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U.S. Patent Apr. 20,2010 Sheet 4 of 4 US 7,700,341 B2
MTSP3 194 LACGKS-- - LKTPRVVGGEEASVDSWPWQVSIQYDKQHVCGGSILD 236
MTSP4-S 396 PQCDGRPDCRDGSDEEHCECGLQGPSSRIVGGAVSSEGEWPWQASLQVRGRHICGGALIA 455
MTSP4-L 540 PQCDGRPDCREK3SDEEHCECGLQGPSSRIVGGAVSSEGEWPWQASLQVRGRHICGGALIA 593
MTSP6 205 TACGHR RGYSSRIVGGNMSLLSQWPWQASLQFQGYHLCGGSVIT 248
MTSP3 237 PHWVLTAAHCFRKHTDVFN--WKVRAGSDKLGS FPSLAVAKIIIIEFN'PMYPKDND 290
MTSP4-S 456 DRWVITAAHCFQEDSMASTVLWTVFLGKVWQNSRWPGEVSFKVSRLLLHPYHEEDSHDYD 515 MTSP4-L 600 Drotitaahcfqeds^stv'lktvfjgkvm'owsrwpgevsfk'/shlllhpyhebdshdyd 713 MTSP5 249 PLWIITAAHOTYDLYLPKS--WTIQVGLVSLLD--NPAPSHLVEK.IVYHSKYKPKRLGKD 304
MTSP3 291 IALMKLQFPLTFSGTVRPICLPFFDEELTPATPLWIIGWGFTKQNGGKMSDILLQASVQV 350
MTSP4-S 516 VALLQLDHPWRSAAVRPVCLPARSHFFEPGLHCWITGWGALRE-GGPISNALQKVDVQL 574
MTSP4-L 660 VALLQLDHPWRSAAVRPVCLPARSHFFEPGLHCWITGWGALRE-GGPISNALQKVDVQL 718
MTSPS 305 IALMKLAGPLTFKEMIQPVCI.PNSEENFPDGKVCWTSGWGATED-GGDASPVLNHAAVPL 363
MTSP3 351 IDSTRCNADDAYQGEVTEKMMCAGIPEGGVDTCQGDSGGPLMYQSDQ--WHWGIVSWGY 408
MTSP4-S 575 IPQDLCS--EVYRYQVTPRMLCAGYRKGKKDACQGDSGGPLVCKALSGRWFLAGLVSWGL 632
MTSP4-L 719 IPQDLCS--EVYRYQVTPRMLCAGYRKGKKDACQGDSGGPLVCKALSGRWFLAGLVSWGL 776
MTSPS 364 ISNKICNHRDWGGIISPSMLCAGYLTGGVDSCQGDSGPLVCQERR-LWKVLVGATSFGI 442
MTSP3 409 GCGGPSTPGVYTKVSAYLNWIYNVWKAEL 437
MTSP4-S 633 GCGRPNYFGVYTRITGVISWIQQWT 658
MTSP4-L 777 GCGRPNYFGVYTRITGVISWIQQWT 802
HTSP6 423 GCAEVNKPGVYTRVTSFLDWIHEQMERDLXT 453
cleavage site + potential glycosylation site * unpaired cysteine
NUCLEIC ACID MOLECULES ENCODING vided are prognostic, diagnostic and therapeutic methods
TRANSMEMBRANE SERINE PROTEASES, using the proteases and domains thereof and the encoding
THE ENCODED PROTEINS AND METHODS nucleic acid molecules.
5 BACKGROUND OF THE INVENTION AND
RELATED APPLICATIONS OBJECTS THEREOF
For U.S. purposes, benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No. 60/179,982, 10 to Edwin L. Madison and Edgar O. Ong, filed Feb. 3, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF"; to U.S. provisional application Ser. No. 60/183,542, to Edwin L. Madison and Edgar O. Ong, filed Feb. 18, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF"; to U.S. provisional application Ser. No. 60/213, 20 124, to Edwin L. Madison and Edgar O. Ong, filed Jun. 22, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF"; to U.S. provisional application Ser. No. 60/220,970, to Edwin L. Madi- 25 son and Edgar O. Ong, filed Jul. 26, 2000, entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF"; and to U.S. provisional applica- 30 tion Ser. No. 60/234,840 to EdwinL. Madison, Edgar O. Ong and Jiunn-Chern Yeh, filed Sep. 22, 2000, entitled "NUCLEIC ACID MOLECULES ENCODING TRANSMEMBRANE SERINE PROTEASES, THE ENCODED PROTEINS AND METHODS BASED THEREON" is 35 claimed herein. Benefit of priority under 35 U.S.C. §120 to U.S. application Ser. No. 09/657,986, to Edwin L. Madison, Joseph Edward Semple, Gary Samuel Coombs, John Eugene Reiner, Edgar O. Ong, Gian Luca Araldi, filed Sep. 8, 2000, 40 entitled "INHIBITORS OF SERINE PROTEASE ACTIVITY OF MATRIPTASE OR MTSP1," now U.S. Pat. No. 6,797,504, is also claimed herein. This application is a continuation-in-part of U.S. application Ser. No. 09/657,986, filed Sep. 8, 2000, now U.S. Pat. No. 6,797,504. For interna- 45 tional purposes, benefit of priority to each of the above-noted applications is claimed herein.
This application is related to U.S. provisional application Ser. No. 60/166,391 to Edwin L. Madison and Edgar O. Ong, 50 filedNov. 18,1999 entitled "NUCLEOTIDE AND PROTEIN SEQUENCES OF PROTEASE DOMAINS OF ENDOTHELIASE AND METHODS BASED THEREON". This application is also related to International PCT application No. PCT/US00/31803, filed Nov. 17, 2000. 55
The above-noted provisional applications, patent application and International PCT application are incorporated by reference in their entirety. All patents, applications, published applications and other publications and sequences from GenBank and other data bases referred to herein are incorporated 60 by reference in their entirety.
FIELD OF INVENTION
Nucleic acid molecules that encode proteases and portions thereof, particularly protease domains are provided. Also pro
Cancer a leading cause of death in the United States, developing in one in three Americans; one of every four Americans dies of cancer. Cancer is characterized by an increase in the number of abnormal neoplastic cells, which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells that metastasize via the blood or lymphatic system to regional lymph nodes and to distant sites.
Among the hallmarks of cancer is a breakdown in the communication among tumor cells and their environment. Normal cells do not divide in the absence of stimulatory signals, cease dividing in the presence of inhibitory signals. Growth-stimulatory and growth-inhibitory signals, are routinely exchanged between cells within a tissue. In a cancerous, or neoplastic, state, a cell acquires the ability to "override" these signals and to proliferate under conditions in which normal cells do not grow.
In order to proliferate tumor cells acquire a number of distinct aberrant traits reflecting genetic alterations. The genomes of certain well-studied tumors carry several different independently altered genes, including activated oncogenes and inactivated tumor suppressor genes. Each of these genetic changes appears to be responsible for imparting some of the traits that, in the aggregate, represent the full neoplastic phenotype.
A variety of biochemical factors have been associated with different phases of metastasis. Cell surface receptors for collagen, glycoproteins such as laminin, and proteoglycans, facilitate tumor cell attachment, an important step in invasion and metastases. Attachment triggers the release of degradative enzymes which facilitate the penetration of tumor cells through tissue barriers. Once the tumor cells have entered the target tissue, specific growth factors are required for further proliferation. Tumor invasion (or progression) involves a complex series of events, in which tumor cells detach from the primary tumor, break down the normal tissue surrounding it, and migrate into a blood or lymphatic vessel to be carried to a distant site. The breaking down of normal tissue barriers is accomplished by the elaboration of specific enzymes that degrade the proteins of the extracellular matrix that make up basement membranes and stromal components of tissues.
A class of extracellular matrix degrading enzymes have been implicated in tumor invasion. Among these are the matrix metalloproteinases (MMP). For example, the production of the matrix metalloproteinase stromelysin is associated with malignant tumors with metastatic potential (see, e.g., Matrisian et al. (1990) Smnrs. in Cancer Biology 1:107-115; McDonnell et al. (1990) Cancer and Metastasis Reviews 9:309-319).
The capacity of cancer cells to metastasize and invade tissue is facilitated by degradation of the basement membrane. Several proteinase enzymes, including the MMPs, have been reported to facilitate the process of invasion of tumor cells. MMPs are reported to enhance degradation of the basement membrane, which thereby permits tumorous cells to invade tissues. For example, two major metalloproteinases having molecular weights of about 70 kDa and 92 kDa appear to enhance ability of tumor cells to metastasize.
Type II Transmembrane Serine Proteases (TTSPs)
In addition to the MMPs, serine proteases have been implicated in neoplastic disease progression. Most serine proteases, which are either secreted enzymes or are sequestered in cytoplasmic storage organelles, have roles in blood coagulation, wound healing, digestion, immune responses and tumor invasion and metastasis. A class cell surface proteins designated type II transmembrane serine proteases, which are membrane-anchored proteins with N-terminal extracellular domains, has been identified. As cell surface proteins, they are positioned to play a role in intracellular signal transduction and in mediating cell surface proteolytic events.
Cell surface proteolysis is a mechanism for the generation of biologically active proteins that mediate a variety of cellular functions. These membrane-anchored proteins, include a disintegrin-like and metalloproteinase (ADAM) and membrane-type matrix metalloproteinase (MT-MMP). In mammals, at least 17 members of the family are known, including seven in humans (see, Hooper et al. (2001) J. Biol. Chem. 276:857-860). These include: corin (accession nos. AF133845 and AB013874; see, Yan et al. (1999) J. Biol. Chem. 274:14926-14938; Tomia et al. (1998) J. Biochem. 124:784-789; Yan et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:8525-8529); enterpeptidase (also designated enterokinase; accession no. U09860 for the human protein; see, Kitamoto et al. (1995) Biochem. 27: 4562-4568; Yahagi et al. (1996) Biochem. Biophys. Res. Commun. 219:806-812; Kitamoto et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:75887592; Matsushima et al. (1994) J. Biol. Chem. 269:1997619982;); human airway trypsin-like protease (HAT; accession no.AB002134; seeYamaoka et al. J. Biol. Chem. 273:1189411901); MTSP1 and matriptase (also called TADG-15; see SEQ ID Nos. 1 and 2; accession nos. AF133086/AF118224, AF04280022; Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:11054-1161; Linetal. (1999) J. Biol. Chem. 274: 18231-18236; Takeuchi et al. (2000) J. Biol. Chem. 275: 26333-26342; and Kim etal. (1999) Immunogenetics 49:420429); hepsin (see, accession nos. Ml8930, AF030065, X70900; Leytus et al. (1988) Biochem. 27: 11895-11901; Vu et al. (1997) J. Biol. Chem. 272:31315-31320; and Farley et al. (1993) Biochem. Biophys. Acta 1173:350-352; and see, U.S. Pat. No. 5,972,616); TMPRS2 (see, Accession Nos. U75329 and AF113596; Paoloni-Giacobino et al. (1997) Genomics 44:309-320; and Jacquinet et al. (2000) FEBSLett. 468: 93-100); and TMPRSS4 (see, Accession No. NM 016425; Wallrapp et al. (2000) Cancer 60:2602-2606).
Serine proteases, including transmembrane serine proteases, have been implicated in processes involved in neoplastic development and progression. While the precise role of these proteases has not been elaborated, serine proteases and inhibitors thereof are involved in the control of many intraand extracellular physiological processes, including degradative actions in cancer cell invasion, metastatic spread, and neovascularization of tumors, that are involved in tumor progression. It is believed that proteases are involved in the degradation of extracellular matrix (ECM) and contribute to tissue remodeling, and are necessary for cancer invasion and metastasis. The activity and/or expression of some proteases have been shown to correlate with tumor progression and development.
For example, a membrane-type serine protease MTSP1 (also called matriptase; see SEQ ID Nos. 1 and 2 from U.S. Pat. No. 5,972,616; and GenBank Accession No. AF118224; (1999) J. Biol. Chem. 274:18231 -18236; U.S. Pat. No. 5,792, 616; see, also Takeuchi (1999) Proc. Natl. Acad. Sci. U.S.A. 96:11054-1161) that is expressed in epithelial cancer and normal tissue (Takeucuhi et al. (1999) Proc. Natl. Acad. Sci.
USA, 96(20): 11054-61) has been identified. Matriptase was originally identified in human breast cancer cells as a major gelatinase (see, U.S. Pat. No. 5,482,848), a type of matrix metalloprotease (MMP). It has been proposed that it plays a 5 role in the metastasis of breast cancer. Its primary cleavage specificity is Arg-Lys residues. Matriptase also is expressed in a variety of epithelial tissues with high levels of activity and/or expression in the human gastrointestinal tract and the prostate.
10 Prostate-specific antigen (PSA), a kallikrein-like serine protease, degrades extracellular matrix glycoproteins fibronectin and laminin, and, has been postulated to facilitate invasion by prostate cancer cells (Webber et al. (1995) Clin. Cancer Res., l(10):1089-94). Blocking PSA proteolytic
15 activity with PSA-specific monoclonal antibodies results in a dose-dependent decrease in vitro in the invasion of the reconstituted basement membrane Matrigel by LNCaP human prostate carcinoma cells which secrete high levels of PSA. Hepsin, a cell surface serine protease identified in
20 hepatoma cells, is overexpressed in ovarian cancer (Tanimoto et al. (1997) Cancer Res., 57(14):2884-7). The hepsin transcript appears to be abundant in carcinoma tissue and is almost never expressed in normal adult tissue, including normal ovary. It has been suggested that hepsin is frequently
25 overexpressed in ovarian tumors and therefore may be a candidate protease in the invasive process and growth capacity of ovarian tumor cells.
A serine protease-like gene, designated normal epithelial cell-specific 1 (NES1) (Liu etal., Cancer Res., 56(14):3371-9
30 (1996)) has been identified. Although expression of the NES1 mRNA is observed in all normal and immortalized nontumorigenic epithelial cell lines, the majority of human breast cancer cell lines show a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to
35 polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this protease-like gene product in the suppression of tumorigenesis.
Hence transmembrane serine proteases appear to be
40 involved in the etiology and pathogenesis of tumors. There is a need to further elucidate their role in these processes and to identify additional transmembrane proteases. Therefore, it is an object herein to provide transmembrane serine protease (MTSP) proteins and nucleic acids encoding such MTSP
45 proteases that are involved in the regulation of or participate in tumorigenesis and/or carcinogenesis. It is also an object herein to provide prognostic, diagnostic, therapeutic screening methods using the such proteases and the nucleic acids encoding such proteases.
SUMMARY OF THE INVENTION
Provided herein are isolated protease domains of the Transmembrane Serine Protease family, particularly the Type II
55 Transmembrane Serine Protease (TTSP) family (also referred to herein as MTSPs), and more particularly TTSP family members whose functional activity differs in tumor cells from non-tumor cells in the same tissue. For example, the MTSPs include those that are activated and/or expressed
60 in tumor cells at different levels, typically higher, from nontumor cells; and those from cells in which substrates therefor differ in tumor cells from non-tumor cells or otherwise alter the specificity of the MTSP.
The MTSP family as intended herein does not include any
65 membrane anchored or spanning proteases that are expressed on endothelial cells. Included among the MTSPs are several heretofore unidentified MTSP family members, designated
herein as MTSP3 and MTSP4 and a new form of a protein designated herein as MTSP6. In addition to the protease domains of each of MTSP3 and MTSP4, the full-length proteins, including those that results from splice variants, zymogens and activated forms, and uses thereof, are also 5 provided.
The protease domains as provided herein are single-chain polypeptides, with an N-terminus (such as IV, W, IL and II) generated at the cleavage site (generally having the consensus sequence RjWGG, RjlVGG, RjlVNG, RjlLGG, 10 RjVGLL, RjlLGG or a variation thereof; an N-terminus RJ, V or Rjl, where the arrow represents the cleavage point) when the zymogen is activated. To identify the protease domain an RI should be identified, and then the following amino acids compared to the above noted motif. 15
The protease domains generated herein, however, do not result from activation, which produces a two chain activated product, but rather are single chain polypeptides with the N-terminus include the consensus sequence jWGG, 1IVGG, jVGLL, jILGG or jIVNG or other such motif at 20 the N-terminus. As shown herein, such polypeptides, although not the result of activation and not double-chain forms, exhibit proteolytic (catalytic) activity. These protease domain polypeptides are used in assays to screen for agents that modulate the activity of the MTSP Such assays are also 25 provided herein. In exemplary assays, the affects of test compounds in the ability of a protease domains to proteolytically cleave a known substrate, typically a fluorescently, chromogenically or otherwise detectably labeled substrate, are assessed. Agents, generally compounds, particularly small 30 molecules, that modulate the activity of the protease domain are candidate compounds for modulating the activity of the MTSP. The protease domains can also be used to produce single-chain protease-specific antibodies. The protease domains provided herein include, but are not limited to, the 35 single chain region having an N-terminus at the cleavage site for activation of the zymogen, through the C-terminus, or C-terminal truncated portions thereof that exhibit proteolytic activity as a single-chain polypeptide in in vitro proteolysis assays, of any MTSP family member, preferably from a mam- 40 mal, including and most preferably human, that, for example, is expressed in tumor cells at different levels from non-tumor cells, and that is not expressed on an endothelial cell. These include, but are not limited to: MTSP1 (or matriptase), MTSP3, MTSP4 and MTSP6. Other MTSP protease domains 45 of interest herein, particularly for use in in vitro drug screening proteolytic assays, include, but are not limited to: corin (accession nos. AF133845 and AB013874; see, Yan et al. (1999) J. Biol. Chem. 274:14926-14938; Tomia et al. (1998) J. Biochem. 124:784-789; Yan et al. (2000) Proc. Natl. Acad. 50 Sci. U.S.A. 97:8525-8529; SEQ ID Nos. 61 and 62 for the human protein); enterpeptidase (also designated enterokinase; accession no. U09860 for the human protein; see, Kitamoto et al. (1995) Biochem. 27: 4562-4568; Yahagi et al. (1996) Biochem. Biophys. Res. Commun. 219:806-812; Kita- 55 moto et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:75887592; Matsushima et al. (1994) J. Biol. Chem. 269:1997619982; see SEQ ID Nos. 63 and 64 for the human protein); human airway trypsin-like protease (HAT; accession no. AB002134; see Yamaoka et al. J. Biol. Chem. 273:11894- 60 11901; SEQ ID Nos. 65 and 66 for the human protein); hepsin (see, accession nos. Ml 8930, AF030065, X70900; Leytus et al. (1988) Biochem. 27: 11895-11901; Vu et al. (1997) J. Biol. Chem. 272:31315-31320; and Farley et al. (1993) Biochem. Biophys. Acta 1173:350-352; SEQ ID Nos. 67 and 68 for the 65 human protein); TMPRS2 (see, Accession Nos. U75329 and AF113596; Paoloni-Giacobino et al. (1997) Genomics
44:309-320; and Jacquinet et al. (2000) FEBS Lett. 468: 93-100; SEQ ID Nos. 69 and 70 for the human protein) TMPRSS4 (see, Accession No. NM 016425; Wallrapp et al. (2000) Cancer 60:2602-2606; SEQ ID Nos. 71 and 72 forthe human protein); and TADG-12 (also designated MTSP6, see SEQ ID Nos. 11 and 12; see International PCT application No. WO 00/52044, which claims priority to U.S. application Ser.No. 09/261,416).
Also provided are muteins of the single chain protease domains and MTSPs, particularly muteins in which the Cys residue in the protease domain that is free (i.e., does not form disulfide linkages with any other Cys residue in the protein) is substituted with another amino acid substitution, preferably with a conservative amino acid substitution or a substition that does not eliminate the activity, and muteins in which a glycosylation site(s) is eliminated. Muteins in which other conservative amino acid substitutions in which catalytic activity is retained are also contemplated (see, e.g., Table 1, for exemplary amino acid substitutions). See, also, FIG. 4, which identifies the free Cys residues in MTSP3, MTSP4 and MTSP6.
Hence, provided herein are members of a family of transmembrane serine protease (MTSP) proteins, and functional domains, especially protease (or catalytic) domains thereof, muteins and other derivatives and analogs thereof. Also provided herein are nucleic acids encoding the MTSPs.
Exemplary MTSPs (see, e.g., SEQ ID No. 1-12,49 and 50) are provided herein, as are the single chain protease domains thereof as follows: SEQ ID Nos. 1, 2, 49 and 50 set forth amino acid and nucleic acid sequences of MTSP1 and the protease domain thereof; SEQ ID No. 3 sets forth the MTSP3 nucleic acid sequence and SEQ ID No. 4 the encoded MTSP3 amino acids; SEQ ID No. 5 MTSP4 a nucleic acid sequence of the protease domain and SEQ ID No. 6 the encoded MTSP4 amino acid protease domain; SEQ ID No. 7 MTSP4-L a nucleic acid sequence and SEQ ID No. 8 the encoded MTSP4-L amino acid sequence; SEQ ID No. 9 an MTSP4-S encoding nucleic acid sequence and SEQ ID No. 10 the encoded MTSP4-S amino acid sequence; and SEQ ID No. 11 an MTSP6 encoding nucleic acid sequence and SEQ ID No. 12 the encoded MTSP6 amino acid sequence. The single chain protease domains of each are delineated below.
Nucleic acid molecules that encode a single-chain protease domain or catalytically active portion thereof are provided. Also provided are nucleic acid molecules that hybridize to such MTSP encoding nucleic acid along their full length and encode the protease domain or portion thereof are provided. Hybridization is preferably effected under conditions of at least low, generally at least moderate, and often high stringency.
Additionally provided herein are antibodies that specifically bind to the MTSPs, cells, combinations, kits and articles of manufacture that contain the nucleic acid encoding the MTSP and/or the MTSP. Further provided herein are prognostic, diagnostic, therapeutic screening methods using MTSPs and the nucleic acids encoding MTSP. Also provided are transgenic non-human animals bearing inactivated genes encoding the MTSP and bearing the genes encoding the MTSP under non-native promotor control. Such animals are useful in animal models of tumor initation, growth and/or progression models.
Provided herein are members of a family of membrane serine proteases (MTSP) that are expressed in certain tumor or cancer cells such lung, prostate, colon and breast cancers. In particular, it is shown herein, that MTSPs, particularly, MTSP3, MTSP4 and MTSP6 are expressed in lung carcinoma, breast carcinoma, colon adenocarcinoma and/or ova7
rian carcinomas as well as in certain normal cells and tissues (see e.g., EXAMPLES for tissue-specific expression profiles of each protein exemplified herein). The MTSPs that are of particular interest herein, are those that are expressed in tumor cells, for example, those that appear to be expressed at 5 different levels in tumor cells from normal cells, or whose functional activity is different in tumor cells from normal cells, such as by an alteration in a substrate therefor, or a cofactor. Hence the MTSP provided herein can serve as diagnostic markers for certain tumors. The level of activated 10 MTSP3, MTSP4 and MTSP6 can be diagnostic of prostate cancer. In addition, MTSP4 is expressed and/or activated in lymphomas, leukemias, lung caner, breast, prostrate and colon cancers. MTSP6 is activated and/or expressed in breast, lung, prostate, colon and ovarian cancers. Furthermore, com- 15 pounds that modulate the activity of these MTSPs, as assessed by the assays provided herein, particularly the in vitro proteolytic assays that use the single chain protease domains, are potential therapeutic candidates for treatment of various malignancies and neoplastic disease. 20
Also provided herein are methods of modulating the activity of the MTSPs and screening for compounds that modulate, including inhibit, antagonize, agonize or otherwise alter the activity of the MTSPs. Of particular interest is the extracellular domain of these MTSPs that includes the proteolytic 25 (catalytic) portion of the protein.
MTSP proteins, including, but not limited to, MTSP3, MTSP4, and MTSP6, including splice variants thereof, and nucleic acids encoding MTSPs, and domains, derivatives and analogs thereof are provided herein. Single chain protease 30 domains, in the N-terminal is that which would be generated by activation of the zymogen, from any MTSP, particularly those that are not expressed in endothelial cells and that are expressed in tumor cells are also provided.
Antibodies that specifically bind to the MTSP, particularly 35 the single chain protease domain, and any and all forms of MTSP3 and MTSP4, and cells, combinations, kits and articles of manufacture containing the MTSP proteins, domains thereof, or encoding nucleic acids are also provided herein. Transgenic non-human animals bearing inactivated 40 genes encoding the MTSP and bearing the genes encoding the MTSP under a non-native promotor control are additionally provided herein. Also provided are nucleic acid molecules encoding each of the MTSPs and domains thereof.
Also provided are plasmids containing any of the nucleic 45 acid molecules provided herein. Cells containing the plasmids are also provided. Such cells include, but are not limited to, bacterial cells, yeast cells, fungal cells, plant cells, insect cells and animal cells.
Also provided is a method of producing a MTSP by grow- 50 ing the above-described cells under conditions whereby the MTSP is expressed by the cells, and recovering the expressed MTSP protein. Methods for isolating nucleic acid encoding other MTSPs are also provided.
Also provided are cells, preferably eukaryotic cells, such 55 as mammalian cells and yeast cells, in which the MTSP protein, preferably MTSP3 and MTSP4, is expressed in the surface of the cells. Such cells are used in drug screening assays to identify compounds that modulate the activity of the MTSP protein. These assays including in vitro binding 60 assays, and transcription based assays in which signal transduction mediated by the MTSP is assessed.
Further provided herein are prognostic, diagnostic and therapeutic screening methods using the MTSP and the nucleic acids encoding MTSP. In particular, the prognostic, 65 diagnostic and therapeutic screening methods are used for preventing, treating, or for finding agents useful in preventing
or treating, tumors or cancers such as lung carcinoma, colon adenocarcinoma and ovarian carcinoma.
Also provided are methods for screening for compounds that modulate the activity of any MTSP. The compounds are identified by contacting them with the MTSP and a substrate for the MTSP. A change in the amount of substrate cleaved in the presence of the compounds compared to that in the absence of the compound indicates that the compound modulates the activity of the MTSP. Such compounds are selected for further analyses or for use to modulate the activity of the MTSP, such as inhibitors or agonists. The compounds can also be identified by contacting the substrates with a cell that expresses the MTSP or the extracellular domain or proteolyrically active portion thereof. For assays in which the extracellular domain or a proteolytically active portion thereof is employed, the MTSP is any MTSP that is expressed on cells, other than endothelial cells, including, but not limited to MTSP1, MTSP3, MTSP4 and MTSP6.
Also provided herein are modulators of the activity of the MTSP, especially the modulators obtained according to the screening methods provide herein. Such modulators may have use in treating cancerous conditions, and other neoplastic conditions.
Pharmaceutical composition containing the protease domains of an MTSP protein, and the MTSP proteins, MTSP3, MTSP4 and MTSP6 are provided herein in a pharmaceutically acceptable carrier or excipient are provided herein.
Also provided are articles of manufacture that contain the MTSP proteins and protease domains of MTSPs in single chain form. The articles contain a) packaging material; b) the polypeptide (or encoding nucleic acid), particularly the single chain protease domain thereof; and c) a label indicating that the article is for using ins assay s for identifying modulators of the activities of an MTSP protein is provided herein.
Conjugates containing a) a MTSP protease domain in single chain from; and b) a targeting agent linked to the MTSP directly or via a linker, wherein the agent facilitates: i) affinity isolation or purification of the conjugate; ii) attachment of the conjugate to a surface; iii) detection of the conjugate; or iv) targeted delivery to a selected tissue or cell, is provided herein. The conjugate can contain a plurality of agents linked thereto. The conjugate can be a chemical conjugate; and it can be a fusion protein.
In yet another embodiment, the targeting agent is a protein or peptide fragment. The protein or peptide fragment can include a protein binding sequence, a nucleic acid binding sequence, a lipid binding sequence, a polysaccharide binding sequence, or a metal binding sequence.
Method of diagnosing a disease or disorder characterized by detecting an aberrant level of an MTSP, particularly an MTSP3, MTSP4 or MTSP 6, in a subject is provided. The method can be practiced by measuring the level of the DNA, RNA, protein or functional activity of the MTSP. An increase or decrease in the level of the DNA, RNA, protein or functional activity of the MTSP, relative to the level of the DNA, RNA, protein or functional activity found in an analogous sample not having the disease or disorder (or other suitable control) is indicative of the presence of the disease or disorder in the subject or other relative any other suitable control.
Combinations are provided herein. The combination can include: a) an inhibitor of the activity of an MTSP; and b) an anti-cancer treatment or agent. The MTSP inhibitor and the anti-cancer agent can be formulated in a single pharmaceutical composition or each is formulated in a separate pharmaceutical composition. The MTSP inhibitor can be an antibody or a fragment or binding portion thereof against the MTSP,