMPs) inhibit metalloproteinases, 
a multigene family of endopeptide hydrolases. The metalloproteinases are 
secreted by connective tissue and hematopoietic cells, use Zn.sup.++ or 
Ca.sup.++ for catalysis, and may be inactivated by metal chelators as well 
as TIMP molecules. 
The matrix metalloproteinases (MMPs) participate in a variety of 
biologically important processes including the degradation of many 
structural components of tissues, particularly the extracellular matrix 
(ECM). This ability is desirable in processes where destruction of 
existing tissues is necessary, eg, in embryo implantation (Reponen P et 
al. (1995) Dev Dyn 202: 388-96), embryogenesis, and tissue remodeling. 
Metalloproteinases have also been implicated in processes where their 
activity is more specifically directed, such as in the movement of cells 
through tissues. Some of these molecules and their known substrates as 
reviewed in Murphy G and A J P Docherty (1992; Am J Respir Cell Mol Biol 
120-125) are summarized below: 
______________________________________ 
MMP-1 Interstitial 
Fibrillar Type X Collagens; Gelatin; 
collagenase 
Proteoglycan 
MMP-2 Gelatinase A 
Collagens IV, V, VII, X, XI; Elastin 
MMP-3 Stromelysin-1 
Proteoglycan; Collagens II, IV, 
IX, X, XI; Fibronectin; 
Procollagen; Laminin; Gelatin; 
Collagenase; Gelatinase B 
MMP-7 Matrilysin Proteoglycan; Collagens II, IV, 
IX, X, XI; Fibronectin; 
Procollagen; Laminin; Gelatin; 
Collagenase; Gelatinase B; Elastin 
MMP-8 Neutrophil Fibrillar Collagens; Gelatin, 
collagenase 
Proteoglycans 
MMP-9 Gelatinase B 
Collagens IV, V, VII, X, XI; Elastin 
MMP-10 Stromelysin-2 
Proteoglycan; Collagens II, IV, 
IX, X, XI; Fibronectin; 
Procollagen; Laminin; Gelatin; 
Collagenase; Gelatinase B 
______________________________________ 
Metalloproteinases also inactivate several members of a class of serine 
protease inhibitors known as serpins. The inactivation of the serpins such 
as alpha-1 protease inhibitor allows serine proteases to destroy a variety 
of biologically important, non-ECM, molecules such as alpha-1 antitrypsin 
(Sorsa T et al. (1993) Agents Actions Suppl 39: 225-9). Therefore, 
controlling the activity of the metalloproteinases can have indirect 
effects on the activity of a range of other potent proteases. 
It is, however, the MMPs which play the most important roles in 
pathological processes. In rheumatoid and other forms of arthritis, 
proteolysis of extracellular matrix components by MMPs is a major cause of 
cartilage and synovial tissue destruction (Firestein G S (1992) Curr Opin 
Rheumatol 4: 348-54). Similarly, the activity of elastinolytic MMPs 
secreted by mononuclear phagocytes has been implicated in the destruction 
of alveolar structure in pulmonary emphysema (Shapiro S D (1994) Am J 
Respir Crit Care Med). 
MMPs are also associated with tumor metastasis. In order to colonize 
secondary sites, the primary tumor cells must both enter and exit the 
vascular system. This movement through tissues and the endothelial 
basement membrane is aided by the activity of metalloproteinases secreted 
by the tumor cells. In addition, the degradation of normal extracellular 
matrix structure is critical for angiogenesis as well as tumor cell 
migration (Ray J M and Stetler-Stevenson W G (1994) Eur Respir J 7: 
2062-72; Mignatti P and Rifkin DB (1993) Physiol Rev 73: 161-95). 
MMPs have also been implicated in periodontal disease where inflammation of 
the periodontium leads to connective tissue degradation and eventually to 
tooth loss. Tissue degradation is largely mediated by neutrophils which 
are attracted to sites of inflammation and secrete the MMPs believed to 
play a major role in tissue destruction (Sodek J and Overall C M (1992) 
Matrix Suppl 1: 352-62). MMPs are also involved in corneal ulcer formation 
following alkali burns and bacterial inflammation (Wentworth J S et al. 
(1992) 33: 2174-9; Burns F R et al. (1992) Matrix Suppl 1: 317-318). 
MMPs participate in both normal and abnormal bone resorption. Coliagenase 
produced by osteoblasts degrades collagen on the surface of the bone and 
affords osteoclasts access to underlying mineralized bone. The osteoclasts 
utilize MMPs and other proteases to resorb bone. When catabolism exceeds 
deposition, the resulting imbalance can lead to demineralization of the 
bone and osteoporosis or osteoarthritis (Hembry R M et al (1995) Ann Rheum 
Dis 54: 25-32; Everts V et al. (1992) J Cell Physiol 150: 221-231; Vaes G 
et al. (1992) Matrix Suppl 1: 383-388). Even though TIMPs are expressed in 
osteoarthritic joints, the amount of inhibitory activity does not 
compensate for the amounts of MMPs carrying out degradation. 
The involvement of MMPs in a wide range of pathological conditions suggests 
that natural inhibitors of MMPs, such as TIMPs, would be therapeutically 
useful for treatment of pathological conditions associated with excessive 
expression of MMPs. 
TIMP Molecules 
The nucleotide and amino acid sequences of three human TIMPs have been 
previously characterized and named TIMP-1 (Docherty A J P et al. (1985) 
Nature 318: 66-69), TIMP-2 (Boone T C et al. (1990) Proc Natl Acad Sci 87: 
2800-2804; Stetler-Stevenson W G et al. (1990) J Biol Chem 265: 13933-38), 
and TIMP-3 (Wilde C G et al. (1994) DNA Cell Biol 13: 711-18). These 
proteins are classified as TIMPs based on their ability to inhibit 
metalloproteinases, structural similarity to each other, the 12 cysteines 
which form disulfide bonds important in secondary structure, and the 
presence of the VIRAK motif which interacts with the metal ion of the 
metalloproteinases. 
Although human TIMPs inhibit a variety of metalloproteinases, the 
expression and specific activity of individual TIMPs do differ. TIMP-1, a 
30 kD protein, is the most commonly expressed molecule and contains two 
asparagine residues which act as carbohydrate binding sites, one in loop 1 
and one in loop 2 (Murphy and Docherty, supra). In addition, a truncated 
form of TIMP-1 which contains only the first three loops of the molecule 
is able to inhibit MMPs. Although TIMP-1 is a better inhibitor of 
interstitial collagenase than TIMP-2 (Howard E W et al. (1991) J Biol Chem 
266: 13070-75), the 23 kD TIMP-2 molecule is the most effective inhibitor 
of gelatinases A and B. TIMP-3 is a 21 kD protein which inhibits 
collagenase 1, stromelysin, and gelatinases A and B (Apte S S et al. 
(1995) J Biol Chem 270: 14313-18) and may be induced by mitogens (Wick et 
al. (1994) J Biol Chem 269: 18953-60). 
There have been reports of other inhibitors of metalloproteinases (IMPs) 
with physical characteristics different from those of the known TIMPs. In 
some cases, these activities result from alternate forms of the known 
TIMPs. For example, one IMP present in the conditioned media of a human 
bladder carcinoma was identified as a partially glycosylated form of 
TIMP-1, and another, as a partially processed/degraded form of TIMP-2 
(Miyazaki K et al. (1993) J Biol Chem 268: 14387-93). Additional reports 
have described sources and characteristics of IMP activity, but active 
molecules have not been identified (Apodaca G et al. (1990) Cancer 
Research 50: 2322-29). 
TIMPs which have been cloned from other species include bovine TIMP-1 
(Freudenstein R et al. (1990) Biochem Biophys Res Comm 171: 250-256) and 
TIMP-2 (Boone T C et al. (1990) Proc Natl Acad Sci 87: 2800-2804); murine 
TIMP-1 (Gewert D R et al. (1987) EMBO J 6: 651-657); rabbit TIMP-1 
(Horowitz S et al. (1989) J Biol Chem 264: 7092-7095); and chicken TIMP-3 
(Pavloff N et al. (1992) J Biol Chem 267: 17321-6). 
SUMMARY 
The present invention relates to a novel tissue inhibitor of 
metalloproteinases, TIMP-4, whose nucleic acid sequence was identified 
among the polynucleotides of a human uterus library and to the use of the 
nucleic acid and amino acid sequences of TIMP-4 in the study, diagnosis, 
prevention and treatment of disease. 
The novel polynucleotide encoding TIMP-4 was first identified within Incyte 
Clone No. 589345 through a computer generated search for nucleotide 
sequence alignments. The clone was resequenced, and the coding region 
determined. The nucleotide sequence encodes a protein of 224 amino acids 
including the 5' signal sequence of 29 residues, M.sub.1 through A.sub.29 
of SEQ ID NO:2. Other significant features of the protein include the 
presence of the VIRAK motif at amino acid residues 47 through 51 of SEQ ID 
NO:2 and the 12 cysteine residues characteristic of known TIMPs. The 
present invention is based, in part, on the facts that TIMP-4 is more 
closely related to TIMP-2 than to the other TIMPs and that both TIMP-4 and 
TIMP-2 have been found in female reproductive tissues. 
The use of TIMP-4, and of the nucleic acid sequences which encode it, is 
based on the amino acid and structural homologies between TIMP-4 and the 
other known TIMPs and on the ability of the known TIMPS to inhibit the 
activities of MMPs. The timing of and amount of expression of MMPs and 
TIMPs are implicated in metastasis of neoplastic cells, anglogenesis and 
growth of tumors such as cervisal, uterine, pancreatic, colon or 
intestinal carcinomas, pregnancy, osteoarthritis, osteoporosis, pulmonary 
emphysema, periodontal disease, rheumatoid arthritis, corneal or diabetic 
ulcers or ulcers/lesions caused by microorganisms. 
The TIMP-4 polynucleotide sequence, oligonucleotides, fragments, portions 
or antisense molecules thereof, may be used in diagnostic assays to detect 
and quantify levels of TIMP-4 mRNA in cells and tissues. For example, 
TIMP-4 polynucleotides, or fragments thereof, may be used in hybridization 
assays of bodily fluids or biopsied tissues to detect the level of TIMP-4 
expression. 
The present invention also relates, in part, to an expression vector and 
host cells comprising nucleic acids encoding TIMP-4. Such transfected host 
cells are useful for the production and recovery of TIMP-4. The present 
invention also encompasses purified TIMP-4. 
The invention further provides diagnostic kits for the detection of 
naturally occurring TIMP-4 and provides for the use of purified TIMP-4 as 
a positive control and to produce anti-TIMP-4 antibodies. These antibodies 
may be used to detect conditions which involve abnormal TIMP-4 expression, 
for example, to diagnose nonhealing corneal or diabetic ulcers or lesions 
caused by microorganisms. 
The invention further provides for methods of treatment of conditions 
associated with excessive expression of MMPs, such as metastasis of 
neoplastic cells, anglogenesis and growth of tumors such as cervical, 
uterine, pancreatic, colon or intestinal carcinomas, osteoarthritis, 
osteoporosis, pulmonary emphysema, periodontal disease, and rheumatoid 
arthritis. In addition, the invention provides for the use of TIMP-4 as a 
means of birth control, ie, to neutralize MMP activity, thereby preventing 
implantation of the embryo. Each of these uses would include 
administration of effective amounts of purified TIMP-4 or vectors 
comprising nucleic acid sequences encoding TIMP-4 to individuals subject 
to these conditions for the purpose of inhibiting MMP expression. 
The invention also provides pharmaceutical compositions comprising vectors 
containing the nucleic acid sequence encoding TIMP-4 or purified TIMP-4 
protein which can be used in the prevention or treatment of various 
conditions including, but not limited to, metastasis of neoplastic cells, 
anglogenesis and growth of tumors such as cervical, uterine, pancreatic, 
colon or intestinal carcinomas, pregnancy, osteoarthritis, osteoporosis, 
pulmonary emphysema, periodontal disease, and rheumatoid arthritis. For 
example, TIMP-4 may be used in inhibiting or neutralizing 
metalloproteinase activity associated with excessive expression of 
gelatinases in the alveolar structure of the lung which leads to pulmonary 
emphysema. Oligonucleotides or fragments of the nucleotide sequence and 
antagonists and inhibitors of the protein may be administered to 
neutralize the activity of TIMP-4 in situations where TIMPs are 
overexpressed, for example, during the healing of conditions such as 
corneal and diabetic ulcers, and ulcers/lesions caused by microorganisms.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a novel tissue inhibitor of 
metalloproteinases, TIMP-4, whose nucleic acid sequence was identified 
among the polynucleotides of a uterine tissue library (UTRSNOT01) and to 
the use of the nucleic acid and amino acid sequences of TIMP-4 in the 
study, diagnosis, prevention and treatment of disease. 
The polynucleotide sequence encoding TIMP-4 was first identified within 
Incyte Clone No. 589345. A BLAST search (Basic Local Alignment Search 
Tool; Altschul S F (1993) J Mol Evol 36: 290-300; Altschul, S F et al 
(1990) J Mol Biol 215: 403-10) comparing the cDNAs of the UTRSNOT01 
library against the primate database of GenBank 91 identified Incyte Clone 
589345 as a nonexact homolog of human metalloproteinase inhibitor. Incyte 
Clone 589345 was resequenced and the deduced amino acid sequence compared 
with a consensus sequence for the known TIMP molecules. The relationships 
of the TIM P molecules, one to another, can be described on the basis of 
their comparison to the amino acid consensus sequence: TIMP-2 shows 62% 
homology to the amino acid consensus sequence; TIMP-4, 61% homology; 
TIMP-3, 56% homology; and TIMP-1, 43% homology. Therefore, in series, 
TIMP-4 is most closely related to TIMP-2, closely related to TIMP-3 and 
related to TIMP-1. The exact relationships of each of the known TIMPs to 
TIMP-4 are shown in FIGS. 2 through 4. 
The present invention is based, in part, on the amount of amino acid 
sequence and structural homology among the TIMP molecules, and on the fact 
that all of the known TIMPs have been found in female reproductive 
tissues. The protein TIMP-4 has a length of 224 amino acids with a 5' 
signal sequence of 29 residues, M.sub.1 through A.sub.29, and displays 30% 
amino acid sequence identity with the deduced sequence of human TIMP-2. 
Other significant features of TIMP-4 include the presence of the VIRAK 
motif, at amino acid residues 47-51 of SEQ ID NO:2, which indicates that 
TIMP-4 interacts with Zn.sup.++ (Williamson R A et al. (1994) Protein Eng 
7: 1035-1040) and the presence of the 12 cysteine residues common to the 
known TIMPs (FIGS. 2-4; Negro A et al. (1995) FEBS Lett 360: 52-56). 
TIMP-4 lacks the 3' NAT glycosylation site which is located five amino 
acid residues before the stop codon of TIMP-3 (FIG. 3), but the mature 
protein which begins at C.sub.30 of SEQ ID NO:2 has five asparagines, at 
least five of which may associate with carbohydrate moieties. 
The use of TIMP-4, and of the nucleic acid sequences which encode it, is 
based on the amino acid and structural homologies between TIMP-4 and the 
other known TIMPs and on the ability of the known TIMPS to inhibit the 
activities of MMPs. The timing of and amount of expression of MMPs and 
TIMPs are implicated in metastasis of neoplastic cells, anglogenesis and 
growth of tumors such as cervical, uterine, pancreatic, colon or 
intestinal carcinomas, osteoarthritis, osteoporosis, pulmonary emphysema, 
periodontal disease, rheumatoid arthritis, corneal or diabetic ulcers or 
lesions caused by microorganisms. In each of the next three situations, 
the level of MMP expression precedes or exceeds the expression of TIMP. 
MMPs play a role in loosening the components of the extracellular matrix of 
veins and arteries and easing the passage of various cell types. In the 
same manner, neoplastic cells enter and exit the vascular system of 
individuals with metastatic carcinomas (Ray J M and Stetler-Stevenson W G, 
(supra) and Mignatti P and Rifkin D B (supra). Supplying purified TIMP-4 
to those individuals would inhibit MMPs and interfere with metastasis of 
neoplastic cells. Similarly, supplying purified TIMP-4 to individuals with 
developing tumors would interfere with MMP-associated anglogenesis, the 
vascularization of the tumor and inhibit tumor growth. 
In reproductive studies, Reponen P et al. (supra) has shown that the 
trophoblast secretes MMPs during implantation of the embryo. The timely 
inhibition of these MMPs, post coitus, by supplying women with recombinant 
TIMP-4 would inhibit the MMPs and prevent loosening of the extracellular 
matrix, implantation and pregnancy. 
In diseases such as osteoarthritis, rheumatoid arthritis, pulmonary 
emphysema, periodontal disease, and osteoporosis, the excess MMPs cause 
inflammation, tissue destruction and impaired function. Delivery of TIMP-4 
to the tissue or disease site would inhibit the activity of the MMPs and 
reduce inflammation and damage. 
Alternatively, induction and overexpression of TIMPs by insult or injury, 
and in the presence of a systemic disease such as diabetes, results in 
recalcitrant wound healing (Wentworth J S et al, supra; Burns F R et al, 
supra). Inhibiting or downregulating TIMP expression through the delivery 
of antisense molecules, antibodies, antagonists or inhibitors to the 
subject's ulcer or lesion would allow the MMPs to carry out normal tissue 
remodeling processes. 
"Nucleic acid sequence" as used herein refers to an oligonucleotide, 
nucleotide or polynucleotide sequence, and fragments or portions thereof, 
and to DNA or RNA of genomic or synthetic origin which may be 
single-stranded or double-stranded, and represent the sense or antisense 
strand. Similarly, amino acid sequence as used refers to an oligopeptide, 
peptide, polypeptide or protein sequence. 
As used herein, "TIMP-4" refers to TIMP-4 protein from any species, 
including, bovine, ovine, porcine, equine, murine and preferably human, in 
a naturally occurring form or from any source, whether natural, synthetic, 
semi-synthetic or recombinant. As used herein, "naturally occurring" 
refers to a molecule, nucleic acid or amino acid sequence, found in 
nature. 
The present invention also encompasses TIMP-4 variants. A preferred TIMP-4 
variant is one having at least 80% amino acid sequence similarity, a more 
preferred TIMP-4 variant is one having at least 90% amino acid sequence 
similarity and a most preferred TIMP-4 variant is one having at least 95% 
amino acid sequence similarity to the TIMP-4 amino acid sequence (SEQ ID 
NO:2). A "variant" of TIMP-4 may have an amino acid sequence that is 
different by one or more amino acid "substitutions". The variant may have 
"conservative" changes, wherein a substituted amino acid has similar 
structural or chemical properties, eg, replacement of leucine with 
isoleucine. More rarely, a variant may have "nonconservative" changes, eg, 
replacement of a glycine with a tryptophan. Similar minor variations may 
also include amino acid deletions or insertions, or both. Guidance in 
determining which and how many amino acid residues may be substituted, 
inserted or deleted without abolishing biological or immunological 
activity may be found using computer programs well known in the art, for 
example, DNASTAR software. 
Similarly, the term "biologically active" refers to a TIMP-4 having 
structural, regulatory or biochemical functions of the naturally occurring 
TIMP-4. Likewise, "immunological activity" is defined as the capability of 
the natural, recombinant or synthetic TIMP-4, or any oligopeptide thereof, 
to induce a specific immune response in appropriate animals or cells and 
to bind with specific antibodies. 
The term "derivative" refers to the chemical modification of the encoded 
TIMP-4. Illustrative of such modifications would be replacement of 
hydrogen by an alkyl, acyl, or amino group. A TIMP-4 derivative would 
encode a polypeptide which retains essential biological characteristics of 
TIMPs such as, for example, the inhibition of MMPs. 
As used herein, the term "purified" refers to molecules, either nucleic or 
amino acid sequences, that are removed from their natural environment and 
isolated or separated from at least one other component with which they 
are naturally associated. 
The TIMP-4 Coding Sequences 
The determined nucleic acid and deduced amino acid sequences of TIMP-4 are 
shown in FIG. 1. In accordance with the invention, any nucleotide sequence 
which encodes the amino acid sequence of TIMP-4 can be used to generate 
recombinant molecules which express TIMP-4. In a specific embodiment 
described herein, TIMP-4 was first isolated and identified within Incyte 
Clone 589345 from human uterus (UTRSNOT01) library, patent application 
Ser. No. 60/005,864 (Incyte Docket No. PD-0084P), "Polynucleotides and 
Polypeptides Derived from Uterus", by Stuart et al, filed Oct. 26, 1995, 
and hereby incorporated by reference 
Methods for DNA sequencing are well known in the art and employ such 
enzymes as the Klenow fragment of DNA polymerase I, Sequenase.RTM. (US 
Biochemical Corp, Cleveland Ohio), Taq polymerase (Perkin Elmer, Foster 
City Calif.), thermostable T7 polymerase (Amersham, Chicago Ill.), or 
combinations of recombinant polymerases and proofreading exonucleases such 
as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg 
Md.). Methods to extend the DNA from an oligonucleotide primer annealed to 
the DNA template of interest have been developed for both single-stranded 
and double-stranded templates. Chain termination reaction products were 
separated using electrophoresis and detected via their incorporated, 
labeled precursors. Recent improvements in mechanized reaction 
preparation, sequencing and analysis have permitted expansion in the 
number of sequences that can be determined per day. Preferably, the 
process is automated with machines such as the Hamilton Micro Lab 2200 
(Hamilton, Reno Nev.), Peltier Thermal Cycler (PTC200; MJ Research, 
Watertown Mass.) and the Applied Biosystems (Foster City Calif.) Catalyst 
800 and 377 and 373 DNA sequencers. 
The quality of any particular cDNA library may be determined by performing 
a pilot scale analysis of the cDNAs and checking for percentages of clones 
containing vector, lambda or E. coli DNA, mitochondrial or repetitive DNA, 
and clones with exact or homologous matches to public databases. 
Extending TIMP-4 Polynucleotide Sequence 
The polynucleotide sequence of TIMP-4 may be extended utilizing partial 
nucleotide sequence and various methods known in the art to detect 
upstream sequences such as promoters and regulatory elements. Gobinda et 
al (1993; PCR Methods Applic 2: 318-22) disclose "restriction-site 
polymerase chain reaction (PCR)" as a direct method which uses universal 
primers to retrieve unknown sequence adjacent to a known locus. First, 
genomic DNA is amplified in the presence of primer to a linker sequence 
and a primer specific to the known region. The amplified sequences are 
subjected to a second round of PCR with the same linker primer and another 
specific primer internal to the first one. Products of each round of PCR 
are transcribed with an appropriate RNA polymerase and sequenced using 
reverse transcriptase. 
Inverse PCR can be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia T et al (1988) Nucleic Acids Res 
16: 8186). The primers may be designed using Oligo 4.0 (National 
Biosciences Inc, Plymouth Minn.), or another appropriate program, to be 
22-30 nucleotides in length, to have a GC content of 50% or more, and to 
anneal to the target sequence at temperatures about 68.degree.-72.degree. 
C. The method uses several restriction enzymes to generate a suitable 
fragment in the known region of a gene. The fragment is then circularized 
by intramolecular ligation and used as a PCR template. 
Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1: 111-19) is a 
method for PCR amplification of DNA fragments adjacent to a known sequence 
in human and yeast artificial chromosome (YAC) DNA. Capture PCR also 
requires multiple restriction enzyme digestions and ligations to place an 
engineered double-stranded sequence into an unknown portion of the DNA 
molecule before PCR. 
Parker J D et al (1991; Nucleic Acids Res 19: 3055-60), teach walking PCR, 
a method for targeted gene walking which permits retrieval of unknown 
sequence. PromoterFinder.TM. a new kit available from Clontech (Palo Alto 
Calif.) uses PCR, nested primers and special libraries to "walk in" 
genomic DNA. This process avoids the need to screen libraries and is 
useful in finding intron/exon junctions. 
Another PCR method, "Improved Method for Obtaining Full Length cDNA 
Sequences" by Guegler et al, patent application Ser. No. 08/487,112, filed 
Jun. 7, 1995 and hereby incorporated by reference, employs XL-PCR.TM. 
(Perkin-Elmer, Foster City Calif.) to amplify and/or extend nucleotide 
sequences. 
Preferred libraries for screening for full length cDNAs are ones that have 
been size-selected to include larger cDNAs. Also, random primed libraries 
are preferred in that they will contain more sequences which contain the 
5' and upstream regions of genes. A randomly primed library may be 
particularly useful if an oligo d(T) library does not yield a full-length 
cDNA. Genomic libraries are useful for extending sequence into the gene's 
promoter binding region. 
A new method for either analyzing the size or confirming the nucleotide 
sequence of sequencing or PCR products is capillary electrophoresis. 
Systems for rapid sequencing are available from Perkin Elmer, Beckman 
Instruments (Fullerton Calif.), and other companies. Capillary sequencing 
employs flowable polymers for electrophoretic separation, four different 
fluorescent dyes (one for each nucleotide) which are laser activated, and 
detection of the emitted wavelengths by a charge coupled device camera. 
Output/light intensity is converted to electrical signal using appropriate 
software (eg, Genotyper.TM. and Sequence Navigator.TM. from Perkin Elmer) 
and the entire process from loading of samples to computer analysis and 
electronic data display is computer controlled. Capillary electrophoresis 
is particularly suited to the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. The 
reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has 
been reported (Ruiz-Martinez M C et al (1993) Anal Chem 65: 2851-8). 
Expression of TIMP-4 Nucleotide Sequence 
In accordance with the present invention, TIMP-4 polynucleotide sequences 
which encode TIMP-4, as well as fragments, fusion proteins or functional 
equivalents thereof, may be used to generate recombinant DNA molecules 
that direct the expression of TIMP-4 in appropriate host cells. Due to the 
inherent degeneracy of the genetic code, other DNA sequences which encode 
substantially the same or a functionally equivalent amino acid sequence, 
may be used to clone and express TIMP-4. As will be understood by those of 
skill in the art, it may be advantageous to produce TIMP-4-encoding 
nucleotide sequences possessing non-naturally occurring codons. Codons 
preferred by a particular prokaryotic or eukaryotic host (Murray E E 
(1989) Nuc Acids Res 17: 477-508) can be selected, for example, to 
increase the rate of TIMP-4 expression or to produce recombinant RNA 
transcripts having desirable properties, such as a longer half-life, than 
transcripts produced from naturally occurring sequence. 
Also included within the scope of the present invention are polynucleotide 
sequences that are capable of hybridizing to the nucleotide sequence of 
SEQ ID NO:1 under conditions of intermediate to maximal stringency. 
Hybridization conditions are based on the melting temperature (Tm) of the 
nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide 
to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic 
Press, San Diego Calif.) incorporated herein by reference, and confer a 
defined "stringency" as explained below. 
"Maximum stringency" typically occurs at about Tm-5.degree. C. (5.degree. 
C. below the Tm of the probe); "high stringency" at about 5.degree. C. to 
10.degree. C. below Tm; "intermediate stringency" at about 10.degree. C. 
to 20.degree. C. below Tm; and "low stringency" at about 20.degree. C. to 
25.degree. C. below Tm. As will be understood by those of skill in the 
art, a maximum stringency hybridization can be used to identify or detect 
identical or allelic polynucleotide sequences while an intermediate (or 
low) stringency hybridization can be used to identify or detect related 
polynucleotide sequences. The term "hybridization" as used herein shall 
include "the process by which a strand of nucleic acid joins with a 
complementary strand through base pairing" (Coombs J (1994) Dictionary of 
Biotechnology, Stockton Press, New York N.Y.) as well as the process of 
amplification has carried out in polymerase chain reaction technologies as 
described in Dieffenbach C W and G S Dveksler (1995, PCR Primer, a 
Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.) and 
incorporated herein by reference. 
As used herein a "deletion" is defined as a change in either nucleotide or 
amino acid sequence in which one or more nucleotides or amino acid 
residues, respectively, are absent. 
As used herein an "insertion" or "addition" is that change in a nucleotide 
or amino acid sequence which has resulted in the addition of one or more 
nucleotides or amino acid residues, respectively, as compared to the 
naturally occurring TIMP-4. 
As used herein "substitution" results from the replacement of one or more 
nucleotides or amino acids by different nucleotides or amino acids, 
respectively. 
Derivative TIMP-4 polynucleotide sequences which may be used in accordance 
with the invention include deletions, insertions or substitutions of 
different nucleotide residues resulting in a polynucleotide that encodes 
the same or a functionally equivalent TIMP-4. The protein may also show 
deletions, insertions or substitutions of amino acid residues which 
produce a silent change and result in a functionally equivalent TIMP-4. 
Variant molecules may be synthesized by deliberate amino acid 
substitution. Such variants are based on the similarity in polarity, 
charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic 
nature of the residues as long as the biological activity of TIMP-4 is 
retained. For example, negatively charged amino acids include aspartic 
acid and glutamic acid; positively charged amino acids include lysine and 
arginine; and amino acids with uncharged polar head groups having similar 
hydrophilicity values include leucine, isoleucine, valine; glycine, 
alanine, asparagine, glutamine; serine, threonine, phenylalanine, and 
tyrosine. 
Included within the scope of the present invention are alleles of TIMP-4. 
As used herein, an "allele" or "allelic sequence" is an alternative form 
of TIMP-4. Alleles result from a mutation, ie, a change in the nucleic 
acid sequence, and generally produce altered mRNAs or polypeptides whose 
structure or function may or may not be altered. Any given gene may have 
none, one or many allelic forms. Common mutational changes which give rise 
to alleles are generally ascribed to deletions, additions or substitutions 
of amino acids. Each of these types of changes may occur alone, or in 
combination with the others, one or more times in a given sequence. 
The nucleotide sequences of the present invention may be engineered in 
order to alter a TIMP-4 coding sequence for a variety of reasons, 
including but not limited to, alterations which modify the cloning, 
processing and/or expression of the gene product. For example, mutations 
may be introduced using techniques which are well known in the art, eg, 
site-directed mutagenesis to insert new restriction sites, to alter 
glycosylation patterns, to change codon preference, etc. 
In another embodiment of the invention, a natural, modified or recombinant 
TIMP-4 sequence may be ligated to a heterologous sequence to encode a 
fusion protein. For example, for screening of peptide libraries for 
inhibitors of TIMP-4 activity, it may be useful to encode a chimeric 
TIMP-4 protein expressing a heterologous epitope that is recognized by a 
commercially available antibody. A fusion protein may also be engineered 
to contain a cleavage site located between a TIMP-4 sequence and the 
heterologous protein sequence, so that the TIMP-4 may be cleaved and 
purified away from the heterologous moiety. 
In an alternate embodiment of the invention, the entire coding sequence of 
TIMP-4, or any part thereof, could be synthesized using chemical methods 
(Sindelar L E and J M Jaklevic (1995) Nuc Acids Res 23: 982-7; Modesti A 
et al (1993) Biochim Biophys Acta 1216: 369-74). Similarly, the protein 
itself could be produced using chemical methods. For example, peptides can 
be synthesized by solid phase techniques, cleaved from the resin, and 
purified by preparative high performance liquid chromatography (eg, 
Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman 
and Co, New York N.Y.). The composition of the synthetic peptides may be 
confirmed by amino acid analysis or sequencing (eg, using the Edman 
degradation procedure and deduced translation from the nucleotide sequence 
as reported in Castalano-Sherman J et al. (1993) J Dental Res 72: 1566-72) 
Direct peptide synthesis can be performed using various solid-phase 
techniques (Roberge J Y et al (1995) Science 269: 202-204) and automated 
synthesis may be achieved, for example, using Applied Biosystems 431A 
Peptide Synthesizer in accordance with the instructions provided by the 
manufacturer. Additionally the amino acid sequence of TIMP-4, or any part 
thereof, may be altered during direct synthesis and/or combined using 
chemical methods with other TIMP sequences, or any part thereof, to 
produce a variant polypeptide. 
Expression Systems for TIMP-4 Nucleotide Sequences 
In order to express a biologically active TIMP-4, the nucleotide sequence 
coding for TIMP-4, or a functional equivalent, is inserted into an 
appropriate expression vector, ie, a vector which contains the necessary 
elements for the transcription and translation of the inserted coding 
sequence. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing a TIMP-4 coding sequence and 
appropriate transcriptional or translational controls. These methods 
include in vitro recombinant DNA techniques, synthetic techniques and in 
vivo recombination or genetic recombination. Such techniques are described 
in Maniatis et al. (1989) Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Press, Plainview N.Y. and Ausubel F M et al. (1989) Current 
Protocols in Molecular Biology, John Wiley & Sons, New York N.Y. 
A variety of expression vector/host systems may be utilized to express a 
TIMP-4 coding sequence. These include but are not limited to 
microorganisms such as bacteria transformed with recombinant 
bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed 
with yeast expression vectors; insect cell systems infected with virus 
expression vectors (eg, baculovirus); plant cell systems transfected with 
virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco 
mosaic virus, TMV) or transformed with bacterial expression vectors (eg, 
Ti or pBR322 plasmid); or animal cell systems. 
The "control elements" or "regulatory sequences" of these systems vary in 
their strength and specificities and are those nontranslated regions of 
the vector, enhancers, promoters, and 3' untranslated regions, which 
interact with host cellular proteins to carry out transcription and 
translation. Depending on the vector system and host utilized, any number 
of suitable transcription and translation elements, including constitutive 
and inducible promoters such as those discussed in Baeuerle Pa. (1995) 
Inducible Gene Expression, Vol 1 and 2, Birkhauser, Cambridge Mass. For 
example, when cloning in bacterial systems, inducible promoters such as 
the hybrid lacZ promoter of the Bluescript.RTM. phagemid (Stratagene, 
LaJolla Calif.) and ptrp-lac hybrids and the like may be used. The 
baculovirus polyhedrin promoter may be used in insect cells. Promoters or 
enhancers derived from the genomes of plant cells (eg, heat shock, 
RUBISCO; and storage protein genes) or from plant viruses (eg, viral 
promoters or leader sequences) may be cloned into the vector. In mammalian 
cell systems, promoters from the mammalian genes or from mammalian viruses 
are most appropriate. If it is necessary to generate a cell line that 
contains multiple copies of the TIMP-4 nucleotide sequence, vectors based 
on SV40 or EBV may be used with an appropriate selectable marker. 
In bacterial systems, a number of expression vectors may be selected 
depending upon the use intended for TIMP-4. For example, when large 
quantities of TIMP-4 are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be desirable. Such vectors include, but are not limited to, 
the E. coli cloning and expression vector Bluescript.RTM. (Stratagene), in 
which the TIMP-4 coding region may be ligated into the vector in frame 
with sequences for the amino-terminal Met and the subsequent 7 residues of 
.beta.-galactosidase so that a hybrid protein is produced; pIN vectors 
(Van Heeke & Schuster (1989) J Biol Chem 264: 5503-5509); and the like. 
pGEX vectors (Promega, Madison Wis.) may also be used to express foreign 
polypeptides as fusion proteins with glutathione S-transferase (GST). In 
general, such fusion proteins are soluble and can easily be purified from 
lysed cells by adsorption to glutathione-agarose beads followed by elution 
in the presence of free glutathione. Proteins made in such systems are 
designed to include heparin, thrombin or factor XA protease cleavage sites 
so that the cloned polypeptide of interest can be released from the GST 
moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase 
and PGH may be used. For reviews, see Ausubel et al (supra) and Grant et 
al (1987) Methods in Enzymology 153: 516-544. 
In cases where plant expression vectors are used, the expression of a 
TIMP-4 coding sequence may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S and 19S promoters of CaMV 
(Brisson et al (1984) Nature 310: 511-514) may be used alone or in 
combination with the cDNA version of the omega leader sequence from TMV 
(Takamatsu et al (1987) EMBO J 6: 307-311). Alternatively, plant promoters 
such as the small subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3: 
1671-1680; Broglie et al (1984) Science 224: 838-843); or heat shock 
promoters (Winter J and Sinibaldi R M (1991) Results Probl Cell Differ 17: 
85-105) may be used. These constructs can be introduced into plant cells 
by direct DNA transformation or pathogen-mediated transfection. For 
reviews of such techniques, see Hobbs S or Murry L E in McGraw Yearbook of 
Science and Technology (1992) McGraw Hill New York N.Y., pp 191-196 or 
Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, 
Academic Press, New York N.Y., pp 421-463. 
An alternative expression system which could be used to express TIMP-4 is 
an insect system. In one such system, Autographa californica nuclear 
polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in 
Spodoptera frugiperda cells or in Trichoplusia larvae. The TIMP-4 coding 
sequence may be cloned into a non-essential region of the virus, such as 
the polyhedrin gene, and placed under control of the polyhedrin promoter. 
Successful insertion of TIMP-4 will render the polyhedrin gene inactive 
and produce recombinant virus lacking coat protein. The recombinant 
viruses are then used to infect S. frugiperda cells or Trichoplusia larvae 
in which TIMP-4 is expressed (Smith et al (1983) J Virol 46: 584; 
Engelhard E K et al (1994) Proc Nat Acad Sci 91: 3224-7). 
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, a 
TIMP-4 coding sequence may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a non-essential E1 or E3 region 
of the viral genome will result in a viable virus capable of expressing 
TIMP-4 in infected host cells. (Logan and Shenk (1984) Proc Natl Acad Sci 
81: 3655-3659). In addition, transcription enhancers, such as the rous 
sarcoma virus (RSV) enhancer, may be used to increase expression in 
mammalian host cells. 
Specific initiation signals may also be required for efficient translation 
of an inserted TIMP-4 coding sequences. These signals include the ATG 
initiation codon and adjacent sequences. In cases where TIMP-4, its 
initiation codon and upstream sequences are inserted into the appropriate 
expression vector, no additional translational control signals may be 
needed. However, in cases where only coding sequence, or a portion 
thereof, is inserted, exogenous transcriptional control signals including 
the ATG initiation codon must be provided. Furthermore, the initiation 
codon must be in the correct reading frame to ensure transcription of the 
entire insert. Exogenous transcriptional elements and initiation codons 
can be of various origins, both natural and synthetic. The efficiency of 
expression may be enhanced by the inclusion of enhancers appropriate to 
the cell system in use (Scharf et al (1994) Results Probl Cell Differ 20: 
125-62; Bittner et al (1987) Methods in Enzymol 153: 516-544). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be important for correct insertion, folding and/or function. 
Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific 
cellular machinery and characteristic mechanisms for such 
post-translational activities and may be chosen to ensure the correct 
modification and processing of the introduced, foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express 
TIMP-4 may be transformed using expression vectors which contain viral 
origins of replication or endogenous expression elements and a selectable 
marker gene. Following the introduction of the vector, cells may be 
allowed to grow for 1-2 days in an enriched media before they are switched 
to selective media. The purpose of the selectable marker is to confer 
resistance to selection and it's presence allows growth and recovery of 
cells which are successfully expressing the introduced sequences. 
Resistant clumps of stably transformed cells can be proliferated using 
tissue culture techniques appropriate to the cell type. 
Any number of selection systems may be used to recover transformed cell 
lines. These include, but are not limited to, the herpes simplex virus 
thymidine kinase (Wigler et al (1977) Cell 11: 223) and adenine 
phosphoribosyltransferase (Lowy et al (1980) Cell 22: 817) genes which can 
be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, 
antimetabolite, antibiotic or herbicide resistance can be used as the 
basis for selection; for example, dhfr which confers resistance to 
methotrexate (Wigler et al (1980) Natl Acad Sci 77: 3567); npt, which 
confers resistance to the aminoglycosides neomycin and G-418 
(Colberre-Garapin et al (1981) J Mol Biol 150: 1) and als or pat, which 
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, 
respectively (Murry, supra). Additional selectable genes have been 
described, for example, trpB, which allows cells to utilize indole in 
place of tryptophan, or hisD, which allows cells to utilize histinol in 
place of histidine (Hartman and Mulligan (1988) Proc Natl Acad Sci 85: 
8047). Recently, the use of visible markers has gained popularity with 
such markers as anthocyanins, .beta. glucuronidase and its substrate, GUS, 
and luciferase and its substrate, luciferin, being widely used not only to 
identify transformants, but also to quantify the amount of transient or 
stable protein expression attributable to a specific vector system (Rhodes 
C A et al (1995) Methods Mol Biol 55: 121-31). 
Identification of Transformants Containing Nucleotides Encoding TIMP-4 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression should be 
confirmed. For example, if the TIMP-4 is inserted within a marker gene 
sequence, recombinant cells containing TIMP-4 can be identified by the 
absence of marker gene function. Alternatively, a marker gene can be 
placed in tandem with a TIMP-4 sequence under the control of a single 
promoter. Expression of the marker gene in response to induction or 
selection will usually result in expression of TIMP-4 as well. 
Alternatively, host cells which contain the coding sequence for TIMP-4 and 
express TIMP-4 may be identified by a variety of procedures known to those 
of skill in the art. These procedures include, but are not limited to, 
DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay 
techniques which include membrane, solution, or chip-based technologies 
for the detection and/or quantification of the nucleic acid or protein. 
The presence of the TIMP-4 polynucleotide sequence can be detected by 
DNA-DNA or DNA-RNA hybridization or amplification using probes, portions 
or fragments of TIMP-4. Nucleic acid amplification based assays involve 
the use of oligonucleotides or oligomers based on the TIMP-4 sequence to 
detect transformants containing TIMP-4 DNA or RNA. As used herein 
"oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at 
least about 10 nucleotides and as many as about 60 nucleotides, preferably 
about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides 
which can be used as a probe or amplimer. 
Sequences for the Expression and Recovery of TIMP-4 Protein 
Host cells transformed with a TIMP-4 nucleotide sequence may be cultured 
under conditions suitable for the expression and recovery of the encoded 
protein from cell culture. The protein produced by a recombinant cell may 
be secreted or may be contained intracellularly depending on the sequence 
and/or the vector used. As will be understood by those of skill in the 
art, expression vectors containing TIMP-4 can be designed with signal 
sequences which direct secretion of TIMP-4 through a particular 
prokaryotic or eukaryotic cell membrane. Other recombinant constructions 
may join TIMP-4 to nucleotide sequence encoding a polypeptide domain which 
will facilitate purification of soluble proteins (Kroll D J et al (1993) 
DNA Cell Biol 12: 441-53; see also above discussion of vectors containing 
fusion proteins). 
TIMP-4 may also be expressed as a recombinant protein with one or more 
additional polypeptide domains added to facilitate protein purification. 
Such purification facilitating domains include, but are not limited to, 
metal chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAGS extension/affinity purification system (Immunex Corp, Seattle 
Wash.). The inclusion of a clearable linker sequences such as Factor XA or 
enterokinase (Invitrogen, San Diego Calif.) between the purification 
domain and TIMP-4 is useful to facilitate purification. 
Detection of the Expressed and Activity of TIMP-4 Protein 
A variety of protocols for detecting and measuring the expression of 
TIMP-4, using either polyclonal or monoclonal antibodies specific for 
TIMP-4 are known in the art. Examples include enzyme-linked immunosorbent 
assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell 
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing 
monoclonal antibodies reactive to two non-interfering epitopes on TIMP-4 
is preferred, but a competitive binding assay may be employed. These and 
other assays are described, among other places, in Hampton R et al (1990, 
Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and 
Maddox D E et al (1983, J Exp Med 158: 1211). 
The activity of TIMP-4 can be assessed chemically by using expressed TIMP-4 
to inhibit the ability of proteolytic enzymes to degrade matrix materials. 
For example, a simple reverse zymogram, patterned after the technique of 
Tyagi S C et al (1993; Mol Cell Biochem 126: 49-59), hereby incorporated 
by reference, would involve the electrophoresis of different 
concentrations of TIMP-4 or media containing TIMP-4. The resulting gel or 
an electroblot of that gel would be coated or soaked in gelatin. Then, 
gelatinase would be used to digest away the gelatin. In those areas of the 
gel or blot where the gelatin was in contact with TIMP-4, digestion would 
be inhibited. The inhibition associated with different concentrations of 
TIMP-4 could be used to quantitatively to assess TIMP-4 activity. Similar 
gels could be run using collagen/collagenase or other extracellular matrix 
components and their enzymes. 
Labeling Nucleotides or Antibodies for TIMP-4 Assays 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and can be used in various nucleic and amino acid 
assays. Means for producing labeled hybridization or PCR probes for 
detecting sequences related to TIMP-4 include oligolabeling, nick 
translation, end-labeling or PCR amplification using a labeled nucleotide. 
Alternatively, the TIMP-4 sequence, or any portion of it, may be cloned 
into a vector for the production of an mRNA probe. Such vectors are known 
in the art, are commercially available, and may be used to synthesize RNA 
probes in vitro by addition of an appropriate RNA polymerase such as T7, 
T3 or SP6 and labeled nucleotides. 
A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega 
(Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supply commercial 
kits and protocols for these procedures. Suitable reporter molecules or 
labels include those radionuclides, enzymes, fluorescent, 
chemiluminescent, or chromogenic agents as well as substrates, cofactors, 
inhibitors, magnetic particles and the like. Patents teaching the use of 
such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 
3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant 
immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567 
incorporated herein by reference. 
Uses of TIMP-4 
The rationale for using TIMP-4 diagnostically and therapeutically is based 
on its amino acid sequence, its structural similarity to other TIMPs, and 
the known activities of TIMP molecules. All of the known TIMPs degrade 
MMPs, therefore TIMP-4 can be used in conditions associated with a surplus 
of MMPs, ie, any condition where the amount of MMPs is excessive and 
amount of TIMP is insufficient to inhibit that quantity of MMPs. Such 
conditions are exemplified by metastasis of neoplastic cells, anglogenesis 
and growth of cervical, uterine, pancreatic, colon or intestinal 
carcinomas, osteoarthritis, osteoporosis, pulmonary emphysema, periodontal 
disease, and rheumatoid arthritis. 
TIMP-4 can also be used to prevent metastasis of neoplastic cells and 
vascularization of tumors. Tumor-produced MMPs loosen the extracellular 
matrix of the tumor allowing neoplastic cells to escape from the original 
tumor site. Then, the neoplastic cells produce MMPs which dissolve a path 
through surrounding tissues and breach the basement membranes and 
endothelial walls of the vascular system. Inhibition of the MMPs by TIMP-4 
can prevent the metastasis of neoplastic cells. Similarly, tumor-produced 
MMPs loosen the extracellular matrix of developing tumors allowing the 
tissue mass to grow and permitting anglogenesis. Inhibition of the MMPs by 
TIMP-4 can prevent tumor growth and anglogenesis. 
TIMP-4 can also be used for post-coital birth control. The trophoblast 
secretes MMPs during implantation of the embryo. The timely inhibition of 
these MMPs by recombinant TIMP-4, post coitus, would prevent loosening of 
the extracellular matrix, implantation of the embryo, and therefore, 
pregnancy. 
Because some conditions associated with high MMP/TIMP ratio, such as 
osteoarthritis, result in inflammation and pain, TIMP-4 also can have a 
role in controlling inflammation and inflammation-associated pain. For 
example, a therapeutic molecule comprising TIMP-4 or vectors encoding 
TIMP-4 can find application in a disease state such as osteoarthritis 
where the excessive expression of MMPs causes erosion of connective 
tissues and can lead to osteoporosis. 
In another embodiment of the invention, antisense molecules, anti-TIMP-4 
antibodies, antagonists or inhibitors capable of neutralizing the activity 
of TIMP-4 can be used as therapeutics for conditions characterized by 
excess expression of TIMP-4. High TIMP-4 expression may interfere with the 
healing process by preventing MMPs from tearing down matrix materials, 
such as in corneal or diabetic ulcers or ulcers/lesions caused by 
microorganisms, such as Helicobacter, Hemophilus, Treponema, or Neisseria. 
In such cases, the exposure to invading microorganisms, mitogens or toxic 
materials induces expression of TIMP-4 early in the disease process. 
Inhibition of the MMPs by TIMP-4 slows removal of dead or damaged cells 
which is necessary to create an environment for healing. Excessive TIMP-4 
prevents the removal of injured matrix and interferes with tissue 
remodeling and healing. 
TIMP-4 Antibodies 
Procedures well known in the art can be used for the production of 
antibodies to TIMP-4. Such antibodies include, but are not limited to, 
polyclonal, monoclonal, chimeric, single chain, Fab fragments and 
fragments produced by a Fab expression library. Neutralizing antibodies, 
ie, those which inhibit biological activity of TIMP-4, are especially 
preferred for diagnostics and therapeutics. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, etc may be immunized by injection with TIMP-4 or any portion, 
fragment or oligopeptide which retains immunogenic properties. Depending 
on the host species, various adjuvants may be used to increase 
immunological response. Such adjuvants include but are not limited to 
Freund's, mineral gels such as aluminum hydroxide, and surface active 
substances such as lysolecithin, pluronic polyols, polyanions, peptides, 
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli 
Calmette-Guerin) and Corynebacterium parvum are potentially useful human 
adjuvants. 
Monoclonal antibodies to TIMP-4 may be prepared using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Koehler and Milstein (1975 Nature 256: 495-497), 
the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4: 
72; Cote et al (1983) Proc Natl Acad Sci 80: 2026-2030) and the 
EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and 
Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96). In addition, 
techniques developed for the production of "chimeric antibodies", the 
splicing of mouse antibody genes to human antibody genes to obtain a 
molecule with appropriate antigen specificity and biological activity can 
be used (Morrison et al (1984) Proc Natl Acad Sci 81: 6851-6855; Neuberger 
et al (1984) Nature 312: 604-608; Takeda et al (1985) Nature 314: 
452-454). Alternatively, techniques described for the production of single 
chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce 
TIMP-4-specific single chain antibodies. 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening recombinant immunoglobulin libraries 
or panels of highly specific binding reagents as disclosed in Orlandi et 
al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C 
(1991; Nature 349: 293-299). 
Antibody fragments which contain specific binding sites for TIMP-4 may also 
be generated. For example, such fragments include, but are not limited to, 
the F(ab').sub.2 fragments which can be produced by pepsin digestion of 
the antibody molecule and the Fab fragments which can be generated by 
reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed to allow rapid 
and easy identification of monoclonal Fab fragments with the desired 
specificity (Huse W D et al (1989) Science 256: 1275-1281). 
TIMP-4-specific antibodies are useful for the diagnosis of conditions and 
diseases associated with aberrant expression of TIMP-4. A variety of 
protocols for competitive binding or immunoradiometric assays using either 
polyclonal or monoclonal antibodies with established specificities are 
well known in the art. Such immunoassays typically involve the formation 
of complexes between TIMP-4 and its specific antibody (or TIMP-4-binding 
molecule) and the measurement of complex formation. A two-site, 
monoclonal-based immunoassay utilizing monoclonal antibodies reactive to 
two noninterfering epitopes on a specific TIMP-4 protein is preferred, but 
a competitive binding assay may also be employed. These assays are 
described in Maddox D E et al (1983, J Exp Med 158: 1211). 
Diagnostic Assays Using TIMP-4 Specific Antibodies 
Particular TIMP-4 antibodies are useful for the diagnosis of conditions or 
diseases characterized by aberrant expression of TIMP-4. Diagnostic assays 
for TIMP-4 include methods utilizing the antibody and a label to detect 
TIMP-4 in human body fluids, cells, tissues or extracts of such tissues. 
The polypeptides and antibodies of the present invention may be used with 
or without modification. Frequently, the polypeptides and antibodies will 
be labeled by joining them, either covalently or noncovalently, with a 
reporter molecule. A wide variety of reporter molecules are known, several 
of which were described above. 
A variety of protocols for measuring TIMP-4, using either polyclonal or 
monoclonal antibodies specific for the respective protein are known in the 
art. Examples include enzyme-linked immunosorbent assay (ELISA), 
radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A 
two-site, monoclonal-based immunoassay utilizing monoclonal antibodies 
reactive to two non-interfering epitopes on TIMP-4 is preferred, but a 
competitive binding assay may be employed. These assays are described, 
among other places, in Maddox, D E et al (1983, J Exp Med 158: 1211). 
In order to provide a basis for the diagnosis of disease, normal or 
standard values for TIMP-4 expression must be established. This is 
accomplished by combining body fluids or cell extracts taken from normal 
subjects, either animal or human, with antibody to TIMP-4 under conditions 
suitable for complex formation which are well known in the art. The amount 
of standard complex formation may be quantified by comparing it with a 
dilution series of positive controls where a known amount of antibody is 
combined with known concentrations of purified TIMP-4. Then, standard 
values obtained from normal samples may be compared with values obtained 
from samples from subjects potentially affected by a disorder or disease 
related to TIMP-4 expression. Deviation between standard and subject 
values establishes the presence of disease state. 
Drug Screening 
TIMP-4, its catalytic or immunogenic fragments or oligopeptides can be used 
for screening therapeutic compounds in any of a variety of drug screening 
techniques. The fragment employed in such a test may be free in solution, 
affixed to a solid support, borne on a cell surface, or located 
intracellularly. The abolition of catalytic activity or the formation of 
binding complexes, between TIMP-4 and the agent being tested, may be 
measured. 
Another technique for drug screening which provides for high throughput 
screening of compounds having suitable binding affinity to the TIMP-4 is 
described in detail in "Determination of Amino Acid Sequence Antigenicity" 
by Geysen H N, WO Application 84/03564, published on Sep. 13, 1984, and 
incorporated herein by reference. In summary, large numbers of different 
small peptide test compounds are synthesized on a solid substrate, such as 
plastic pins or some other surface. The peptide test compounds are reacted 
with fragments of TIMP-4 and washed. Bound TIMP-4 is then detected by 
methods well known in the art. Purified TIMP-4 can also be coated directly 
onto plates for use in the aforementioned drug screening techniques. 
Alternatively, non-neutralizing antibodies can be used to capture the 
peptide and immobilize it on a solid support. 
This invention also contemplates the use of competitive drug screening 
assays in which neutralizing antibodies capable of binding TIMP-4 
specifically compete with a test compound for binding TIMP-4. In this 
manner, the antibodies can be used to detect the presence of any peptide 
which shares one or more antigenic determinants with TIMP-4. 
Uses of TIMP-4 Polynucleotide 
A TIMP-4 polynucleotide, or any part thereof, may be used for diagnostic 
and/or therapeutic purposes. For diagnostic purposes, the TIMP-4 of this 
invention may be used to detect and quantitate gene expression in 
conditions or diseases in which TIMP-4 activity may be implicated. These 
specifically include, but are not limited to, metastasis of neoplastic 
cells, anglogenesis and growth of tumors, cervical, uterine, pancreatic, 
colon or intestinal carcinomas, pregnancy, osteoarthritis, osteoporosis, 
pulmonary emphysema, periodontal disease, rheumatoid arthritis, corneal 
and diabetic ulcers, or ulcers/lesions caused by microorganisms. Included 
in the scope of the invention are oligonucleotide sequences, antisense RNA 
and DNA molecules and ribozymes, which function to inhibit translation of 
a TIMP-4. 
Another aspect of the subject invention is to provide for hybridization or 
PCR probes which are capable of detecting polynucleotide sequences, 
including genomic sequences, encoding TIM P-4 or closely related 
molecules. The specificity of the probe, whether it is made from a highly 
conserved region, eg, 10 unique nucleotides in the 5' regulatory region, 
or a less conserved region, eg, between cysteine residues especially in 
the 3' region, and the stringency of the hybridization or amplification 
(high, intermediate or low) will determine whether the probe identifies 
only naturally occurring TIMP-4 or related sequences. 
Diagnostics 
TIMP-4 polynucleotide sequences may be used for the diagnosis of conditions 
or diseases resulting from aberrant expression of TIMP-4. For example, 
polynucleotide sequences encoding TIMP-4 may be used in hybridization or 
PCR assays of tissues from biopsies or autopsies to detect abnormalities 
in TIMP-4 expression. The form of such qualitative or quantitative methods 
may include Southern or northern analysis, dot blot or other 
membrane-based technologies; PCR technologies; dip stick, pin, chip and 
ELISA technologies. All of these techniques are well known in the art, and 
are in fact the basis of many commercially available diagnostic kits. 
Such assays may be tailored to evaluate the efficacy of a particular 
therapeutic treatment regime and may be used in animal studies, in 
clinical trials, or in monitoring the treatment of an individual patient. 
In order to provide a basis for the diagnosis of disease, a normal or 
standard profile for TIMP-4 expression must be established. This is 
accomplished by combining body fluids or cell extracts taken from normal 
subjects, either animal or human, with TIMP-4 or a portion thereof, under 
conditions suitable for hybridization or amplification. Standard 
hybridization may be quantified by comparing the values obtained for 
normal subjects with a dilution series of positive controls run in the 
same experiment where a known amount of purified TIMP-4 is used. Standard 
values obtained from normal samples may be compared with values obtained 
from samples from subjects potentially affected by a disorder or disease 
related to TIMP-4 expression. Deviation between standard and subject 
values establishes the presence of the disease state. 
If disease is established, an existing therapeutic agent is administered, 
and a treatment profile may be generated. Finally, the assay may be 
repeated on a regular basis to evaluate whether the values in the profile 
progress toward or return to the normal or standard pattern. Successive 
treatment profiles may be used to show the efficacy of treatment over a 
period of several days or several months. 
PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 
provides additional uses for oligonucleotides based upon the TIMP-4 
sequence. Such oligomers are generally chemically synthesized, but they 
may be generated enzymatically or produced from a recombinant source. 
Oligomers generally comprise two nucleotide sequences, one with sense 
orientation (5'-&gt;3') and one with antisense (3'&lt;-5') employed under 
optimized conditions for identification of a specific gene or condition. 
The same two oligomers, nested sets of oligomers, or even a degenerate 
pool of oligomers may be employed under less stringent conditions for 
detection and/or quantitation of closely related DNA or RNA sequences. 
Additionally methods to quantitate the expression of a particular molecule 
include radiolabeling (Melby P C et al 1993 J Immunol Methods 159: 235-44) 
or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, 
coamplification of a control nucleic acid, and standard curves onto which 
the experimental results are interpolated. Quantitation of multiple 
samples may be speeded up by running the assay in an ELISA format where 
the oligomer-of-interest is presented in various dilutions and a 
spectrophotometric or colorimetric response gives rapid quantitation. For 
example, upregulation of TIMP-4 in lung may indicate that the expression 
of MMPs needs to be monitored since the unopposed activity of MMPs can 
result in the destruction that is associated with pulmonary emphysema. A 
definitive diagnosis may allow health professionals to treat the patient 
and prevent further worsening of the condition. Similarly, assays known to 
those of skill in the art can be used to monitor the progress of a patient 
displaying a TIMP-4 associated disease state during therapy. 
Therapeutics 
A TIMP-4 sequence may be useful in the treatment of various conditions or 
diseases. By introducing TIMP-4 sequence into cells, gene therapy can be 
used to treat conditions characterized by underexpression of TIMP-4. In 
some instances, the sequence encoding a TIMP-4 is intended to replace or 
act in the place of a functionally deficient naturally occurring gene. 
Expression vectors derived retroviruses, adenovirus, herpes or vaccinia 
viruses, or from various bacterial plasmids, may be used for delivery of 
recombinant TIMP-4, sense or antisense molecules, to the targeted cell 
population. Methods which are well known to those skilled in the art can 
be used to construct recombinant vectors containing TIMP-4. See, for 
example, the techniques described in Maniatis et al (supra) and Ausubel et 
al (supra). Alternatively, recombinant TIMP-4 can be delivered to target 
cells in liposomes. 
Conditions or diseases characterized by excess TIMP-4 can be treated by 
using the same gene therapy techniques to introduce recombinant antisense 
constructs. The successful delivery and expression of such sequences will 
modulate or inhibit the transcription of TIMP-4 mRNA. 
The full length cDNA sequence and/or its regulatory elements enable 
researchers to use TIMP-4 as a tool in sense (Youssoufian H and H F Lodish 
1993 Mol Cell Biol 13: 98-104) or antisense (Eguchi et al (1991) Annu Rev 
Biochem 60: 631-652) investigations or regulation of gene function. 
Oligonucleotides, designed from the cDNA or control sequences obtained 
from the genomic DNA can be used in vitro or in vivo to inhibit 
expression. Such technology is now well known in the art, and sense or 
antisense oligomers, or larger fragments, can be designed from various 
locations along the coding or control regions. 
TIMP-4 can be turned off by transfecting a cell or tissue with expression 
vectors which express high levels of a TIMP-4 fragment. Such constructs 
can flood cells with untranslatable sense or antisense sequences. Even in 
the absence of integration into the DNA, such vectors may continue to 
transcribe RNA molecules until all copies are disabled by endogenous 
nucleases. Such transient expression may last for a month or more with a 
non-replicating vector (Mettler I, personal communication) and even longer 
if appropriate replication elements are part of the vector system. 
On the other hand, stable transformation of appropriate germ line cells, or 
preferably a zygote, with a vector containing the TIMP-4 fragments may 
produce a transgenic organism (U.S. Pat. No. 4,736,866, 12 Apr. 1988), 
which produces enough copies of the sense or antisense sequence to 
significantly compromise or entirely eliminate activity of the naturally 
occurring TIMP-4 gene. 
As mentioned previously, modifications of gene expression can be obtained 
by designing antisense sequences to the control regions of the TIMP-4 
gene, ie, the promoters, enhancers, and introns. Oligonucleotides derived 
from the transcription initiation site, eg, between -10 and +10 regions of 
the leader sequence, are preferred. Antisense RNA and DNA molecules may 
also be designed to block translation of mRNA by preventing the transcript 
from binding to ribosomes. Similarly, inhibition can be achieved using 
Hogeboom base-pairing methodology, also known as "triple helix" base 
pairing. Triple helix pairing compromises the ability of the double helix 
to open sufficiently for the binding of polymerases, transcription 
factors, or regulatory molecules. 
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific 
cleavage of RNA. The mechanism of ribozyme action involves sequence 
specific hybridization of the ribozyme molecule to complementary target 
RNA, followed by a endonucleolytic cleavage. Within the scope of the 
invention are engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of TIMP-4 
RNA sequences. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites which include the following sequences, GUA, GUU and GUC. Once 
identified, short RNA sequences of between 15 and 20 ribonucleotides 
corresponding to the region of the target gene containing the cleavage 
site may be evaluated for secondary structural features which may render 
the oligonucleotide sequence inoperable. The suitability of candidate 
targets may also be evaluated by testing accessibility to hybridization 
with lo complementary oligonucleotides using ribonuclease protection 
assays. 
Both antisense RNA and DNA molecules and ribozymes of the invention may be 
prepared by any method known in the art for the synthesis of RNA 
molecules. These include techniques for chemically synthesizing 
oligonucleotides such as solid phase phosphoramidite chemical synthesis. 
Alternatively, RNA molecules may be generated by in vitro and in vivo 
transcription of DNA sequences encoding the antisense RNA molecule. Such 
DNA sequences may be incorporated into a wide variety of vectors with 
suitable RNA polymerase promoters such as T7 or SP6. Alternatively, 
antisense cDNA constructs that synthesize antisense RNA constitutively or 
inducibly can be introduced into cell lines, cells or tissues. 
DNA molecules may be modified to increase intracellular stability and 
half-life. Possible modifications include, but are not limited to, the 
addition of flanking sequences of the 5' and/or 3' ends of the molecule or 
the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase 
linkages within the backbone of the molecule. 
Methods for introducing vectors into cells or tissue include those methods 
discussed infra. In addition, several of these transformation or 
transfection methods are equally suitable for the ex vivo therapy, the 
introduction of vectors into stem cells taken from the patient and 
clonally propagated for autologous transplant as in U.S. Pat. Nos. 
5,399,493 and 5,437,994, disclosed herein by reference. 
Furthermore, the TIMP-4 polynucleotide sequences disclosed herein may be 
used in molecular biology techniques that have not yet been developed, 
provided the new techniques rely on properties of nucleotide sequences 
that are currently known, including but not limited to such properties as 
the triplet genetic code and specific base pair interactions. 
Detection and Mapping of Polynucleotide Sequences Related to TIMP-4 
The nucleic acid sequence for TIMP-4 can also be used to generate 
hybridization probes for mapping the naturally occurring genomic sequence. 
The sequence may be mapped to a particular chromosome or to a specific 
region of the chromosome using well known techniques. These include in 
situ hybridization to chromosomal spreads (Verma et al (1988) Human 
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), 
flow-sorted chromosomal preparations, or artificial chromosome 
constructions such as YACs, bacterial artificial chromosomes (BACs), 
bacterial P1 constructions or single chromosome cDNA libraries. 
In situ hybridization of chromosomal preparations and physical mapping 
techniques such as linkage analysis using established chromosomal markers 
are invaluable in extending genetic maps. Examples of genetic maps can be 
found in Science (1995; 270: 410f and 1994; 265: 1981f). Often the 
placement of a gene on the chromosome of another mammalian species may 
reveal associated markers even if the number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once a disease or syndrome, 
such as ataxia telangiectasia (AT), has been crudely localized by genetic 
linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti 
et al (1988) Nature 336: 577-580), any sequences mapping to that area may 
represent associated or regulatory genes for further investigation. The 
nucleotide sequence of the subject invention may also be used to detect 
differences in the chromosomal location due to translocation, inversion, 
etc. between normal, carrier or affected individuals. 
Pharmaceutical Compositions 
The present invention comprises pharmaceutical compositions which may 
comprise all or portions of TIMP-4 or inhibitors of TIMP-4 including 
antibodies and antagonists, alone or in combination with at least one 
other agent, such as stabilizing compound, which may be administered in 
any sterile, biocompatible pharmaceutical carrier, including, but not 
limited to, saline, buffered saline, dextrose, and water. 
TIMP-4 can be administered to a patient alone, or in combination with other 
TIMPs, agents, drugs or hormones in pharmaceutical compositions where it 
is mixed with excipient(s) or pharmaceutically acceptable carriers. In one 
embodiment of the present invention, the pharmaceutically acceptable 
carrier is pharmaceutically inert. Since TIMP-4 is a secreted protein 
which works extracellularly to inhibit secreted MMPs, the preferred route 
for administration of TIMP-4 or its inhibitors is by diffusion from the 
site of administration. 
TIMP-4 may be used alone or in combination with other TIMP molecules to 
prevent metastasis of neoplastic cells, growth or vascularization of 
tumors and with other chemotherapy for treating cervical, uterine, 
pancreatic, colon or intestinal carcinomas. TIMP-4 is administered 
intra-arterially in a sufficient amount to inhibit tumor-produced 
metalloproteinases from loosening the tumor tissue for anglogenesis, 
dissolving a path through surrounding tissues, and breaching the basement 
membranes and endothelial walls of the vascular system, thereby preventing 
the metastasis of neoplastic cells. 
Administration of compounds to treat osteoarthritis has been local or 
systemic; however, TIMP-4 could also be injected directly into a swollen 
joint or delivered in combination with a penetrating agent, by patch 
applied over the joint. Systemic delivery for osteoarthritis or 
osteoporosis could be via such means as intranasal spray, patch or 
subcutaneous injection. 
TIMP-4 may be used alone or in combination with other TIMP molecules to 
treat periodontal disease. Administration of a penetrating treatment in a 
gel, toothpaste, mouthwash, spray or lozenger form could prevent the 
destruction of connective tissue which eventually results in the loss of 
teeth. Similarly, administration of TIMP-4 via inhaler could slow or 
prevent the destruction of connective tissue that alters the alveoli and 
results in pulmonary emphysema. 
An effective amount of TIMP-4, alone or in combination with other TIMPs, 
may be administered to females as a means for post-coital birth control. 
TIMPs may be administered from a foam, vaginal sponge, or suppository over 
approximately a week's time following suspected conception. TIMPs can 
inactivate the MMPs secreted by trophoblastic cells to begin implantation, 
thereby preventing pregnancy. This lavage of TIMPs would work prior to and 
supplement the usual TIMPS which are produced at the implantation site. 
Alternatively, for treatment of conditions or diseases characterized by the 
excessive expression of TIMP-4, such as corneal or diabetic ulcers and 
ulcers/lesions produced by infectious microorganisms, administration of 
antisense molecules, antagonists or inhibitors of TIMP-4 may be delivered 
to wound sites using irrigation, salve or dermal patches or bandages and 
appropriate penetrant. These molecules can aid in the healing of corneal 
or diabetic ulcers and ulcers/lesions attributable to microorganisms, 
alkali burns or other insults or injuries. 
Further details on techniques for formulation and administration may be 
found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack 
Publishing Co, Easton Pa.). Although local delivery is desirable, there 
are other means, for example, oral; parenteral delivery, including 
intra-arterial (directly to the tumor), intramuscular, subcutaneous, 
intramedullary, intrathecal, intraventricular, intravenous, 
intraperitoneal, or intranasal administration. 
For injection, the pharmaceutical compositions of the invention may be 
formulated in aqueous solutions, preferably in physiologically compatible 
buffers such as Hanks's solution, Ringer's solution, or physiologically 
buffered saline. For tissue or cellular administration, penetrants 
appropriate to the particular barrier to be permeated are used in the 
formulation. Such penetrants are generally known in the art. 
The pharmaceutical compositions can be formulated using pharmaceutically 
acceptable carriers well known in the art in dosages suitable for oral 
administration. Such carriers enable the pharmaceutical compositions to be 
formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, 
suspensions and the like, for oral or nasal ingestion by a patient to be 
treated. 
Pharmaceutical compositions suitable for use in the present invention 
include compositions wherein the active ingredients are contained in an 
effective amount to achieve the intended purpose. The determination of an 
effective dose is well within the capability of those skilled in the art, 
especially in light of the disclosure provided below. 
In addition to the active ingredients these pharmaceutical compositions may 
contain suitable pharmaceutically acceptable carriers comprising 
excipients and auxiliaries which facilitate processing of the active 
compounds into preparations which can be used pharmaceutically. The 
preparations formulated for oral administration may be in the form of 
tablets, dragees, capsules, or solutions. 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is itself known, eg, by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. 
Pharmaceutical formulations for parenteral administration include aqueous 
solutions of the active compounds in water-soluble form. Additionally, 
suspensions of the active compounds may be prepared as appropriate oily 
injection suspensions. Suitable lipophilic solvents or vehicles include 
fatty oils such as sesame oil, or synthetic fatty acid esters, such as 
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions 
may contain substances which increase the viscosity of the suspension, 
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, 
the suspension may also contain suitable stabilizers or agents which 
increase the solubility of the compounds to allow for the preparation of 
highly concentrated solutions. 
Pharmaceutical preparations for oral use can be obtained by combining the 
active compounds with solid excipient, optionally grinding a resulting 
mixture, and processing the mixture of granules, after adding suitable 
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable 
excipients are carbohydrate or protein fillers such as sugars, including 
lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, 
potato, etc; cellulose such as methyl cellulose, 
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a 
salt thereof such as sodium alginate. 
Dragee cores are provided with suitable coatings such as concentrated sugar 
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer 
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or 
pigments may be added to the tablets or dragee coatings for product 
identification or to characterize the quantity of active compound, ie, 
dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating such as glycerol or sorbitol. The push-fit capsules can 
contain the active ingredients mixed with a filler or binders such as 
lactose or starches, lubricants such as talc or magnesium stearate, and, 
optionally, stabilizers. In soft capsules, the active compounds may be 
dissolved or suspended in suitable liquids, such as fatty oils, liquid 
paraffin, or liquid polyethylene glycol with or without stabilizers. 
Compositions comprising a compound of the invention formulated in a 
pharmaceutical acceptable carrier may be prepared, placed in an 
appropriate container, and labeled for treatment of an indicated 
condition. For administration of TIMP-4, such labeling would include 
amount, frequency and method of administration. 
The pharmaceutical composition may be provided as a salt and can be formed 
with many acids, including but not limited to hydrochloric, sulfuric, 
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more 
soluble in aqueous or other protonic solvents that are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% 
mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to 
use. 
For any compound used in the method of the invention, the therapeutically 
effective dose can be estimated initially from cell culture assays, eg, of 
neoplastic cells. Then, preferably, dosage can be formulated in animal 
models affected with the neoplasm to achieve a desirable concentration 
range and route of administration that inhibits MMPs. Such information can 
be used to determine useful doses and route of administration in humans. 
A therapeutically effective dose refers to that amount of TIMP-4 or its 
inhibitor which ameliorates symptoms, eg, reduces or prevents metastasis 
of neoplastic cells or prevents implantation. Toxicity and therapeutic 
efficacy of such compounds can be determined by standard pharmaceutical 
procedures in cell cultures or experimental animals, eg, for determining 
the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose 
therapeutically effective in 50% of the population). The dose ratio 
between toxic and therapeutic effects is the therapeutic index, and it can 
be expressed as the ratio LD50/ED50. Compounds, TIMP variants or 
fragments, which exhibit large therapeutic indices are preferred. The data 
obtained from these cell culture assays and additional animal studies can 
be used in formulating a range of dosage for human use. The dosage of such 
compounds lies preferably within a range of circulating concentrations 
that include the ED50 with little or no toxicity. The dosage varies within 
this range depending upon the dosage form employed, sensitivity of the 
patient, and the route of administration. 
The exact dosage is chosen by the individual physician in view of the 
patient to be treated. Dosage and administration are adjusted to provide 
sufficient levels of the active moiety or to maintain the desired effect. 
Additional factors which may be taken into account include the severity of 
the disease state, eg, tumor size and location; age, weight, and gender of 
the patient; diet, time and frequency of administration, drug 
combination(s), reaction sensitivities, and tolerance/response to therapy. 
Long acting pharmaceutical compositions might be administered every 3 to 4 
days, every week, or once every two weeks depending on half-life and 
clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. 
Those skilled in the art will employ different formulations for TIMP-4 
than for the inhibitors of TIMP-4. Similarly administration of TIMP-4 to a 
tumor will necessitate delivery in a manner different from that of a 
TIMP-4 inhibitor being applied to an external ulcer. 
It is contemplated that conditions or diseases associated with an 
insufficiency or excess of TIMP are treatable with either TIMP-4 or 
inhibitors of TIMP-4, respectively. Conditions associated with production 
of excess MMPs include, but are not limited to, metastasis of neoplastic 
cells; anglogenesis and growth of tumors, particularly cervical, uterine, 
pancreatic, colon or intestinal carcinomas; pregnancy, osteoarthritis, 
osteoporosis, pulmonary emphysema, periodontal disease, rheumatoid 
arthritis; whereas conditions or diseases associated with untimely or 
excessive TIMP-4 may include, but are not limited to, corneal and diabetic 
ulcers, and ulcers/lesions caused or perpetuated by the presence of 
microorganisms such as Helicobacter, Hemophilus, Treponema, or Neisseria. 
The assays previously discussed may be used to diagnose the conditions or 
diseases and to monitor treatment. 
The examples below are provided to illustrate the subject invention. These 
examples are provided by way of illustration and are not included for the 
purpose of limiting the invention. 
EXAMPLES 
I Uterus cDNA Library Construction 
The UTRSNOT01 library was constructed using tissue isolated from the uterus 
of a 59 year old, Caucasian female; (Lot#94-072, Keystone Skin Bank, 
International Institute for the Advancement of Medicine, Exton Pa.) who 
died following myocardial infarction. The frozen tissue was ground in a 
mortar and pestle and lysed immediately in a buffer containing guanidinium 
isothiocyanate. The lysate was centrifuged over a 5.7M CsCl cushion using 
an SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) 
for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted 
once with acid phenol, pH 4.0, precipitated using 0.3M sodium acetate and 
2.5 volumes of ethanol, resuspended in water and DNase treated for 15 min 
at 37.degree. C. The RNA was isolated using the Qiagen Oligotex kit 
(QIAGEN Inc, Chatsworth Calif.). 
The poly-A+ RNA was handled according to the recommended protocols in the 
SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning 
(Catalog#18248-013; Gibco/BRL). First strand cDNA synthesis was 
accomplished using oligo d(T) priming and second strand synthesis was 
performed using a combination of DNA polymerase I, E. coli ligase and 
RNase H. The cDNA was blunted with T4 polymerase, and a Sal I linker was 
added to the blunt ended cDNA. The Sal I adapted, double-stranded cDNAs 
were the digested with Not I and fractionated on a Sepharose CL4B column 
(Catalog#275105, Pharmacia). Those cDNAs exceeding 400 bp were ligated 
into pSport I which was subsequently transformed into DH5a.TM. competent 
cells (Catalog#18258-012, Gibco/BRL). 
II Isolation and Sequencing of cDNA Clones 
Plasmid DNA was purified using the Miniprep Kit (catalog #77468, Advanced 
Genetic Technologies Corporation, Gaithersburg Md.), a 96-well block kit 
with reagents for 960 purifications. The recommended protocol included 
with the kit was employed except for the following changes. Each of the 96 
wells was filled with only 1 ml of sterile Terrific Broth (Catalog #22711, 
LIFE TECHNOLOGIES.TM.) with carbenicillin at 25 mg/L and glycerol at 0.4%. 
After the wells were inoculated, the bacteria were cultured for 24 hours 
and lysed with 60 .mu.l of lysis buffer. A centrifugation step (Beckman 
GS-6R @2900 rpm for 5 min; Beckman Instruments) was performed before the 
contents of the block were added to the primary filter plate. The optional 
step of adding isopropanol to TRIS buffer was not routinely performed. 
After the last step in the protocol, samples were transferred to a Beckman 
96-well block for storage. 
The cDNAs were sequenced by the method of Sanger F and A R Coulson (1975; J 
Mol Biol 94: 441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno Nev.) 
in combination with four Peltier Thermal Cyclers (PTC200 from MJ Research, 
Watertown Mass.) and Applied Biosystems 377 or 373 DNA Sequencing Systems 
(Perkin Elmer), and reading frame was determined. 
III Homology Searching of cDNA Clones and Their Deduced Proteins 
Each cDNA was compared to sequences in GertBank using a search algorithm 
developed by Applied Biosystems and incorporated into the INHERIT.TM. 670 
Sequence Analysis System. In this algorithm, Pattern Specification 
Language (TRW Inc, Los Angeles Calif.) was used to determine regions of 
homology. The three parameters that determine how the sequence comparisons 
run were window size, window offset, and error tolerance. Using a 
combination of these three parameters, the DNA database was searched for 
sequences containing regions of homology to the query sequence, and the 
appropriate sequences were scored with an initial value. Subsequently, 
these homologous regions were examined using dot matrix homology plots to 
distinguish regions of homology from chance matches. Smith-Waterman 
alignments were used to display the results of the homology search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT.TM. 670 Sequence Analysis System in a way similar to that used in 
DNA sequence homologies. Pattern Specification Language and parameter 
windows were used to search protein databases for sequences containing 
regions of homology which were scored with an initial value. Dot-matrix 
homology plots were examined to distinguish regions of significant 
homology from chance matches. 
BLAST, which stands for Basic Local Alignment Search Tool (Altschul S F 
(1993) J Mol Evol 36: 290-300; Altschul, S F et al (1990) J Mol Biol 215: 
403-10), was used to search for local sequence alignments. BLAST produces 
alignments of both nucleotide and amino acid sequences to determine 
sequence similarity. Because of the local nature of the alignments, BLAST 
is especially useful in determining exact matches or in identifying 
homologs. BLAST is useful for matches which do not contain gaps. The 
fundamental unit of BLAST algorithm output is the High-scoring Segment 
Pair (HSP). 
An HSP consists of two sequence fragments of arbitrary but equal lengths 
whose alignment is locally maximal and for which the alignment score meets 
or exceeds a threshold or cutoff score set by the user. The BLAST approach 
is to look for HSPs between a query sequence and a database sequence, to 
evaluate the statistical significance of any matches found, and to report 
only those matches which satisfy the user-selected threshold of 
significance. The parameter E establishes the statistically significant 
threshold for reporting database sequence matches. E is interpreted as the 
upper bound of the expected frequency of chance occurrence of an HSP (or 
set of HSPs) within the context of the entire database search. Any 
database sequence whose match satisfies E is reported in the program 
output. 
IV Extension of TIMP-4 to Recover Regulatory Elements 
The nucleic acid sequence of full length TIMP-4 (SEQ ID NO:1) may be used 
to design oligonucleotide primers for obtaining 5' sequences from genomic 
libraries. One primer is synthesized to initiate extension in the 
antisense direction (XLR) and the other is synthesized to extend sequence 
in the sense direction (XLF). The primers allowed the known TIMP-4 
sequence to be extended "outward" generating amplicons containing new, 
unknown nucleotide sequence for the control region of interest. The 
initial primers may be designed from the cDNA using Oligo 4.0 (National 
Biosciences Inc, Plymouth Minn.), or another appropriate program, to be 
22-30 nucleotides in length, to have a GC content of 50% or more, and to 
anneal to the target sequence at temperatures about 68.degree.-72.degree. 
C. Any stretch of nucleotides which would result in hairpin structures and 
primer-primer dimerizations is avoided. 
A human genomic library is used to extend and amplify 5' upstream sequence. 
If necessary, a second set of primers is designed to further extend the 
known region. 
By following the instructions for the XL-PCR kit (Perkin Elmer) and 
thoroughly mixing the enzyme and reaction mix, high fidelity amplification 
is obtained. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR is performed using 
the Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.) and the 
following parameters: 
______________________________________ 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.l aliquot of the reaction mixture is analyzed by electrophoresis 
on a low concentration (about 0.6-0.8%) agarose mini-gel to determine 
which reactions were successful in extending the sequence. The largest 
products or bands were selected and cut out of the gel. Further 
purification involves using a commercial gel extraction method such as 
QIAQuick.TM. (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was 
used to trim single-stranded, nucleotide overhangs creating blunt ends 
which facilitate religation and cloning. 
After ethanol precipitation, the products are redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase are added, and the mixture is incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C. Competent E. coli 
cells (in 40 .mu.l of appropriate media) are transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook J et al, 
supra). After incubation for one hour at 37.degree. C., the whole 
transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et 
al, supra) containing 2.times. Carb. The following day, several colonies 
are randomly picked from each plate and cultured in 150 .mu.l of liquid 
LB/2.times. carb medium placed in an individual well of an appropriate, 
commercially-available, sterile 96-well microtiter plate. The following 
day, 5 .mu.l of each overnight culture is transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
is transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer 
and one or both of the gene specific primers used for the extension 
reaction are added to each well. Amplification is performed using the 
following conditions: 
______________________________________ 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
______________________________________ 
Aliquots of the PCR reactions are run on agarose gels together with 
molecular weight markers. The sizes of the PCR products are compared to 
the original partial cDNAs, and appropriate clones are selected, ligated 
into plasmid and sequenced. 
V Labeling of Hybridization Probes 
Hybridization probes derived from SEQ ID NO:1 may be employed to screen 
cDNAs, mRNAs or genomic DNAs. Although the labeling of oligonucleotides, 
consisting of about 20 base-pairs, is specifically described, essentially 
the same procedure may be used with larger cDNA fragments. 
Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 
mCi of [.gamma.-.sup.32 P] adenosine triphosphate (Amersham, Chicago Ill.) 
and T4 polynucleotide kinase (DuPont NEN.RTM., Boston Mass.). The labeled 
oligonucleotides are purified with Sephadex G-25 super fine resin column 
(Pharmacia). A portion containing 10.sup.7 counts per minute of each is 
used in a typical membrane based hybridization analysis of human genomic 
DNA digested with one of the following endonucleases (Ase I, Bgl II, EcoR 
I, Pst I, Xba 1, or Pvu II; DuPont NEN.RTM.). 
The DNA from each digest is fractionated on a 0.7 percent agarose gel and 
transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham 
N.H.). Hybridization is carried out for 16 hours at 40.degree. C. To 
remove nonspecific signals, blots are sequentially washed at room 
temperature under increasingly stringent conditions up to 0.1.times. 
saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. 
film (Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimager 
cassette (Molecular Dynamics, Sunnyvale Calif.) for several hours, 
hybridization patterns are compared visually. 
VI Antisense Molecules 
The TIMP-4 sequence, or any part thereof, provide the basis for the design 
of antisense molecules which may be used to inhibit in vivo or in vitro 
expression of naturally occurring TIMP-4. Although use of antisense 
oligomers, consisting of about 20 base-pairs, is specifically described, 
essentially the same procedure may be used with larger cDNA fragments. An 
oligonucleotide based on the coding sequence of TIMP-4 may be used to 
inhibit expression of naturally occurring TIMP-4. The complementary 
oligonucleotide can be designed from the conserved 5' sequence and used 
either to inhibit transcription by preventing promoter binding to the 
upstream nontranslated sequence or translation of a TIMP-4 transcript by 
preventing the ribosome from binding to the -10 to +10 region of the 
leader/signal sequence. 
VII Expression of TIMP-4 
Expression of the TIMP-4 may be accomplished by subcloning the cDNAs into 
appropriate vectors and transfecting the vectors into host cells. The 
vector, pBluescript, is used to express TIMP-4 in E. coli., strain 
XL1-BlueMRF.TM. (Stratagene). Upstream of the cloning site, this vector 
contains a promoter for .beta.-galactosidase, followed by sequence 
containing the amino-terminal Met and the subsequent 7 residues of 
.beta.-galactosidase. Immediately following these eight residues is a 
bacteriophage promoter useful for transcription and a linker containing a 
number of unique restriction sites. 
Induction of an isolated, transfected bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
seven residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length TIMP-4. The signal sequence directs the secretion of 
TIMP-4 into the bacterial growth media which can be used directly in 
assays for activity as exemplified in Section VIII. 
VIII TIMP-4 Activity 
TIMP-4 activity is measured using reverse zymography patterned after the 
method of Tyagi S C et al (1993; Mol Cell Biochem 126: 49-59). Reverse 
zymography involves electrophoresis of different concentrations of TIMP-4 
or a media containing secreted TIMP-4. The non-denaturing gel in which 
TIMP-4 has been size separated from other proteins is soaked in gelatin. 
Then, a time course of digestion with gelatinase is used to assess the 
activity and effective concentration of TIMP-4 in inhibiting digestion of 
the gelatin. The control lane shows complete digestion and would be 
transparent when stained with Comassie Blue, while those lanes containing 
TIMP-4 show various degrees of gelatin dissolution and differential 
staining. Similar gels could be run using collagen/collagenase or other 
extracellular matrix components and their enzymes. 
IX Production of TIMP-4 Specific Antibodies 
Although TIMP-4 purified using PAGE electrophoresis (Maniatis, supra) can 
be used to immunize rabbits using standard protocols, a monoclonal 
approach is more commonly employed. The amino acid sequence translated 
from TIMP-4 is analyzed using DNASTAR software (DNASTAR Inc) to determine 
regions of high immunogenicity and a corresponding oligopeptide is 
synthesized and used to raise antibodies by means known to those of skill 
in the art. Analysis to select appropriate epitopes, such as those near 
the C-terminus or in adjacent hydrophilic regions is described by Ausubel 
F M et al (supra). 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry, 
and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with 
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel F M et al, 
supra). Rabbits are immunized with the oligopeptide-KLH complex in 
complete Freund's adjuvant. The resulting antisera are tested for 
antipeptide activity, for example, by binding the peptide to plastic, 
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting 
with radioiodinated, goat anti-rabbit IgG. 
X Purification of Naturally occurring TIMP-4 Using Specific Antibodies 
Naturally occurring or recombinant TIMP-4 can be purified by immunoaffinity 
chromatography using antibodies specific for TIMP-4. An immunoaffinity 
column is constructed by covalently coupling TIMP-4 antibody to an 
activated chromatographic resin such as CnBr-activated Sepharose 
(Pharmacia Biotech). After the coupling, the resin is blocked and washed 
according to the manufacturer's instructions. 
Media containing TIMP-4 is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of TIMP-4 (eg, high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/TIMP-4 binding 
(eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as 
urea or thiocyanate ion), and TIMP-4 is collected. 
XI Identification of Molecules Which Interact with TIMP-4 
TIMP-4, or biologically active fragments thereof, is labeled with .sup.125 
I Bolton-Hunter reagent (Bolton, A E and Hunter, W M (1973) Biochem J 133: 
529). Candidate inhibitory molecules previously arrayed in the wells of a 
96 well plate are incubated with the labeled TIMP-4, washed and any wells 
with labeled TIMP-4 complex are assayed. Data obtained using different 
concentrations of TIMP-4 are used to calculate values for the number, 
affinity, and association of TIMP-4 with the candidate inhibitory 
molecules. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 675 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: METALLOPROTEINASES 
(B) CLONE: 589345 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGCCTGGGAGCCCTCGGCCCGCGCCAAGCTGGGTGCTGTTGCTGCGGCTGCTGGCGTTG60 
CTGCGGCCCCCGGGGCTGGGTGAGGCATGCAGCTGCGCCCCGGCGCACCCTCAGCAGCAC120 
ATCTGCCACTCGGCACTTGTGATTCGGGCCAAAATCTCCAGTGAGAAGGTAGTTCCGGCC180 
AGTGCAGACCCTGCTGACACTGAAAAAATGCTCCGGTATGAAATCAAACAGATAAAGATG240 
TTCAAAGGGTTTGAGAAAGTCAAGGATGTTCAATATATCTATACGCCTTTTGACTCTTCC300 
CTCTGTGGTGTGAAACTAGAAGCCAACAGCCAGAAGCAGTATCTCTTGACTGGTCAGGTC360 
CTCAGTGATGGAAAAGTCTTCATCCATCTGTGCAACTACATCGAGCCCTGGGAGGACCTG420 
TCCTTGGTGCAGAGGGAAAGTCTGAATCATCACTACCATCTGAACTGTGGCTGCCAAATC480 
ACCACCTGCTACACAGTACCCTGTACCATCTCGGCCCCTAACGAGTGCCTCTGGACAGAC540 
TGGCTGTTGGAACGAAAGCTCTATGGTTACCAGGCTCAGCATTATGTCTGTATGAAGCAT600 
GTTGACGGCACCTGCAGCTGGTACCGGGGCCACCTGCCTCTCAGGAAGGAGTTTGTTGAC660 
ATCGTTCAGCCCTAG675 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 224 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: METALLOPROTEINASES 
(B) CLONE: 589345 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProGlySerProArgProAlaProSerTrpValLeuLeuLeuArg 
151015 
LeuLeuAlaLeuLeuArgProProGlyLeuGlyGluAlaCysSerCys 
202530 
AlaProAlaHisProGlnGlnHisIleCysHisSerAlaLeuValIle 
354045 
ArgAlaLysIleSerSerGluLysValValProAlaSerAlaAspPro 
505560 
AlaAspThrGluLysMetLeuArgTyrGluIleLysGlnIleLysMet 
65707580 
PheLysGlyPheGluLysValLysAspValGlnTyrIleTyrThrPro 
859095 
PheAspSerSerLeuCysGlyValLysLeuGluAlaAsnSerGlnLys 
100105110 
GlnTyrLeuLeuThrGlyGlnValLeuSerAspGlyLysValPheIle 
115120125 
HisLeuCysAsnTyrIleGluProTrpGluAspLeuSerLeuValGln 
130135140 
ArgGluSerLeuAsnHisHisTyrHisLeuAsnCysGlyCysGlnIle 
145150155160 
ThrThrCysTyrThrValProCysThrIleSerAlaProAsnGluCys 
165170175 
LeuTrpThrAspTrpLeuLeuGluArgLysLeuTyrGlyTyrGlnAla 
180185190 
GlnHisTyrValCysMetLysHisValAspGlyThrCysSerTrpTyr 
195200205 
ArgGlyHisLeuProLeuArgLysGluPheValAspIleValGlnPro 
210215220 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 220 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: METALLOPROTEINASES 
(B) CLONE: TIMP-2 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetGlyAlaAlaAlaArgThrLeuArgLeuAlaLeuGlyLeuLeuLeu 
151015 
LeuAlaThrLeuLeuArgProAlaAspAlaCysSerCysSerProVal 
202530 
HisProGlnGlnAlaPheCysAsnAlaAspValValIleArgAlaLys 
354045 
AlaValSerGluLysGluValAspSerGlyAsnAspIleTyrGlyAsn 
505560 
ProIleLysArgIleGlnTyrGluIleLysGlnIleLysMetPheLys 
65707580 
GlyProGluLysAspIleGluPheIleTyrThrAlaProSerSerAla 
859095 
ValCysGlyValSerLeuAspValGlyGlyLysLysGluTyrLeuIle 
100105110 
AlaGlyLysAlaGluGlyAspGlyLysMetHisIleThrLeuCysAsp 
115120125 
PheIleValProTrpAspThrLeuSerThrThrGlnLysLysSerLeu 
130135140 
AsnHisArgTyrGlnMetGlyCysGluCysLysIleThrArgCysPro 
145150155160 
MetIleProCysTyrIleSerSerProAspGluCysLeuTrpMetAsp 
165170175 
TrpValThrGluLysAsnIleAsnGlyHisGlnAlaLysPhePheAla 
180185190 
CysIleLysArgSerAspGlySerCysAlaTrpTyrArgGlyAlaAla 
195200205 
ProProLysGlnGluPheLeuAspIleGluAspPro 
210215220 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 211 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: METALLOPROTEINASES 
(B) CLONE: TIMP-3 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetThrProTrpLeuGlyLeuIleValLeuLeuGlySerTrpSerLeu 
151015 
GlyAspTrpGlyAlaGluAlaCysThrCysSerProSerHisProGln 
202530 
AspAlaPheCysAsnSerAspIleValIleArgAlaLysValValGly 
354045 
LysLysLeuValLysGluGlyProPheGlyThrLeuValTyrThrIle 
505560 
LysGlnMetLysMetTyrArgGlyPheThrLysMetProHisValGln 
65707580 
TyrIleHisThrGluAlaSerGluSerLeuCysGlyLeuLysLeuGlu 
859095 
ValAsnLysTyrGlnTyrLeuLeuThrGlyArgValTyrAspGlyLys 
100105110 
MetTyrThrGlyLeuCysAsnPheValGluArgTrpAspGlnLeuThr 
115120125 
LeuSerGlnArgLysGlyLeuAsnTyrArgTyrHisLeuGlyCysAsn 
130135140 
CysLysIleLysSerCysTyrTyrLeuProCysPheValThrSerLys 
145150155160 
AsnGluCysLeuTrpThrAspMetLeuSerAsnPheGlyTyrProGly 
165170175 
TyrGlnSerLysHisTyrAlaCysIleArgGlnLysGlyGlyTyrCys 
180185190 
SerTrpTyrArgGlyTrpAlaProProAspLysSerIleIleAsnAla 
195200205 
ThrAspPro 
210 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 207 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: METALLOPROTEINASES 
(B) CLONE: TIMP-1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetAlaProPheGluProLeuAlaSerGlyIleLeuLeuLeuLeuTrp 
151015 
LeuIleAlaProSerArgAlaCysThrCysValProProHisProGln 
202530 
ThrAlaPheCysAsnSerAspLeuValIleArgAlaLysPheValGly 
354045 
ThrProGluValAsnGlnThrThrLeuTyrGlnArgTyrGluIleLys 
505560 
MetThrLysMetTyrLysGlyPheGlnAlaLeuGlyAspAlaAlaAsp 
65707580 
IleArgPheValTyrThrProAlaMetGluSerValCysGlyTyrPhe 
859095 
HisArgSerHisAsnArgSerGluGluPheLeuIleAlaGlyLysLeu 
100105110 
GlnAspGlyLeuLeuHisIleThrThrCysSerPheValAlaProTrp 
115120125 
AsnSerLeuSerLeuAlaGlnArgArgGlyPheThrLysThrTyrThr 
130135140 
ValGlyCysGluGluCysThrValPheProCysLeuSerIleProCys 
145150155160 
LysLeuGlnSerGlyThrHisCysLeuTrpThrAspGlnLeuLeuGln 
165170175 
GlySerGluLysGlyPheGlnSerArgHisLeuAlaCysLeuProArg 
180185190 
GluProGlyLeuCysThrTrpGlnSerLeuArgSerGlnIleAla 
195200205 
__________________________________________________________________________