[go: up one dir, main page]

WO1995005478A1 - Timp-3 humain - Google Patents

Timp-3 humain Download PDF

Info

Publication number
WO1995005478A1
WO1995005478A1 PCT/US1994/009188 US9409188W WO9505478A1 WO 1995005478 A1 WO1995005478 A1 WO 1995005478A1 US 9409188 W US9409188 W US 9409188W WO 9505478 A1 WO9505478 A1 WO 9505478A1
Authority
WO
WIPO (PCT)
Prior art keywords
timp
human
human timp
cells
polypeptide
Prior art date
Application number
PCT/US1994/009188
Other languages
English (en)
Inventor
Susan P. Hawkes
Narendra S. Kishnani
Te-Tuan Yang
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU78277/94A priority Critical patent/AU7827794A/en
Publication of WO1995005478A1 publication Critical patent/WO1995005478A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is related to the field of protein biochemistry. Specifically, the invention relates to human tissue inhibitor of metalloproteinases (human TIMP-3) .
  • Matrix metalloproteinases are a family of enzymes that includes interstitial collagenase (MMP- 1) , 72-kDa and 92-kDa gelatinases (MMP-2 and MMP-9) , stromelysin (MMP-3) and atrilysin (MMP-7) . These enzymes are secreted by cells within tissues and by infiltrating inflammatory cells. Collectively, they are capable of degrading most of the proteins in the ECM. MMPs display different substrate specificities yet have several properties in common. They are all zinc- containing enzymes that require calcium for activity.
  • zymogens secreted as zymogens and activated in situ, usually by release of an inhibitory N-terminal pro-piece containing a single cysteine residue.
  • the attached pro- piece is believed to coordinate with the zinc in the active site of the proteinase, thereby suppressing the proteolytic activity.
  • Activation may be accompanied by additional proteolytic cleavages that can generate active enzymes of lower molecular weights.
  • All members of the MMP family have a short conserved region consisting of the HEXGH motif that provides two Zn-coordinating histidine residues and a glutamic acid residue that is considered part of the catalytic site.
  • the MMPs are inhibited by members of the family of tissue inhibitors of metalloproteinases (TIMPs, e.g., TIMP-1, TIMP-2, and TIMP-3) , which bind at the active site and block access to substrate.
  • TIMPs tissue inhibitors of metalloproteinases
  • Matrix remodelling which occurs during various normal and pathological processes, depends upon a critical balance between activated MMPs inhibiting TIMPs.
  • TIMP-1 and TIMP-2 The tissue inhibitors of matrix metalloproteinases (TIMP) also comprise a family of proteins that includes TIMP-1, TIMP-2 and TIMP-3.
  • TIMP-1 is a glycoprotein that was originally characterized as a mammalian collagenase inhibitor with a molecular mass of about 28 kDa. Cawston et al. (1981) Biochem. J. 195:159-165; and Murphy et al. (1991). TIMP- 1 was the first inhibitor to be cloned from human tissue.
  • TIMP-1 was subsequently cloned from murine, rabbit, bovine and porcine tissues.
  • TIMP-2 has been previously described. DeClerck et al. (1989) J. Biol. Chem. 164 . : 17445-17453; Goldberg et al. (1989) Proc. Natl. Acad. Sci. USA 8j5:8207-8211; and Stetler-Stevenson et al. (1989) J. Biol. Chem. 264:17374- 17378.
  • TIMP-2 is a protein of approximately 21 kDa which is not N-glycosylated. TIMP-2 has been cloned and sequenced from human, bovine and murine sources. Boone et al. (1990) Proc. Natl. Acad. Sci.
  • TIMP-1 and TIMP-2 from various species share identity in approximately 32% of residues.
  • the first 22 N-terminal amino acids of the mature protein i.e., following the site of signal peptide cleavage
  • This region appears to contai.n an acti.ve si.te, with His 7 and Gin 9 likely being particularly important in the presumed interaction with the zinc at the active site.
  • Also highly conserved are 12 cysteine residues.
  • TIMP-1 six disulfide bonds
  • TIMP-1 and TIMP-2 form complexes with different inactive pro-enzymes: TIMP-1 with the pro-form of gelatinase B (MMP-9) (Wilhelm et al. (1989) J. Biol. Chem. 264: 17213-17221; and Goldberg et al. (1992) J. Biol. Chem. 267:4583-4591) and TIMP-2 with the pro-form of gelatinase A (MMP-2) . Goldberg et al. (1992) J. Biol. Chem. 267:4583-4591; and Stetler- Stevenson et al. (1990) .
  • TIMPs are thought to play physiological roles in a variety of contexts: tissue remodeling and wound repair, including the prevention and repair of blood vessel injury and aneurysms, rheumatoid arthritis, various ulcerated conditions, e.g., resulting in the cornea as the result of alkali burns or as a result of infection by Pseudomonas aeruginosa, Acanthamoeba, Herpes simplex, and vaccinia viruses; periodontal disease; epidermolysis bullosa; promoting the growth and/or proliferation of, e.g., erythroid progenitors and a variety of cultured cells; tissue vascularization; embryogenesis and blastocyst implantation; lactation; bone remodeling; oncogenic transformation; and cell migration, including the invasion and metastasis of cancer cells.
  • tissue remodeling and wound repair including the prevention and repair of blood vessel injury and aneurysms, rheumatoid arthritis, various ulcerated conditions, e.g
  • ChIMP-3 was originally described as a protein whose synthesis is stimulated during the early stages of oncogenic transformation or after treatment of normal cells with phorbol myristate acetate. Blenis and Hawkes (1983) Proc. Natl. Acad. Sci. USA 0:770-774; and Blenis and Hawkes (1984) J. Biol. Chem. 259:11563-11570.
  • This 21 kDa protein was purified from the extracellular matrix (ECM) of fibroblasts. Its NH 2 -terminal sequence shows a strong similarity to the sequences of several mammalian TIMPs and it displays metalloproteinase inhibitor activity. Staskus et al. (1991) . Based on these and other biochemical data, it was proposed that the protein was a TIMP variant or a third member of the TIMP family.
  • a human TIMP-3 heretofore unknown would be a useful addition to the TIMP family and help in modulating and fine-tuning metalloproteinase inhibition.
  • Means for diagnosing and treating conditions involving the activity of MMPs are highly desirable. The present invention addresses these and other needs.
  • the present invention provides an isolated human TIMP-3 polypeptide having substantially the same amino acid residue sequence given in Figure 11.
  • the present invention also includes the nucleotide sequence of human TIMP-3 as depicted in Figure 11.
  • the present invention also provides antibodies specific for human TIMP-3, preferably monoclonal antibodies specific for human TIMP-3.
  • the present invention also includes a method of detecting the presence of human TIMP-3 in a sample, the method comprising the steps of: (a) contacting the sample with an antibody specific for human TIMP-3, under conditions suitable for the formation of a human TIMP-3- antibody complex; and (b) detecting the presence of the complex.
  • the present invention provides a method of detecting elevated levels of human TIMP-3 in a test sample, the method comprising the steps of: (a) contacting the test sample with an antibody specific for human TIMP-3 , under conditions suitable for the formation of a human TIMP-3-antibody complex; (b) contacting a control sample with the antibody, under conditions suitable for human TIMP-3-antibody complex formation; (c) detecting the level of human TIMP-3- antibody complex formation in the control sample and in the test sample; and (d) comparing the levels of complex formation in the control and test samples.
  • the present invention also provides a pharmaceutical composition comprising a human TIMP-3 polypeptide in a pharmaceutically acceptable excipient.
  • Figure 1 shows the primary structure of ChlMP- 3.
  • I, II, and III are potential sequences for the synthesis of peptides to be used for antibody production.
  • a and B are targets for site-directed mutagenesis to introduce glycosylation sites into ChIMP-3.
  • Aspartate 16 (enclosed in box) is also a target for conversion to alanine.
  • FIG. 2 shows protease/substrate SDS-PAGE analysis of human and chicken ECM.
  • Lanes 2 and 3 are concentrated conditioned media (CM) from normal whole embryo cells (FHs 173We) equivalent to 300 ⁇ l and 150 ⁇ l, respectively.
  • Lanes 4-11 contain ECM from human cell lines, as indicated.
  • Lanes 4 and 5 are FHs 173We cells; lanes 6 and 7 are human embryonic kidney cells transformed by adenovirus type 5 DNA (293) ; lanes 8 and 9 are cells derived from a metastatic human neuroblastoma (SK-N-SH) ; lanes 10 and 11 are fibrosarcoma cells (Hs 913T) ; and lane 12 is ECM from approximately 4 x 10 4 chicken embryo fibroblasts (CEF) (lane 12) .
  • SK-N-SH metastatic human neuroblastoma
  • lanes 10 and 11 are fibrosarcoma cells (Hs 913T)
  • lane 12 is ECM from approximately 4 x 10 4 chicken embryo fibroblasts (CEF) (lane 12) .
  • Figure 3 shows protease/substrate SDS-PAGE analysis of conditioned media (left) and ECM (right) from the following cell lines: LA24-CEF (lane 1), SK-N-SH (lane 2), 293 (lane 3), mouse 3T3 (lane 5) , and rat pheochromocytoma cells derived from an adrenal gland tumor (PC12) (lane 6) .
  • Molecular weight standards are in lane 4.
  • Figure 4 shows protease/substrate SDS-PAGE analysis of TIMPs present in conditioned media and ECM of mouse 3T3 cells and transforming chicken LA24-CEF cells. Lanes 1 and 3, CM; lanes 2 and 4, ECM; lane 5, molecular weight standards.
  • FIG. 5 shows the nucleotide sequence of mouse
  • TIMP-3 cDNA aligned with that of ChIMP-3.
  • Figure 6 shows a comparison of the deduced amino acid sequence of mTIMP-3 with TIMP-1 from bovine, pig, human, rabbit, and mouse TIMP-1 (two sequences) ; with TIMP-2 from human, mouse and bovine; and with TIMP-3 from chicken.
  • the mature mTIMP-3 protein begins with the "CTCSPS" as marked.
  • Figure 7 shows a Western blot of human ECM and CM probed with anti-human TIMP-1, anti-ChIMP-3 and anti- human TIMP-2.
  • Lanes 2, 3 , and 5 ECM from human FHs 173We cells (approximately 2.4 x 10 6 cells) ; lanes 1 and 6: dialyzed and concentrated CM (equivalent to 60 ⁇ l CM) from FHs 173We cells; lane 4 is ECM from CEF cells (approximately 4 x 10 6 cells) .
  • the data in this figure were derived from three different gels and therefore the mobilities of the proteins cannot be compared directly.
  • the apparent molecular weights of the reduced proteins are as follows: lane 1, TIMP-1 (30.4 kDa); lane 3, human TIMP-3 (24.7 kDa) , minor band (28.5 kDa); lane 4, ChIMP-3 (24.5 kDa) ; lane 6, TIMP-2 (24.8 kDa) .
  • Figure 8 is a Western blot of human TIMP-3 in a reduced and unreduced state probed with an antibody to the NH 2 -terminus of ChIMP-3, showing the effect of ⁇ - mercaptoethanol on the electrophoretic migration of human TIMP-3 on SDS polyacrylamide gels.
  • Lanes 1-4 contain ECM from approximately 2.4 x 10 6 FHs 173We cells; lane 6 contains ECM from approximately 4 x 10 6 CEF; lane 5 contains sample loading buffer only. (+) indicates addition of /--mercaptoethanol to a final concentration of
  • Figure 9 shows protease/substrate SDS-PAGE analysis of ECM from human SK-N-SH cells, either untreated (-) or treated with N-glycosidase-F (+) .
  • Figure 10 shows a comparison of the N-terminal amino acid sequence of human TIMP-3 with chicken and mouse TIMP-3s and consensus sequences of TIMP-1 and TIMP- 2.
  • Figure 11 shows cDNA nucleotide and amino acid sequences of human TIMP-3. The end of the signal peptide is denoted by the line and indication of mature TIMP-3.
  • Human TIMP-3 was identified on the basis of its ECM localization, its molecular weight, lack of N-linked glycosylation, NH 2 - terminal sequence, amino acid composition, and recognition by antibodies to pure ChIMP-3. Degradation of the ECM by matrix metalloproteinases is highly controlled in normal physiological processes but appears to be accelerated in some pathological conditions.
  • Tissue inhibitors of Metalloproteinases e.g., TIMP-1 and TIMP-2
  • TIMP-1 and TIMP-2 can be found free in the conditioned media of cultured cells.
  • ChlMP- 3 thicken inhibitor of metalloproteinases-3
  • ChIMP-3 is located exclusively in the ECM.
  • the ChIMP-3 cDNA sequence has recently been elucidated and indicates that, although the protein is structurally related to TIMP-1 and TIMP-2, with 42% sequence identity to a consensus sequence of TIMP-2 and 28% to TIMP-1, it is clearly a distinct TIMP- 3. Pavloff et al. (1992).
  • the protein (M r approximately 22 kDa, reduced) is intermediate in molecular weight between TIMP-1 and TIMP-2 and is not N-glycosylated.
  • ChIMP-3 is a multifunctional protein which stimulates the division of serum-deprived cells. Yang and Hawkes (1992) Proc. Natl. Acad. Sci. USA 29:10676-10680. Furthermore, it promotes the expression of the transformed phenotype by enhancing detachment of transforming cells from the ECM and accelerating morphological transformation (Yang and Hawkes (1992)), properties which have not been described for TIMP-1 and TIMP-2. Additional distinct characteristics which set this protein apart from other members of the family include its relative insolubility and its specific localization in the ECM. Blenis and Hawkes (1983) ; Blenis and Hawkes (1984) ; and Staskus et al. (1991) J.
  • TIMP-1 and TIMP-2 are soluble proteins found in body fluids and conditioned media of cultured cells and tissues.
  • Murphy and Sellers (1980) In: Collagenases in Normal and Pathological Connective Tissues, eds. Woolley and Evanson, John Wiley & Sons Ltd. , London, pp. 65-81; Cawston et al. (1981) ; Murphy et al. (1991) ; Reynolds et al. (1981) In: Cellular Interactions, eds. Dingle and
  • Human TIMP- 3 polypeptides, nucleic acids, and antibodies specific for human TIMP-3 polypeptides are useful for a host of uses, including the diagnosis, therapy, or prophylaxis of a variety of conditions characterized by excess or unwanted MMP activity.
  • TIMP-3 refers to a polypeptide or protein which (1) shows preferential association with the ECM; (2) displays TIMP activity as determined by protease/substrate SDS polyacrylamide gel electrophoresis; (3) is the only major band on protease/substrate SDS gel between 21 and 31 kDa; and (4) is recognized by antisera which specifically binds to TIMP-3, i.e., is capable of distinguishing TIMP-3 from other members of the TIMP family.
  • "human TIMP-3” is a polypeptide, or fragment thereof, substantially homologous to the mature TIMP-3 depicted in Figure 11. Human TIMP-3 may be isolated in its native form from human tissue or produced by recombinant DNA techniques.
  • a polypeptide produced by the recombinant expression of a nucleic acid which encodes human TIMP-3 polypeptide is also considered a human TIMP-3, even if such a polypeptide is expressed in a non-human host cell.
  • the endogenous signal peptide may be used if expressed in eukaryotic expression systems.
  • Human TIMP-3 may also be produced with any functional signal peptide, preferably another TIMP signal peptide in the case of eukaryotic expression systems.
  • Recombinant human TIMP-3 may also be produced without a signal peptide.
  • human TIMP-3 is associated with the ECM (and not with conditioned media) and has an apparent molecular weight of about 24 kDa. It is understood by those skilled in the art that this molecular weight is only approximate because it depends on a number of variables.
  • the amino acid sequence of the mature polypeptide is 188 amino acids.
  • TIMP-3 The bulk of human TIMP-3 appears to be non-N- glycosylated; however, there appears to be a glycosylation site near the C-terminus.
  • Protease/ substrate gel analysis also showed in the ECM of human cells minor TIMP species of 28-29 kDa in addition to the major 24 kDa species. These minor species appear to be N-glycosylated, and one or more may be N-glycosylated TIMP-3, which is also included in the invention.
  • TIMP-3 appears to contain intramolecular disulfide bonds because it migrates differently in the presence of reducing agents.
  • human TIMP-3 encompasses non-glycosylated and glycosylated forms of the polypeptide. Preferably, the glycosylated forms are N-linked.
  • Encompassed by the claimed human TIMP-3 polypeptides are variants of human TIMP-3 in which there have been trivial substitutions, deletions, insertions or other modifications of the native human TIMP-3 polypeptide which substantially retain human TIMP-3 characteristics, particularly silent or conservative substitutions.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • polypeptides of the present invention may be coupled to a solid phase support, e.g., nitrocellulose, nylon, column packi•ng materials (e.g., Sepharose beads) , magnetic beads, glass wool, cells, or other substrates.
  • a solid phase support e.g., nitrocellulose, nylon, column packi•ng materials (e.g., Sepharose beads) , magnetic beads, glass wool, cells, or other substrates.
  • isolated The terms “isolated,” “pure,” “substantially pure,” and “substantially homogeneous” are used interchangeably to describe human TIMP-3 which has been separated from components which naturally accompany it.
  • a monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel or chemically determining the N- ter inal sequence by Ed an degradation.
  • higher resolution may be provided by using reversed-phase high performance liquid chromatography (HPLC) or other means well known in the art for purification.
  • HPLC reversed-phase high performance liquid chromatography
  • Human TIMP-3 is substantially free of naturally associated components when it is separated from the contaminants which accompany it in its natural state.
  • a polypeptide which is chemically synthesized or expressed as a recombinant protein in a host cell different from the cell from which it naturally originates is substantially free of its naturally associated components.
  • a polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an isolated polypeptide, as used herein, even if expressed in a homologous cell type. Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
  • the present invention describes the purification of human TIMP-3 from human cells.
  • TIMP-3 may be accomplished by various methods well known in the art.
  • human TIMP-3 may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • Various methods of protein purification are well known in the art, and include those described, e.g., in Guide to Protein Purification, ed. Academic Press, Inc.: San Diego, 1990) and Scopes, Protein Purification: Principles and Practice (Springer-Verlag: New York, 1982) .
  • Protein sequence determination To determine the actual amino acid sequence or to obtain polypeptide fragments of human TIMP-3 , the protein may be digested with enzymes such as trypsin, clostripain, or Staphylococcus protease or with chemical agents such as cyanogen bromide, O-iodosobenzoate, hydroxylamine or 2- nitro-5-thiocyanobenzoate. Peptide fragments may be separated by HPLC and analyzed by gas-phase sequencing. Other sequencing methods known in the art may also be used.
  • enzymes such as trypsin, clostripain, or Staphylococcus protease
  • chemical agents such as cyanogen bromide, O-iodosobenzoate, hydroxylamine or 2- nitro-5-thiocyanobenzoate.
  • Peptide fragments may be separated by HPLC and analyzed by gas-phase sequencing. Other sequencing methods known in the art may also be used.
  • TIMP-3 polypeptides or fragments thereof which are substantially homologous to the primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids.
  • Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, and labelling, e.g. , with radionuclides and various enzymatic modifications.
  • labelling polypeptides and of substituents or labels useful for such purposes, including radioactive isotopes such as 32 P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • polypeptides are well known in the art. See, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol. 1-3, ed. Sambrook, et al.. Cold Spring Harbor Laboratory Press (1989) ; or Current Protocols in Molecular Biology, ed. Ausubel et al. , Greene Publishing and Wiley-Interscience: New York, 1987 and periodic updates.
  • the present invention provides fragments of human TIMP-3 which retain at least one of the biological activities characteristic of human TIMP-3 , such as antigenic fragments useful for raising human TIMP-3-specific antibodies.
  • Segment is a stretch of amino acid residues of at least about 7-17 amino acids (or the minimum size retaining an antigenic determinant of human TIMP-3 and capable of raising human TIMP-3-specific antibodies) and preferably substantially full length.
  • Significant biological activities include immunological activity, MMP inhibition, and other biological activities characteristic of human TIMP-3.
  • human TIMP-3 encompasses such fragments.
  • Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for a human TIMP-3 epitope.
  • tandemly repeated human TIMP-3 fragments may be used as immunogens, thereby producing highly antigenic proteins.
  • polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for human TIMP-3 is described below.
  • the present invention also provides for fusion polypeptides comprising human TIMP-3.
  • Homologous polypeptides may be fusions between two or more human TIMP-3 sequences or between the sequences of human TIMP-3 and a related protein.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be "swapped" between different new fusion polypeptides or fragments.
  • Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding.
  • Fusion partners include immunoglobulins, bacterial j ⁇ -galactosidase, trpE, protein A, /--lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See, e.g., Godowski et al. (1988) Science 241:812-816.
  • Fusion proteins are typically made by either recombinant nucleic acid methods, as described below, but may be chemically synthesized. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156.
  • Figure 1 shows the primary structure of ChIMP-3 based on a model for TIMP-1 by Woessner (1991) .
  • the numbering of the loops is according to Murphy et al. (1991).
  • I, II, and III in Figure 1 are potential sequences for the synthesis of peptides to be used for antibody production.
  • a and B are targets for site- directed mutagenesis to introduce glycosylation sites into ChIMP-3.
  • Aspartate 16 (enclosed in box) is also a target for conversion to alanine.
  • knowledge of the structure of human TIMP-3 based on the present disclosure will enable those skilled in the art to design peptides for antibody production, to introduce glycosylation sites or to perform site-directed mutagenesis, as desired.
  • human TIMP-3 when applied to a nucleic acid refers to a nucleic acid which encodes a human TIMP-3 polypeptide.
  • the nucleic acids of the present invention will possess a sequence which is either derived or substantially similar to the native human TIMP-3-encoding gene as shown in Figure 11.
  • the DNA compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or contain on- natural or derivatized nucleotide bases.
  • Recombinant nucleic acids comprising sequences otherwise not naturally occurring are also provided by this invention.
  • the wild type sequence may be employed, the wild type sequence will often be altered, e.g., by deletion, substitution, or insertion.
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired receptors. Phage libraries are normally preferred, but plasmid libraries may also be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.
  • the DNA sequences used in this invention will usually comprise at least about 5 codons (15 nucleotides) , more usually at least about 7 to 15 codons, and most preferably at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required to hybridize specifically with a human TIMP-3-encoding sequence.
  • nucleic acid sequences used to produce the proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
  • nuclei.c aci.d i.s a nuclei.c aci.d, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other DNA sequences which naturally accompany a native human sequence, e.g., ribosomes, polymerases, and many other human genome sequences.
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • Encode A nucleic acid is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the
  • the anti-sense strand of such a nucleic acid is also said to encode the sequence.
  • operably linked A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • Recombinant nucleic acid is one which is not naturally occurring, or is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • Probes and primers Nucleic acid probes and primers may be prepared based on the sequence of the human TIMP-3 cDNA sequences provided by the present invention.
  • the probe or primer may comprise an isolated nucleic acid, and, especially in the case of probes, are typically attached to a label or reporter molecule.
  • Probes may be used to identify the presence of a hybridizing nucleic acid sequence, e.g., a human TIMP-3 mRNA in a tissue or other sample or a human TIMP-3 cDNA or genomic clone in a library.
  • Primers may be used, for example, for amplification of nucleic acid sequences, e.g., by the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the preparation and use of probes and primers is described, e.g., in Sambrook et al. (1989) or Ausubel et al. (1987).
  • Antisense nucleic acids capable of specifically binding to human TIMP-3 sequences are also useful for interfering with gene expression and therefore may be useful for therapeutic or prophylactic compositions. See, e.g., EPO publication 431523A2.
  • Nucleic acids encoding human TIMP-3 include not only a native or wild-type human TIMP-3 sequence but also any sequence capable of encoding a human TIMP-3 polypeptide, which may be synthesized by making use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., silent or conservative changes thereby producing various endonuclease restriction sites or to optimize expression for a particular system.
  • the nucleic acids of the present invention may be derived from naturally occurring or recombinant single or double stranded nucleic acids or may be chemically synthesized.
  • Portions of the DNA sequence having at least about 15 contiguous nucleotides from a DNA sequence encoding a human TIMP-3 are preferred as probes or primers.
  • nucleic acids of the present invention may be produced by replication in a suitable host cell.
  • the natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell.
  • DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction into, with and without and integration within the genome, cultured mammalian or plant or other eukaryotic cell lines.
  • the purification of nucleic acids produced by the methods of the present invention are described, e.g., in Sambrook et al. (1989) or Ausubel et al. (1987) .
  • the nucleic acids of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22 . : 1859-1862 or the triester method according to Matteucci et al. (1981) J. Am. Chem. Soc. 103 :3185-3191, and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate condi•ti•ons or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • DNA constructs prepared for introduction into a prokaryotic or eukaryotic host typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression vectors include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • ARS origin of replication or autonomously replicating sequence
  • Secretion signals may also be included where appropriate, whether from a native human TIMP-3 protein or from other receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell.
  • Such vectors are prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al. (1989) or Ausubel et al. (1987) .
  • An appropriate promoter and other necessary vector sequences is selected so as to be functional in the host, and may, when appropriate, include those naturally associated with human TIMP-3 genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) or Ausubel et al. (1987); see also, e.g., Metzger et al.
  • 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.
  • Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al. EP 73,657A.
  • Appropriate non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al.
  • the construct can be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene are made.
  • amplifiable gene e.g., DHFR
  • Expression and cloning vectors generally contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures the growth of only those host cells which express the inserts.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli.
  • the choice of the proper selectable marker depends on the host cell, and appropriate markers for different hosts are well known in the art.
  • the vectors containing the nucleic acids of interest can be transcribed in vitro and the resulting RNA introduced into the host cell by well known methods (e.g., by injection; see, Kubo et al. (1988) FEBS Lett. 241:119-125) .
  • the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome) ; and other methods. See generally, Sambrook et al. (1989) and Ausubel et al. (1987) .
  • the cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
  • nucleic acids and polypeptides of the present invention can be prepared by expressing the human TIMP-3 nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
  • prokaryotic hosts are strains of Escherichia coli. although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, are also useful for production of the proteins of the present invention.
  • Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, or others which may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features.
  • Transformed cells are selected by using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different DNA molecule, preferably the same DNA molecule.
  • the transformant can be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity can also serve as an appropriate marker.
  • Prokaryotic or eukaryotic cells transformed with the nucleic acids of the present invention are useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of human TIMP-3 polypeptides.
  • Two constructs of ChIMP-3 cDNA have been expressed as fusion proteins in Escherichia coli. In Western blot analysis of induced bacterial proteins in four strains, polyclonal anti-ChIMP-3 specifically detects proteins at approximately 32-35 kDa, the" expected molecular weight for ChIMP-3 plus the fusion partner.
  • Antibodies The present invention also provides polyclonal and/or monoclonal antibodies capable of specifically binding to human TIMP-3 produced by in vitro or in vivo techniques well known in the art. Such antibodies are raised against human TIMP-3 and are capable of distinguishing human TIMP-3 from other polypeptides, including human TIMP-1 or TIMP-2. Preferably, such antibodies also are capable of distinguishing human TIMP- 3 from TIMP-3 of other species under conditions suitable for complex formation.
  • Peptide fragments suitable for raising antibodies may be prepared by chemical synthesis, and are commonly coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected into a mammal, e.g., rabbits, over several months. The rabbit sera is tested for immunoreactivity to human TIMP-3. Monoclonal antibodies are made by injecting the animal, e.g., mice with the protein, polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies are screened by ELISA and tested for specific immunoreactivity with human TIMP-3. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Laboratories. These antibodies are useful in diagnostics such as immunoassays as well as in pharmaceuticals.
  • a carrier molecule e.g., keyhole limpet hemocyanin
  • an appropriate target immune system is selected, typically a mouse or rabbit.
  • the substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and other parameters well known to immunologists. Typical sites for injection are in the footpads, intramuscularly, intraperitoneally, or intradermally.
  • An immunological response is usually assayed with an immunoassay.
  • immunoassays involve some purification of a source of antigen, for example, produced by the same cells and in the same fashion as the antigen was produced.
  • a variety of immunoassay methods are well known in the art. See, e.g. , Harlow and Lane (1988) ; and Goding (1986) Monoclonal Antibodies: Principles and Practice. 2d ed, Academic Press, New York.
  • Monoclonal antibodies with affinities of 10 8 M "1 preferably 10 9 to 10 10 , or stronger are typically made by standard procedures as described, e.g., in Harlow and Lane (1988) or Goding (1986) .
  • spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter the cells are clonally separated and the supernatants of each clone are tested for their production of an appropriate antibody specific for the desired region of the antigen.
  • the polypeptides and antibodies are labeled by joining, either covalently or noncovalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are * well known. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Patent 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, see Cabilly, U.S. Patent No. 4,816,567. Methods of Use: Drug Screening
  • the present invention encompasses the use of human TIMP-3 in a variety of drug screening techniques.
  • human TIMP-3 is provided free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing human TIMP-3, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One measures for example, the formation of complexes between human TIMP-3 and the agent being tested, or examines the degree to which the formation of a complex between human TIMP-3 and a MMP is interfered with by the agent being tested.
  • the present invention provides methods of screening candidate drugs comprising contacting a sample containing the candidate drug with human TIMP-3 and assaying (i) for the presence of a complex between the drug and human TIMP-3 , or (ii) for the presence of a complex between human TIMP-3 and a MMP.
  • human TIMP-3 is typically labeled. Free human TIMP-3 is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to human TIMP-3 or its interference with TIMP-3:MMP binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to human TIMP-3 and is described, e.g., in Geysen, European Patent Application 84/03564. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are reacted with labeled human TIMP-3 and washed. Bound human TIMP-3 is then detected.
  • the present invention also contemplates the use of competitive drug screening assays in which antibodies capable of specifically binding to human TIMP-3 compete with a test compound for binding to human TIMP-3. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic determinants of human TIMP-3.
  • the goal of rational drug design is to produce structural analogues of biologically active polypeptides of interest or of small molecules with which they interact, e.g., agonists, antagonists, inhibitors, in order to fashion drugs which are, e.g., more active or stable forms of the polypeptide, or which, e.g. , enhance or interfere with the function of a polypeptide in vivo.
  • drugs which are, e.g., more active or stable forms of the polypeptide, or which, e.g. , enhance or interfere with the function of a polypeptide in vivo.
  • Hodgson (1991) Bio/Technology 9:19-21 See, e.g., Hodgson (1991) Bio/Technology 9:19-21.
  • one first determines the three-dimensional structure of a protein of interest or, for example, of a protein-inhibitor complex, by X-ray crystallography, by computer modelling or, most typically, by a combination of the two approaches.
  • the selected peptides would then act as the pharmacore.
  • drugs which have, e.g., improved TIMP-3 activity or stability or which act as inhibitors, agonists, antagonists, etc. of TIMP-3.
  • sufficient amount of polypeptide may be made available to perform such analytical studies as X-ray crystallography.
  • the knowledge of the human TIMP-3 protein sequences provided herein will guide those employing computer modelling techniques in place of or in addition to X-ray crystallography.
  • TIMP-3 activity is induced, for example, by inflammatory cytokines and growth factors at a site of injury. TIMP-3 activity is also increased at an early stage of oncogenic transformation.
  • the present invention thus encompasses the diagnosis of the above named conditions and others characterized by excess or undesired MMP activity.
  • Antibodies specific for human TIMP-3 may be used to determine levels of human TIMP-3 in fixed tissue sections by immunohistochemical methods or in a body fluid or tissue sample by ELISA or radioimmunoassays, for example. Oligonucleotide probes or primers based on the human
  • TIMP-3 sequences may be useful for assaying elevated transcription of TIMP-3, e.g., by probing Northern blots, in situ hybridization or quantitative nucleic acid amplification methods such as the polymerase chain reaction (PCR) , for example.
  • PCR polymerase chain reaction
  • the claimed anti-human TIMP-3 antibodies are useful in monitoring therapy or prophylaxis regimens involving the pharmaceutical compositions of the present invention.
  • Suitable samples such as those derived from biopsied tissues, blood, serum, urine, or saliva, can be tested for the presence of the administered inhibitor at various times during the treatment protocol using standard immunoassay techniques which employ the claimed antibody preparations.
  • the ability of human TIMP-3 to bind MMPs may be exploited to determine the locations of excess amounts of one or more MMPs in situ.
  • the claimed antibodies specific for human TIMP-3 may be used to map the location of TIMP-3.
  • human TIMP-3 When coupled to labels such as scintigraphic labels, e.g., technetium 99 or 1-131, by standard coupling methods, human TIMP-3 may be administered to subjects for this purpose. These techniques can also be employed in histological procedures and in competitive immunoassays.
  • Human TIMP-3 polypeptides and nucleic acids are likewise useful for the treatment or prophylaxis of these conditions. These include, but are not limited to, neoplasias such as fibrosarcomas, teratomas and neoplasias of epithelial origin, tumor metastasis, inflammatory disorders such as rheumatoid arthritis, ulcerations, particularly of the cornea, reaction to infection, periodontal disease, osteoporosis, and the like.
  • the present invention encompasses pharmaceutical compositions comprising human TIMP-3 in one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants. Other active ingredients may also be included in the compositions of the invention. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences. Mack Publishing Co. ,
  • Such pharmaceutical formulations may be applied to a wound or ulcer or injected into a cancer or an arthritic joint, thereby inhibiting MMP activity.
  • Anti- sense nucleic acids comprising sequences derived from a human TIMP-3 gene or antibodies specific for that gene are also useful in inhibiting human TIMP-3 expression.
  • the administration of human TIMP-3 is beneficial in tissue and wound repair (for example, in ulcers of the eye) ; promoting growth and/or proliferation of erythroid progenitors and a variety of cultured cells; tissue vascularization; and embryogenesis, blastocyst formation and other stages of pregnancy as well as lactation.
  • the potency of the claimed pharmaceutical compositions as inhibitors of MMP activity may be determined as described in U.S. Patent No. 5,183,900, U.S. Patent No. 4,743,587, and WO 92/13831, which reference Cawston et al. (1979) Anal. Biochem. 99:340- 345; and Weinberg et al. (1986) Biochem. Biophys. Res. Comm. 139:1184-1187; or other tests of MMP inhibition known in the art.
  • Isolated MMP enzymes can be used to confirm the inhibiting activity of the claimed human TIMP-3 compositions, or crude extracts which contain the range of enzymes exhibiting MMP activity.
  • Human TIMP-3 activity may also be assayed using the cell detachment and cell proliferation assays provided in the Examples below. For example, see Yang and Hawkes (1992) .
  • compositions of the present invention may be formulated for administration by any route, depending on the disease being treated.
  • the compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions.
  • Tablets and capsules for oral administration may be in a form suitable for unit dose presentation and may contain conventional excipients.
  • binding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone
  • fillers such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine
  • tableting lubricants such as magnesium stearate, talc, polyethylene glycol or silica
  • disintegrants such as potato starch
  • acceptable wetting agents such as sodium lauryl sulfate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, e.g.
  • sorbitol, syrup methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats, emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p- hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.
  • non-aqueous vehicles including edible oils
  • almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol
  • preservatives such as methyl or propyl p- hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.
  • the drug may be made up into a cream, lotion, or ointment using conventional formulations.
  • the drug may be made up into a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle.
  • Additives may also be included, e.g., buffers such as sodium metabisulphite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.
  • the claimed pharmaceutical compositions may also be administered parenterally in a sterile medium.
  • the drug may be dissolved or suspended in the vehicle, depending of the vehicle or concentration used.
  • Adjuvants such as local anesthetics, preservatives, and buffering agents can also be dissolved in the vehicle.
  • Commonly used excipients for injectable forms of the pharmaceutical compositions of the present invention include physiological saline, Hank's solution, Ringer's solution, and the like. Injection can be, e.g., intravenous, intramuscular, intraperitoneal, or subcutaneous.
  • the claimed pharmaceutical compositions may be administered orally or injected into the affected joint.
  • the claimed pharmaceutical compositions may also be administered by transdermal or transmucosal . . . delivery by including agents which effect penetration of these tissues, such as bile salts, fusidic acid derivatives, cholic acid, and the like.
  • targeting ligands allows the claimed pharmaceutical compositions to be delivered to specific locations. For example, to focus human TIMP-3 to inhibit the activity of matrix metalloproteinases in a tumor, the
  • TIMP-3 may be conjugated to an antibody or fragment thereof which is immunoreactive with a tumor marker.
  • the targeting ligand can also be a ligand which is specifically bound by a receptor which is present at the target site.
  • TIMP-3 are well known.
  • the dosage unit required can be determined by one of skill in the art and depends, for example, on the condition treated, nature of the formulation, nature of the condition, embodiment of the claimed pharmaceutical compositions, mode of administration, and condition and weight of the patient.
  • Polyclonal anti-ChIMP-3 antibodies were raised against ChIMP-3 from transforming chicken embryo fibroblasts purified to homogeneity by the methods described in Staskus et al. (1991) ; and Yang and Hawkes (1992) .
  • the polyclonal antibodies to ChIMP-3 were raised in a single white New Zealand rabbit following two injections of ChIMP-3 (20 ⁇ g protein/immunization) .
  • the antibodies recognized denatured and denatured/reduced ChIMP-3 on Western blots, performed as described below.
  • the anti-ChIMP-3 antiserum detected a single, strong band at about 24 kDa on blots of chicken reduced ECM proteins from transforming chicken embryo fibroblasts (CEF) . Occasionally, weakly reacting bands at approximately 26 kDa, 42 kDa, and 90-95 kDa were detected. Additional evidence indicates the human TIMP-3 has a propensity for aggregation, so the presence of multimers is not unexpected. Electroelution of the 42 kDa band from a preparative gel and subsequent analysis on a protease/substrate gel detects inhibitor activity in a band of protein at an M r of about 24,000.
  • the 90-95 kDa band could represent a complex between ChIMP-3 and the gelatinase that it inhibits. This assumes that such a complex would be resistant to dissociation in SDS. Indeed, such complexes are maintained in the presence of SDS if the samples are not heated prior to electrophoresis. De Clerck et al. (1991) J. Biol. Chem. 266:3893-3899.
  • ChIMP-3 antisera also recognized mouse TIMP-3 on Western blots of samples of ECM from mouse C3H 10T1/2 cells.
  • the antibody reacts positively with a band at approximately 23-24 kDa (the only band recognized between 21 and 31 kDa) as well as a minor but recognizable band at approximately 55 kDa.
  • ChIMP-3 polyclonal antisera When used to probe Western blots, ChIMP-3 polyclonal antisera also recognized human TIMP-3 and an unidentified protein with a molecular weight of approximately 28-29 kDa.
  • Example 2 Polyclonal Antisera against an N-terminal ChIMP-3 Peptide
  • ChIMP-3 and ChlMP-a on Western blots of ECM proteins from transforming CEF. As the NH 2 -termini of this family of proteins are so similar, this result is not surprising. Whether these anti-peptide antibodies recognize either "native" ChIMP-3 or ChlMP-a has not yet been tested.
  • This antibody also recognized human TIMP-3 and an unidentified protein with a molecular weight of approximately 28-29 kDa.
  • CM conditioned media
  • the protein set was denatured in 6 M guanidine HCl, reduced with ⁇ - mercaptoethanol, and acetylated with iodoacetamide to block sulfhydryl groups.
  • a variety of cultured human cells have been screened by protease/substrate SDS-PAGE analysis (modified reverse zymography) in search of the human counterpart for ChIMP-3.
  • a unique metalloproteinase inhibitor has been detected that is localized specifically in the ECM of the following normal, transformed and cancer cell lines: normal whole embryo (FHs 173We) , normal gingival fibroblasts (GF11 and 1292) , transformed primary embryonic kidney (293), neuroblastoma (SK-N-SH) , carcinoma (HeLa S3) , colon adenocarcinoma (Caco-2) , ileocecal adenocarcinoma (HCT-8) and fibrosarcomas (SW 684 and Hs 913T) .
  • FIG. 2 shows protease/substrate SDS-PAGE analysis of ECM from a representative sample of human cell lines and from chicken.
  • Lanes 2 and 3 are concentrated conditioned media (CM) from FHs 173We cells equivalent to 300 ⁇ l and 150 ⁇ l, respectively.
  • Lanes 4- 11 contain ECM from human cell lines, as indicated.
  • Lanes 4 and 5 are FHs 173We cells; lanes 6 and 7 are 293 cells; lanes 8 and 9 are SK-N-SH cells; lanes 10 and 11 are Hs 913T cells. In each set of two lanes the sample on the left was produced by approximately 8 x 10 3 cells and the one on the right by 4 x 10 3 cells. Lane 12 is
  • ECM from approximately 4 x 10 4 chicken embryo fibroblasts (CEF) .
  • FIG. 2 shows that these human cells generally express at least three ECM inhibitors.
  • the major band of inhibitor activity has an apparent molecular weight of approximately 24-25 kDa, roughly corresponding in molecular weight to ChIMP-3.
  • the major band, human TIMP- 3 is most consistent and is found exclusively in the ECM (lanes 4-11) and not in conditioned media (lanes 2 and 3) . Its molecular weight is intermediate between that of TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa) , both of which were detected in samples of conditioned media (lanes 2 and 3) .
  • Human TIMP-3 is expressed at varying levels in different cell lines. There are in addition two minor inhibitors at approximately 29 and 30 kDa, which are designated IMP- a and IMP-b. These two, clearly seen in the ECM of FHs i73We cells (lanes 4 and 5) , are less consistent than
  • TIMP-3 are either smaller (lanes 6 and 7) or larger (lane 10) by approximately 1-2 kDa than the most frequently observed molecular weight of approximately 29 . . . and 30 kDa (lane 4) .
  • This variability may be due to different extents of glycosylation.
  • a fourth inhibitor, which migrates slightly ahead of TIMP-3, is occasionally seen in some samples (see Figure 9) .
  • Figure 3 provides an analysis of conditioned media (left) and matrix (right) of transforming chicken embryo fibroblasts (LA24-CEF) (lane 1) and four mammalian cell lines: SK-N-SH (lane 2) , 293 (lane 3) , mouse 3T3 cells (lane 5) , and rat pheochromocytoma cells derived from an adrenal gland tumor (PC12) (lane 6) .
  • SK-N-SH SK-N-SH
  • 293 lane 3T3 cells
  • PC12 rat pheochromocytoma cells derived from an adrenal gland tumor
  • PC12 adrenal gland tumor
  • All four mammalian cells express an inhibitor which co-migrates with ChIMP-2, the chicken equivalent of TIMP-2.' Two bands of activity (labelled “A” and “B” to the right of the gel) migrate with an apparent molecular weight of approximately 28-29 kDa, close to the expected mobility for TIMP-1. Significantly, all four mammalian cells express a matrix-specific activity (labelled "TIMP- 3" to the right of the gel) whose apparent molecular weight is similar, although slightly larger, than ChIMP-3 (lane 1) .
  • mouse TIMP-3 has a deduced molecular weight of 21.7 kDa versus 21.8 kDa for ChIMP-3; however, mammalian TIMP-3 has an apparent molecular weight of approximately 24 kDa on protease/substrate gels.
  • Figure 4 compares TIMPs present in conditioned media and ECM of mouse 3T3 cells and transforming chicken embryo fibroblasts (LA24-CEF) by protease/substrate gel electrophoresis.
  • the cells were seeded at 2 x 10 6 /100 mm dish.
  • the 3T3 cells were grown at 37°C for 36 hours before harvest.
  • LA24-infected cells were cultured at 41°C and transformed by transfer to 35°C for 10 hours before harvest.
  • conditioned media were removed and ECM prepared as described by Staskus et al. (1991) .
  • Lanes 1 and 3 are media samples and lanes 2 and 4 are ECM samples. Molecular weight standards were run in lane 5.
  • TIMP-3 the ECM of 3T3 cells contains a major band of activity.
  • This protein is clearly different from the media protein C (probably TIMP-2) and the matrix proteins A and B (lane 2) , which migrate in positions close to that expected for TIMP-1.
  • the matrix IMPs from 3T3 and also from SK-N-SH cells are not N-glycosylated, as shown by their resistance to PNGase F treatment.
  • 3T3 and SK-N-SH cells express a matrix-specific IMP which is non-glycosylated.
  • Example 4 Cloning of Mouse TIMP-3 A mouse cDNA library was screened with a full- length ChIMP-3 probe (Pavloff et al. (1992)) at reduced stringency. Five cDNA clones were obtained. Restriction mapping defined the regions of these cDNAs that cross- hybridized with the ChIMP-3 probe. Four of the clones likely represent overlapping versions of the same sequence; the fifth has an additional EcoRl site towards the 3'-end, suggesting it may result from an incompletely processed or alternatively spliced form.
  • a 1300 bp fragment which includes the region of homology with ChIMP-3 was sequenced.
  • the sequence contains a single open reading frame that encodes a protein that is 82% identical with mature ChIMP-3 at the amino acid level and 7.0% identical at the nucleotide sequence level overall, with blocks of identity occurring throughout the predicted coding region.
  • sequences downstream of the open reading frame of the mouse gene were very similar to the 3 '-untranslated region of ChIMP-3 : one AT-rich stretch of 47 bp is missing only one nucleotide from the corresponding region of ChIMP-3 and contains a 5'-AATGAAA-3' motif previously noted as being present in human and rabbit TIMP-1. In contrast, the 5'-untranslated sequences of the two genes are poorly conserved.
  • the 5'- and 3'-untranslated regions of the cDNA are the longest so far described for a member of the TIMP family.
  • the translation start site of the open reading frame is 1 kb from the 5'-terminus of the longest cDNA (clone 7.1) and the 3 '-untranslated sequence is an estimated 2.7 kb in length.
  • the mature cDNA is 1 kb from the 5'-terminus of the longest cDNA (clone 7.1) and the 3 '-untranslated sequence is an estimated 2.7 kb in length.
  • the mature cDNA is 1 kb from the 5'-terminus of the longest cDNA (clone 7.1) and the 3 '-untranslated sequence is an estimated 2.7 kb in length.
  • the mature cDNA is 1 kb from the 5'-terminus of the longest cDNA (clone 7.1) and the 3 '-untranslated sequence is an estimated 2.7 kb in length.
  • TIMP-1 transcript in mouse and human cells is only 0.9 kb in length.
  • mouse gene product (1) displays TIMP activity as determined by protease/substrate SDS-PAGE; (2) shows preferential ECM association, a hallmark of ChIMP-3; and (3) is recognized by antisera raised against ChIMP-3.
  • the nucleotide sequence of mouse TIMP-3 cDNA, aligned with that of ChIMP-3 is shown in Figure 5.
  • Figure 6 the deduced amino acid sequence of mTIMP-3 is compared with TIMP-1 from bovine, pig, human, rabbit, and mouse TIMP-1 (two sequences) ; with TIMP-2 from human, mouse and bovine; and with TIMP-3 from chicken.
  • the mature mTIMP-3 protein begins with the "CTCSPS" as marked.
  • mTIMP-3 was compared with that of mTIMP-1 and mTIMP-2 in fibroblasts and adult mouse tissues by Northern blot analysis.
  • a single major transcript class at around 4.5 kb was found.
  • There is apparently molecular weight heterogeneity because the 3.5 kb TIMP-2 transcript runs on the same blots as a much tighter species.
  • Smaller minor RNAs are also present, but it is unclear whether these are functional mRNAs or breakdown products of the larger form. No transcripts have been observed in the 0.9-1.0 kb range that are characteristic of other murine TIMPs.
  • mTIMP-3 Transcription of mTIMP-3 in mouse fibroblasts is highly inducible by stimuli that affect ECM remodelling. Expression of mTIMP-3 was induced in confluent serum-deprived mouse C3H 10T1/2 fibroblasts by exposure to PMA and TGF/.1. The combination of these two agents led to a superinduction of mRNA at early times (i.e., 2-4.5 hr) after exposure. This response is similar to that displayed by mTIMP-l and distinct from that of mTIMP-2, which is expressed in an essentially constitutive fashion in mouse fibroblasts. Leco et al. (1992). Like TIMP-1, the PMA- and cytokine-mediated induction of mTIMP-3 depends upon ongoing protein synthesis.
  • mTIMP-3 expression was stimulated by dexamethasone treatment alone and co-administration with either EGF or PMA augmented the induction seen with these agents separately.
  • Gingival fibroblasts (GF11 and 1292) were obtained as a gift from Dr. D. Richards at UCSF ' The following cells were purchased from American Type Culture Collection: transformed primary embryonal kidney (293) , neuroblastoma cells (SK-N-SH) , normal whole embryo (FHs 173We) , cervix epithelioid carcinoma (HeLa S3), colon adenocarcinoma (Caco-2) , ileocecal adenocarcinoma (HCT-8) and fibrosarcoma cells (SW 684 and Hs 913T) .
  • FBS fetal bovine serum
  • SW 684 cells SW 684 cells in Leibovitz L-15
  • GF11 and 1292 in MEM- ⁇
  • FHs 173We and Hs 913T Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/1 glucose and without sodium pyruvate
  • HCT in RPMI 1640
  • Caco-2 Caco-2 in MEM with non-essential amino acids
  • HeLa S3 in F12.
  • confluent cultures were reseeded 1:2 to 1:3 in their corresponding media without serum and cultured for 48 hours before harvesting ECM as described by Blenis and Hawkes (1983) .
  • FCS fetal calf serum
  • FHs 173We cells normal, human whole embryo - first trimester, ATCC No. HTB 1578 were initially seeded under reduced serum conditions (5% FCS) and maintained at
  • ECM was prepared from the 48 hr cells essentially as described by Blenis and Hawkes (1983) . Briefly, the cells were removed from the culture dishes following a 15-20 min incubation in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS) , containing 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride and 1 mg/ml e-amino-n- caproic acid (pH 7.4) (buffer A). After several rinses in PBS containing the protease inhibitors (above) and finally cold (4°C) distilled water, the ECM was solubilized in Laemmli sample buffer (without /_- mercaptoethanol) .
  • PBS Ca 2+ - and Mg 2+ -free phosphate-buffered saline
  • Samples were electrophoresed on sodium dodecyl sulfate- polyacrylamide gels (15%) (SDS-PAGE) (1.5 mm thick), the separating gel having been previously aged at room temperature for at least 24 hr.
  • Sodium thioglycolate was added to the cathode reservoir buffer at a concentration of 0.2 mM.
  • Proteins which electrophoresed as bands in the apparent molecular weight range of 20 to 30 kDa were excised and eluted in 50 mM ammonium bicarbonate, 0.5% SDS and 5 mM dithiothreitol (DTT) at 37°C with occasional shaking for at least 24 hr.
  • DTT dithiothreitol
  • the proteins were concentrated by centrifugation of the eluate in microconcentrators with a 30-kDa cut-off (Filtron) .
  • the reduced 24 kDa human TIMP-3 protein was concentrated and retained by membranes with cut-off values of 10 to 50 kDa and partially retained by membranes with cut-off values of 100 to 1000 kDa. This is postulated to be a function of the protein's "stickiness" rather than differences in apparent molecular weight.
  • the concentration of SDS in the retained fraction was monitored by the colorimetric assay of Waite and Wang (1976) Anal. Biochem. 22:279-280.
  • the retained protein was solubilized in Laemmli sample buffer without SDS, to avoid retention of this detergent in the concentrated protein sample.
  • the concentrated protein sample was then re-electrophoresed on a second preparative gel exactly as above.
  • An alternative concentration procedure includes precipitation of proteins from ammonium bicarbonate elution buffer by the addition of cold (-20°C) methanol (to a final concentration of 90% v/v) and storage overnight at -20°C. Precipitated protein was collected by centrifugation at 10,000 x g for 30 min. The protein was solvated in Laemmli sample buffer containing ⁇ - mercaptoethanol (final concentration 5% v/v) and electrophoresed as above.
  • Proteins resolved on the second preparative gel were transferred electrophoretically to a polyvinylidene difluoride (PVDF - Immobilon-P, Millipore) membrane using 10 mM 3-(-cyclohexylamino) -1-propanesulfonic acid, methanol (10% v/v), pH 11.0, as described by Matsudaira (1987) J. Biol. Chem. 262 : 10035-10038. Proteins were transferred at 300 A constant current for two hours. A representative section of the blot was probed with a polyclonal antibody to ChIMP-3 as described in detail below, while the remainder of the blot was stained with Coomassie Blue. The human TIMP-3 was identified on the
  • ChIMP-3 antibody-probed blot as a single band migrating with an apparent molecular weight of approximately 24 kDa. This band was used to locate the protein on the adjacent Coomassie-stained section of the blot.
  • the blot was incubated with the primary antibody (e.g., 1/500 dilution of anti-ChIMP-3 in TBS containing 1% BSA) for 12 hr at 4°C and rinsed extensively with TBS (see below) . It was then incubated with secondary antibody, goat anti-rabbit IgG, conjugated to alkaline phosphatase (Vector Laboratories) (1/1000 dilution in TBS containing 1% BSA) for 2 hr at room temperature. After further rinsing in TBS (see below) , the im unoblot was developed by adding substrate for alkaline phosphatase (Kit II, Vector Laboratories) .
  • the primary antibody e.g., 1/500 dilution of anti-ChIMP-3 in TBS containing 1% BSA
  • secondary antibody goat anti-rabbit IgG conjugated to alkaline phosphatase (Vector Laboratories) (1/1000 dilution in TBS containing 1% B
  • TBS once
  • TBS 0.1% Tween 20, 0.5 M NaCl (once)
  • TBS 0.1% Tween 20
  • TBS twice
  • Electrophoretic transfer to PVDF membrane was performed using 10 mM CAPS (3- [cyclohexylamino]-l-propanesulfonic acid) buffer, pH 11.0 containing methanol (10% v/v) according to the method described by Matsudaira (1987) J. Biol. Chem. 262:10035- 10038. Transfer was accomplished at 300 A for 2 h.
  • CAPS 3- [cyclohexylamino]-l-propanesulfonic acid
  • Non-specific binding sites on the membrane were blocked with 1% w/v nonfat dry milk powder (Carnation) in TBS, 10 mM Tris-Cl, pH 7.4, 0.9% NaCl, 0.1% normal goat serum for 12 h at 4°C.
  • the blot was then incubated with the primary antibodies (diluted 1:500 in TBS containing 1% w/v BSA) either overnight at 4°C or at room temperature for at least 8 h.
  • the blot was then rinsed extensively in the following sequence: one rinse with TBS, one rinse with TBS containing 0.1% v/v Tween 20 and 0.5 M NaCl, two rinses with TBS with 0.1% v/v Tween 20 and two rinses with TBS.
  • the blots were then incubated with the secondary antibody, goat anti-rabbit IgG conjugated to alkaline phosphatase (BioRad Laboratories) (diluted
  • lanes 2, 3 , and 5 contain ECM from human FHs 173We cells (approximately 2.4 x 10 6 cells). Lanes 1 and 6 contain dialyzed and concentrated CM (equivalent to 60 ⁇ l CM) from FHs 173We cells. Lane 4 contains ECM from CEF cells (approximately 4 x 10 6 cells) .
  • the data in this figure were derived from three different gels and therefore the mobilities of the proteins cannot be compared directly'.
  • the apparent molecular weights for the reduced proteins recognized by each antibody were determined by reference to molecular weight standards on each individual gel (not shown) .
  • anti-ChIMP-3 recognizes a major protein of approximately 24 kDa, intermediate in molecular weight between TIMP-1 and TIMP-2 and migrating slightly more slowly than the control ChIMP-3 in lane 4, together with a minor band of approximately 29 kDa.
  • anti-human TIMP-1 (lane 2) and anti-human TIMP-2 (lane 5) do not recognize any proteins in the ECM samples, although they recognize authentic TIMP-1 (lane 1) and TIMP-2 (lane 6) in samples of conditioned media from the whole embryo cells.
  • polyclonal anti-ChIMP-3 recognizes human TIMP-3 but not human TIMP-1 or TIMP-2.
  • Human TIMP-3 represents a relatively small percentage of ECM proteins in all human cell lines that have been screened. This is in contrast to ChIMP-3, which is a major component of the ECM of chicken embryo fibroblasts during the early stages of transformation.
  • Figure 8 shows a Western blot of human TIMP-3 in a reduced and unreduced state showing the effect of ⁇ - ercapt'oethanol on the electrophoretic migration of human TIMP-3 on SDS polyacrylamide gels.
  • Samples of ECM were electrophoresed on SDS polyacrylamide gels.
  • the proteins were transferred electrophoretically to PVDF membrane and probed with an antibody to the NH 2 -terminus of ChIMP-3.
  • Lanes 1-4 contain ECM from approximately 2.4 x 10 6 FHs l73We cells; lane 6 contains ECM from approximately 4 x 10 6 CEF; lane 5 contains sample loading buffer only.
  • (+) indicates addition of /--mercaptoethanol to a final concentration of 5% v/v; (-) indicates no addition of ⁇ - mercaptoethanol.
  • Human TIMP-3 when reduced with ⁇ - mercaptoethanol, runs more slowly than the unreduced control and at approximately the same apparent molecular weight as reduced ChIMP-3 (in lane 6, reduced ChlMP-a clearly resolves from reduced ChIMP-3) .
  • lane 2 where there is a gradient of reducing agent between lane 1 (no j8-mercaptoethanol) and lanes 3 and 4 (with ⁇ - mercaptoethanol) , the protein shows an altered pattern of mobility that is reminiscent of that observed for ChlMP-
  • ECM was prepared from human neuroblastoma (SK- N-SH) cells that had been cultured with 5% serum and then maintained for 24 hours in serum-free media.
  • the ECM was solubilized in Laemmli buffer without reducing agent and dialyzed extensively against 0.2 M sodium phosphate, pH 8.0, containing 10 mM EDTA, 0.05% w/v SDS at 4°C using Spectro/Por-3 tubing (3,500 molecular weight cut-off. Spectrum Medical Industries) .
  • the sample volumes were reduced by coating the dialysis tubing with carboxymethylcellulose (Aquacide I, Calbiochem) to absorb water.
  • IMP-a and/or IMP-b may be an N-glycosylated form of TIMP-3.
  • Example 9 Cell Detachment Assay First, in order to prepare culture dishes, uncoated plastic dishes are coated with the control protein histone HI (5 ⁇ g/dish) , electrophoretically purified human TIMP-3 , or a "mock" sample isolated from a blank gel under identical conditions. Uncoated dishes are used as an additional control. Proteins are dissolved in 10 mM sodium phosphate buffer, pH 7.0/9 M urea and sterilized by passage through 0.2 ⁇ m membranes (Acrodisc, Gelman) . Protein solutions (0.15 ml) are distributed to 60-mm culture dishes and diluted 10-fold by addition of Tris diluent (137 mM NaCl/5 mM KCl/5 mM
  • LA24-infected chicken embryo fibroblast (CEF) cells may be used.
  • transforming mammalian cells particularly human cells
  • the cells (1 x 10 6 per 60 mm dish in 5 ml of medium 2-2-1+ (Yang and Hawkes (1992)) are incubated at the nonpermissive temperature (41°C) for 6 hr to promote attachment to the culture dishes.
  • Controls include untreated plastic dishes, dishes coated with histone and dishes treated with a sample eluted from a blank gel.
  • Duplicate cultures are then assayed for cell detachment.
  • the assay for cell detachment from the ECM is a modification of published procedures. Johnson and Pastan (1972) Nature 236:247-249; and Shields and Pollock (1974) Cell 2:31-38.
  • the medium is decanted ' and the cell monolayer is washed twice with Ca 2+ -, Mg 2+ -free phosphate- buffered saline (pH 7.4) and incubated in the same buffer containing 5 mM EGTA (2 ml per 60 mm dish) for 10 min at 41°C.
  • the dishes are agitated on a rotary shaker (Braun Thermonix Shaker 1460) at 120 rpm for 5 min at room temperature.
  • EGTA-released cells are removed and the remaining cells are detached by trypsin (0.05% wt/vol, 41°, 15 min) .
  • the number of cells in each sample is determined by counting in a Coulter Counter. Cells detached by EGTA treatment are expressed as a percentage of the total cell number.
  • LA24-infected CEF cells or preferably, mammalian cells, and particularly human cells such as FHs 173We may be used, as for Example 9.
  • the cells are seeded at 2 x 10 s per 60-mm dishes in low serum medium (2-0-0.1+) , cultured at 35°C or 41°C, detached with trypsin (as above) , and counted at 24-hr intervals.
  • the population doubling time is determined from a graph of logarithm of cell number versus time in culture. Values are calculated as averages of triplicate determinations.
  • Example 11 Expression of Human TIMP-3 mRNA in Human Tissues
  • ChIMP-3 cDNA was used to probe the mRNA of a large number of cultured mammalian cells. Evidence was found of the expression of TIMP-3 in rat, mouse, monkey, and human cells.
  • ChIMP-3 cDNA (P483, including the coding region) was random primed with 32 P and used to probe RNA dot blots. Hybridization was observed with RNA from human endometrial adenocarcinoma cells (HEC-l-B) , human newborn foreskin cells (Hs27) , human breast adenocarcinoma cells (MCF-7) , two lines of mutant human fibroblasts (GM03123 and GM00110B) , as well as African green irtonkey kidney cells (CV-1) .
  • HEC-l-B human endometrial adenocarcinoma cells
  • Hs27 human newborn foreskin cells
  • MCF-7 human breast adenocarcinoma cells
  • GM03123 and GM00110B two lines of mutant human fibroblasts
  • CV-1 African green irtonkey kidney cells
  • Northern blots of RNA from chicken cells (CEF) probed with a 32 P-labeled PCR probe from the ChIMP-3 cDNA (P483) showed transcripts at approximately 4500-5500 nt, 2300-2900 nt, and 1000 nt.
  • ECM was prepared from confluent FHs 173We cells using a total of 45 culture dishes (100 mm) .
  • the material was solubilized in Laemmli sample buffer containing /.-mercaptoethanol (final concentration 5% v/v) and electrophoresed in three lanes through a preparative
  • the blot was rinsed extensively with double distilled water and a section of the blot containing a small aliquot of ECM from FHs 173We cells was excised and probed with the polyclonal antibody to the ChIMP-3 peptide, as described above, to locate TIMP- 3.
  • the remainder of the blot was stained for one minute with Coomassie Blue R-250 in acetic acid/methanol/water (1:50:49), destained in methanol/water (50:50) , rinsed extensively with water, dried, and stored at -20°C.
  • the antibody-probed blot was then aligned with the Coomassie- stained blot and used to locate the TIMP-3 bands.
  • TIMP-3 indicates, for those residues determined, that its NH 2 -terminus is identical to that of ChIMP-3 (Pavloff et al. (1992)) and the recently cloned mouse TIMP-3 (Leco, K.J., Khokha, R. , Pavloff, N. , Hawkes, S.P., and Edwards, D.R. (1993), submitted) ( Figure 10). Of those positions where the chicken and mouse sequences differ (valine/serine at position 4 and isoleucine/serine at position 6 for ChIMP-3/mTIMP-3) , no assignments could be made.
  • hTIMP-3 is human TIMP-3; ChIMP-3 is chicken TIMP-3 (Pavloff et al. (1992)) ; and mTIMP-3 is mouse TIMP-3 (Leco et al. (1993)) .
  • TIM-l con is a consensus sequence for TIMP-1 from human (Docherty et al. (1985); Gasson et al. (1985) ; and Carmichael et al. (1986)); murine (Edwards et al. (1986); Gewert et al.
  • TIMP-2 con is a consensus sequence for TIMP-2 from human (Boone et al. (1990) ; and Stetler-Stevenson et al. (1990)); bovine (Boone et al. (1990)); murine (Leco et al. (1992); and Shimizu et al. (1992)) ; rat (Roswit et al.
  • the amino acid composition of human TIMP-3 is similar to ChIMP-3.
  • the major discrepancy is the high glycine content in the chicken protein (23-34 residues compared to 17 in human TIMP-3) . This was previously attributed to contamination with this amino acid from buffers during purification. Staskus et al. (1991) . Indeed, the deduced amino acid composition from ChIMP-3 cDNA analysis indicates 15 glycine residues from a total of 188 amino acids in the mature protein. Pavloff et al. (1992) .
  • TIMP-3 protein which was identified on PVDF with anti-ChIMP-3 antibody, was eluted in 50 mM Tris-HCl buffer, pH 9.0, containing 2% SDS/1% Triton X-100 and analyzed by protease/substrate gel electrophoresis. A single metalloproteinase inhibitory activity was detected as a band which co- migrated with human TIMP-3 present in a sample of total
  • RNA Isolation RNA was isolated by the guanidinium thiocyanate method according to the method described by Chirgwin et al. (1979) Biochem. 18:5294-5299 with modifications according to the method described by Freeman et al. (1983) Proc. Natl. Acad. Sci. USA 80:4094- 4098. 'poly(A) + RNA was purified by a single fractionation over oligo(dT) -cellulose according to the method described by Aviv and Leder (1972) Proc. Natl. Acad. Sci. USA 22:1408-1412. Total RNA from human heart was obtained from Clontech (Palo Alto, CA) .
  • cDNA library A ⁇ gtlO human heart 5' stretch cDNA library was purchased from Clontech. A human placenta cDNA library was a generous gift from Dr. N. Lomri at UCSF, San Francisco, CA. Oligonucleotide Synthesis. Oligonucleotide primers for PCR and probes were synthesized by the phosphoramidite method with an Applied Biosystems model 394 automated DNA synthesizer and purified by PAGE and desalted on Sep-Pak (Millipore Corp.) using standard protocols.
  • Primer TIMP-4 was designed to bind to the noncoding strand of TIMP-3 cDNA corresponding to amino acids 82-87 of ChIMP-3 (YIYTEA, Pavloff et al. (1992)), with amino acid 82 substituted by Y or F to cover other possible TIMP sequences and amino acid 84 substituted by Y (chicken sequence) or H (mouse sequence) .
  • This sequence was chosen for the relatively small codon degeneracy and for the least possible sequence similarity to other TIMPs.
  • the sequence of the 26-base primer consists of 17 bases specific for TIMP-3 , a 6-base EcoRl restriction site and three extra bases at the 5' end (underlined) .
  • Primer TIMP-5 was designed to bind to the coding strand corresponding to amino acids 110-115 of ChIMP-3 (YEGKVY, Pavloff et al. (1992)), with amino acid 114 substituted by M (mouse TIMP-3) and amino acid 115 substituted by Y or H to cover other possible TIMP-3 sequences. This sequence was chosen for the relatively small codon degeneracy and for the least possible sequence similarity to other TIMPs.
  • the sequence of the 26-base primer consists of 17 bases specific for TIMP-3, a 6-base Hindlll restriction site and three extra bases at the 5' end (underlined) .
  • Primer TIMP-3-9-3' was designed on the basis of partial sequence information on human TIMP-3. It was used to clone the 3' end of TIMP-3 and included a 6-base Hindlll restriction site and six extra bases at the 5' end (underlined) . Primer TIMP-3 -9-3 ' :
  • TIMP-3 TIMP-3.
  • Primer 29 was designed to bind to the noncoding strand corresponding to 18 bases in the 3'-noncoding region of ChIMP-3 which is identical in mouse TIMP-3 (nucleotides 831-851, Pavloff et al. (1992)) . This sequence was chosen because the sequence AATGAAA
  • nucleotides 840-846 in ChIMP-3) which we noted was a variant of a poly(A) signal, is also present at the 3' end of human (Docherty et al. (1985) ; Carmichael et al. (1986) ), • and rabbit (Horowitz et al. (1989)) TIMP-1 cDNA and precedes the putative polyadenylation consensus sequence signal, AATAAA.
  • the sequence of the 26-base primer consists of 18 bases specific for ChIMP-3, a 6-base BamHI restriction site and two extra bases at the 5' end (underlined) .
  • Primer TIMP-3-1-67 was designed on the basis of partial sequence of human TIMP-3 and was used as a probe to screen a ⁇ gtlO human heart cDNA library purchased from Clontech.
  • Primer TIMP-3-1-67 5'-GACGCGACCTGTCAGCAGGTACTGGTACTTGTTGACCTCCAGCTTAAGGCC ACAGAGACTCTCGGAA-3 '
  • PCR Polymerase Chain Reaction
  • the DNA templates used in PCR were the following: Human concanavalin A-activated peripheral blood lymphocytes cDNA, U 937 cDNA, WI-L2-729HF2 cDNA, cDNA from human heart, human placenta cDNA library, and genomic DNA ' from Clontech.
  • the PCR was run as described by Perkin Elmer Cetus using Ampliwax. Initially, primer TIMP-4 was used along with primer TIMP-5.
  • the PCR was run in a DNA thermal cycler (Perkin Elmer Cetus) for 35 cycles. The first 10 cycles consisted of heating at 94°C for 1 min., and annealing at 36°C for 2 min. The last 25 cycles consisted of heating at 94°C for 1 min., annealing at 55°C for 1 min., and polymerization at 72°C for 1 min.
  • This reaction consistently yielded a single 114 bp product (P114) detected on a 7% acrylamide gel representing a partial human TIMP-3 cDNA present in all the DNA tested.
  • P114 obtained from all these PCR products was digested with EcoRl and Hindlll, fractionated on a 7% acrylamide gel and cloned into M13 mpl9. Three independent subclones were selected.
  • a specific primer (TIMP-3-9-3' ) was designed to determine the 3'-end sequence of the cDNA. This primer and primer 29 (described above) were used to amplify cDNA from the ⁇ gtlO library.
  • the PCR was run for 30 cycles, each consisting of heating at 94°C for 1 min., annealing at 55°C for 2 min. and polymerization at 72°C for 3 min.
  • the resulting single 450 bp fragment (P450) was cloned in pCRScriptTMSK(+) (Stratagene) .
  • a 32 P-labeled probe, TIMP-3-1-67 was generated by the kinase reaction for subsequent screening of the ⁇ gtlO library. Unincorporated nucleotides were removed on a Sephadex G-25 column (Boehringer Mannheim
  • Double-stranded DNA cloned into pCR-ScriptTMSK(+) and single-stranded DNA cloned ' into M13mpl9 were sequenced by the dideoxy terminator method (Sanger et al. (1977) Proc. Natl. Acad. USA 74:3463-3467) using sequencing kits purchased from United States Biochemicals (Sequenase version 2.0). Each cDNA subclone was sequenced using an M13 universal primer and a reverse sequencing primer.
  • Figure 11 shows nucleotide sequences of human TIMP-3 cDNA corresponding to 18 amino acids of the signal peptide and the entire (188 amino acids) mature protein.
  • the nucleotide sequence includes additional sequences from the 3'-noncoding region.
  • a comparison of the nucleotide sequence of ChIMP-3 (amino acids 88-212) with the 3' end coding sequence of human TIMP-3 indicates that 292 nucleotides are identical out of the 378 total. This represents 77% nucleotide sequence identity.
  • a comparison of the 125 deduced amino acid residues of human TIMP-3 with the deduced amino acid sequence of ChIMP-3 indicates that 102 amino acid residues are conserved. This represents about 82% amino acid sequence identity. There was not much homology in the 75 noncoding nucleotides.
  • the complete sequence of the mature region of human TIMP-3 has been determined by screening the human cDNA library using 32 P-labeled TIMP-1-67 as a probe. Seven positive clones were isolated and are being purified. Obtaining a genomic sequence for human TIMP-3 is readily accomplished by using the human TIMP-3 cDNAs as probes or by designing PCR primers using sequences from the cDNA which are likely to distinguish TIMP-3 from TIMP-1 and -2, as discussed above, particularly from the noncoding regions of the gene.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne l'inhibiteur tissulaire humain des métalloprotéases (TIMP-3 humain) isolé. Le polypeptide, des anticorps préparés contre lui et des compositions pharmaceutiques comprenant ces polypeptides et ces anticorps sont utiles, par exemple dans les domaines du diagnostic, de la thérapie et de la prophylaxie des pathologies caractérisées par une activité excessive ou non désirée d'une métalloprotéinase matricielle.
PCT/US1994/009188 1993-08-12 1994-08-12 Timp-3 humain WO1995005478A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU78277/94A AU7827794A (en) 1993-08-12 1994-08-12 Human timp-3

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10526393A 1993-08-12 1993-08-12
US08/105,263 1993-08-12
US16746393A 1993-12-13 1993-12-13
US08/167,463 1993-12-13

Publications (1)

Publication Number Publication Date
WO1995005478A1 true WO1995005478A1 (fr) 1995-02-23

Family

ID=26802409

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/009188 WO1995005478A1 (fr) 1993-08-12 1994-08-12 Timp-3 humain

Country Status (2)

Country Link
AU (1) AU7827794A (fr)
WO (1) WO1995005478A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038314A1 (fr) * 1996-04-09 1997-10-16 British Biotech Pharmaceuticals Limited Diagnostic et surveillance des affections neurologiques
WO2004085617A3 (fr) * 2003-03-21 2006-04-06 Cleveland Clinic Foundation Inhibiteur de vegf en tant que timp3
US8431396B2 (en) 2003-03-21 2013-04-30 The Cleveland Clinic Foundation Anti-angiogenic peptides
WO2016033212A1 (fr) * 2014-08-27 2016-03-03 Amgen Inc. Variants d'inhibiteur tissulaire de la métalloprotéinase de type trois (timp-3), compositions et procédés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011287A1 (fr) * 1989-03-21 1990-10-04 The United States Of America, Represented By The Secretary, United States Department Of Commerce Peptides inhibiteurs de metalloproteinases matricielles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990011287A1 (fr) * 1989-03-21 1990-10-04 The United States Of America, Represented By The Secretary, United States Department Of Commerce Peptides inhibiteurs de metalloproteinases matricielles

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 54, issued 15 April 1994, J.A. URIA et al., "Structure and Expression in Breast Tumors of Human TIMP-3, a New Member of the Metalloproteinase Inhibitor Family", pages 2091-2094. *
DNA AND CELL BIOLOGY, Volume 13, No. 7, issued July 1994, C.G. WILDE et al., "Cloning and Characterization of Human Tissue Inhibitor of Metalloproteinases-3", pages 711-718. *
FASEB JOURNAL, Volume 8, No. 4, issued 15 March 1994, N.S. KISHINANI et al., "Interactions of TIMP-3 With ECM Components Are Influenced by Charged Polymers", page A47, Abstract No. 271. *
FASEB JOURNAL, Volume 8, No. 5, issued 18 March 1994, C.G. WILDE et al., "Identification of Human TIMP-3 by High-Throughput cDNA Cloning and Database Discovery", page A936, Abstract No. 5423. *
GENE, Volume 114, issued May 1992, S. SHIMIZU et al., "Cloning and Sequencing of the cDNA Encoding a Mouse Tissue Inhibitor of Metalloproteinase-2", pages 291-292. *
GENE, Volume 141, issued April 1994, S.M. SILBIGER et al., "Cloning of cDNAs Encoding Human TIMP-3, a Novel Member of the Tissue Inhibitor of Metalloproteinase Family", pages 293-297. *
GENOMICS, Volume 19, issued January 1994, S.S. APTE et al., "Cloning of the cDNA Encoding Human Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) and Mapping of the TIMP3 Gene to Chromosome 22", pages 86-90. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, No. 24, issued 25 August 1992, N. PAVLOFF et al., "A New Inhibitor of Metalloproteinases from Chicken: ChIMP-3", pages 17231-17236. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 269, No. 29, issued 22 July 1994, M. WICK et al., "A Novel Member of the Human Tissue Inhibitor of Metalloproteinases (TIMP) Gene Family is Regulated During G1 Progression, Mitogenic Stimulation, Differentiation and Senescence", pages 18953-18960. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 87, issued April 1990, T.C. BOONE et al., "cDNA Cloning and Expression of a Metalloproteinase Inhibitor Related to Tissue Inhibitor of Metalloproteinases", pages 2800-2804. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued November 1992, T. YANG et al., "Role of the 21-kDa Protein TIMP-3 in Oncogenic Transformation of Cultured Chicken Embryo Fibroblasts", pages 10676-10680. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038314A1 (fr) * 1996-04-09 1997-10-16 British Biotech Pharmaceuticals Limited Diagnostic et surveillance des affections neurologiques
WO2004085617A3 (fr) * 2003-03-21 2006-04-06 Cleveland Clinic Foundation Inhibiteur de vegf en tant que timp3
US7183256B2 (en) 2003-03-21 2007-02-27 Cleveland Clinic Foundation TIMP3 as VEGF inhibitor
US8431396B2 (en) 2003-03-21 2013-04-30 The Cleveland Clinic Foundation Anti-angiogenic peptides
US9498509B2 (en) 2003-03-21 2016-11-22 The Cleveland Clinic Foundation TIMP3 as VEGF inhibitor
WO2016033212A1 (fr) * 2014-08-27 2016-03-03 Amgen Inc. Variants d'inhibiteur tissulaire de la métalloprotéinase de type trois (timp-3), compositions et procédés
CN107001445A (zh) * 2014-08-27 2017-08-01 美国安进公司 3型金属蛋白酶组织抑制剂(timp‑3)的变体、组合物及方法
JP2017532004A (ja) * 2014-08-27 2017-11-02 アムジエン・インコーポレーテツド 組織メタロプロテイナーゼ阻害物質３型（ｔｉｍｐ−３）の変異体、組成物、及び方法
EP3575316A1 (fr) * 2014-08-27 2019-12-04 Amgen, Inc Variants d'inhibiteur tissulaire de la métalloprotéinase de type trois (timp-3), compositions et procédés
US11149078B2 (en) 2014-08-27 2021-10-19 Amgen Inc. Variants of tissue inhibitor or metalloprotienase type three (TIMP-3), compositions and methods

Also Published As

Publication number Publication date
AU7827794A (en) 1995-03-14

Similar Documents

Publication Publication Date Title
US5859206A (en) Antibodies specific for heregulin 2-α
CA2108473C (fr) Structure, production et utilisation de l'hereguline
AU708829B2 (en) Human tissue inhibitor of metalloproteinase-4
US6521227B1 (en) Polynucleotides encoding prostatic growth factor and process for producing prostatic growth factor polypeptides
US7741055B2 (en) Prostatic growth factor
US6232291B1 (en) Cytostatin III
US20130095569A1 (en) Pregnancy-associated plasma protein-a2 (papp-a2) polynucleotides
CA2051676A1 (fr) Recepteur d'un activateur de la plasminogene de type urokinase
WO1992022320A1 (fr) Variantes de l'inhibiteur c1 et traitement des reactions inflammatoires avec l'inhibiteur c1
US5484703A (en) Assay using recombinant histidyl-tRNA synthetase
US6313267B1 (en) Calcium-binding proteins
WO1996018730A1 (fr) Facteur de croissance prostatique
JP2002501365A (ja) 組織型プラスミノーゲンアクチベーターの脳関連インヒビター
WO1995005478A1 (fr) Timp-3 humain
EP0555286B1 (fr) Inhibiteurs de croissance cellulaire
US6329169B1 (en) Nucleic acid molecules encoding cytostatin II
US6312688B1 (en) Tyrosine-phosphatase-related protein
WO1995009913A1 (fr) Inhibiteur de metalloproteases derive de monocytes/macrophages humains
WO1995009913A9 (fr) Inhibiteur de metalloproteases derive de monocytes/macrophages humains
JP2001500251A (ja) Ｈｋ２ポリペプチドを検出するための方法
US6800473B1 (en) Human cathepsin L2 protein, gene encoding said protein and use thereof
JP4346540B2 (ja) 絨毛外栄養膜細胞特異的蛋白質
GB2169295A (en) Process for the production of a metalloproteinase inhibitor
JP3232415B2 (ja) モノクローナル抗体，その製造法および用途
WO1999006555A1 (fr) Nouvelle proteine humaine mmp-20 et son utilisation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK ES FI GB GE HU JP KE KG KP KR KZ LK LT LU LV MD MG MN MW NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase