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WO1998011238A9 - Protease m, une nouvelle serine protease - Google Patents

Protease m, une nouvelle serine protease

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Publication number
WO1998011238A9
WO1998011238A9 PCT/US1997/016175 US9716175W WO9811238A9 WO 1998011238 A9 WO1998011238 A9 WO 1998011238A9 US 9716175 W US9716175 W US 9716175W WO 9811238 A9 WO9811238 A9 WO 9811238A9
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WIPO (PCT)
Prior art keywords
protease
protein
nucleic acid
cell
seq
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PCT/US1997/016175
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English (en)
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WO1998011238A2 (fr
WO1998011238A3 (fr
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Priority to AU44807/97A priority Critical patent/AU4480797A/en
Publication of WO1998011238A2 publication Critical patent/WO1998011238A2/fr
Publication of WO1998011238A3 publication Critical patent/WO1998011238A3/fr
Publication of WO1998011238A9 publication Critical patent/WO1998011238A9/fr

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Definitions

  • Tumor suppresser genes can be divided into two classes. Class I, in which a loss of function results from a mutation or deletion and class II, in which a loss of function results from a regulatory block to expression (Lee et al. 1991. Proc. Natl. Acad. Sci. 88:2825). Thus, both activation and loss of genes can lead to unregulated cell proliferation and to the accumulation of genetic errors which ultimately will result in the development of cancer (Pardee, Science 246:603-608, 1989).
  • Metastasis is a multistage process involving numerous aberrant functions of the tumor cell. These aberrant functions include tumor angiogenesis, attachment, adhesion to the vascular basement membrane, local proteolysis, degradation of extracellular matrix components, migration through the vasculature, invasion of the basement membrane, and proliferation at secondary sites (Poste, G. and Fidler, I.J. (1980) Nature 283:139-146; Liotta, L.A. et al. (1991) Cell 64:327-336).
  • Increased proteolytic potential is one documented feature of the metastatic phenotype. This increased potential is thought to result from the combined aberrant regulation of proteolytic enzymes (e.g., metalloproteinases and serine, cysteine and aspartyl proteinases) and their endogenous inhibitors (for a review, see e.g., Sloane, B.F. and Honn, K.V. (1984) Cancer Metastasis Rev. 3:249-263).
  • proteolytic enzymes e.g., metalloproteinases and serine, cysteine and aspartyl proteinases
  • endogenous inhibitors for a review, see e.g., Sloane, B.F. and Honn, K.V. (1984) Cancer Metastasis Rev. 3:249-263.
  • proteolytic enzymes e.g., metalloproteinases and serine, cysteine and aspartyl proteinases
  • endogenous inhibitors for a review, see e.g.
  • Serine proteases are protein cleaving enzymes, which contain a serine residue in their active sites, and which play important roles in diverse physiological processes, including digestion (e.g. trypsin, chymotrypsin) and blood clotting (e.g. plasminogen activator, thrombin) Serine proteases also act as regulators of a variety of processes by proteolytic activation of precursor proteins.
  • the kallikreins are a sub-family of serine proteases originally defined as cleaving vasoactive peptides (kinins) from kininogen ( Schachter M. (1980) Pharmacol. Rev. 31: 1 -1 7.).
  • kinins vasoactive peptides
  • Schachter M. (1980) Pharmacol. Rev. 31: 1 -1 7. cleaving vasoactive peptides
  • the kallikreins comprise a large, multi-gene family in rodents, although only three members of this family are known in humans. These genes clustered on chromosome 19ql3.2-ql3.4 (Reigman PH, et al.
  • Genomics 14:6-11 are hKLKl, hKLK2, and hKLK3 which encode the proteins hKl (pancreatic/renal kallikrein), hK2 (glandular kallikrein), and hK3 (prostate specific antigen) respectively (Berg T, et al. (1992) Agents Actions 38 (Suppl l):19-25).
  • the hKl protein is secreted from pancreas, kidney, and salivary glands (Fukushima D,et al. (1985) Biochemistry 24:8037-8043), and is the only member of the family having true kallikrein activity. Its major function is the generation of kinins from kininogens and the regulation of blood pressure (Schachter , supra).
  • the hK2 protein has yet to be detected in human tissue or fluids, but its sequence has been inferred from a genomic clone ( Schedlich LJ, et al. (1987) DNA 6:429-437) as well as cDNA clones isolated from prostate libraries ( Schedlich LJ, et al., (1987)DN4 6:429-437).
  • hK2 expression is specific for prostate and is regulated by androgens (Schedlich et al. supra). Determining the function for this protein and evaluating its usefulness as a marker for prostate cancer will have to await the identification and isolation of the protein.
  • the hK3 protein is PSA, the prostate specific antigen.
  • PSA is produced predominantly in males by prostate epithelial cells and secreted into the seminal fluid where it serves to degrade the gel-like seminogelin protein and increase sperm motility (Lilja H. ( 1985) J. Clin. Invest. 76:1899-1903; Lilja H, et al. (1987) J. Clin. Invest. 80:281 -285).
  • PSA is produced at higher levels in normal than in malignant prostate tissue, a defect in the malignant tissues ultimately results in the leakage of PSA into the bloodstream (McCormack RT, et al. (1995) Urology 45:729), forming the basis of the use of PSA as a marker for prostate cancer.
  • Serine proteases may accomplish matrix degradation during metastases by activating metalloproteases (Alexander and Werb. 1991. Extracellular Matrix Degradation. In Cell Biology of Extracellular Matrix. Ed by Hay, E.D. New York. Plenum Press. 1991 :255).
  • the principal serine proteases known implicated in matrix degradation mediate the plasminogen activation cascade. Included in this group are the urokinase plasminogen activator-receptor (uPA-uPAR) , leukocyte elastase, and tumor assocaited trypsin (Chen. 1992. Curr. Opin. Cell Biol. 4:802). Both uPA and tPA can activate serum protein plasminogen , yielding the broad-specificity protease plasmin by cleavage of one bond. Plasmin participates in fibrinolysis, tissue remodeling and tumor invasion (Chen, supra).
  • proteases While proteases have been thought to promote tissue invasion and metastases, the development of metastatic potential appears to be more complicated. For example, overexpression of the protease inhibitors PAI-1 and PAI-2, which negatively regulate plasminogen activator, has also been found to be assocaited with certain types of cancers (Sumiyoshi et al. 1991 Thromb. Res., 63:59; Reilly et al. 1990. Biochem. Soc. Transact. 18:354). Janicke et al.
  • PAI-1 secretion by tumor cells may enhance cell migration by upsetting the protease- antiprotease equilibrium near the cell surface of a tumor cell, perhaps via a mechanism involving urokinase plasminogen activator receptor clearance (Janicke et al. 1994. Cancer Res. 54:2527).
  • Protease M a novel member of the serine protease family, referred to as Protease M.
  • a partial Protease M cDNA was originally identified by its differential expression in a primary ductal breast carcinoma and its reduced expression in a pleural metastasis from the same patient using the differential display method. Subsequently, a full-length cDNA of 1 ,526 nucleotides was isolated from a normal breast epithelial cell cDNA library and was sequenced. Expression studies indicate that expression of the Protease M gene is downregulated in metastatic breast cancer cell lines and is upregulated in primary breast cancer cell lines and ovarian cancer tissues and tumor cell lines.
  • this invention pertains to isolated nucleic acid molecules comprising a nucleotide sequence encoding a Protease M protein or a biologically active portion thereof.
  • the invention features an isolated nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1.
  • an isolated nucleic acid molecule of the present invention is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
  • a nucleic acid molecule of the present invention comprises the coding region of the nucleotide sequence of SEQ ID NO: 1.
  • the invention provides for isolated nucleic acid molecules which encode proteins containing amino acid sequences which are homologous to the sequence shown in SEQ ID NO:2.
  • protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2.
  • the protein is at least about 70 %, preferably at least 80 % homologous, or more preferebly at least 90 % homologous to the amino acid sequence of SEQ ID NO: 2.
  • an isolated nucleic acid molecule of the invention encodes the amino acid sequence of SEQ ID NO: 2.
  • an isolated nucleic acid molecule encodes a Protease M fusion protein.
  • an isolated nucleic acid molecule of the invention is antisense to the nucleic acid molecule of claim 1.
  • an isolated nucleic acid is antisense to a coding region of the coding strand of the nucleotide sequence of SEQ ID NO: 1.
  • an isolated nucleic acid molecule of the invention is antisense to a noncoding region of the nucleotide sequence of SEQ ID NO: 1.
  • an isolated nucleic acid molecule which encodes a Proteinase M polypeptide is isoloated using at least a portion of the nucleotide sequence of SEQ ID NO:l as a probe or a primer.
  • vectors e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention.
  • Such vectors can encode a protein comprising the amino acid sequence of SEQ ID NO: 2.
  • a vector is provided which comprises the coding region of the nucleotide sequence of SEQ ID NO: 1.
  • such a host cell is used to produce Protease M protein by culturing the host cell in a suitable medium. If desired, Protease M protein can be then isolated from the medium or the host cell.
  • Still another aspect of the invention pertains to isolated Protease M protein.
  • an isolated Protease M protein is encoded by the nucleic acid shown in SEQ ID No:l .
  • the Protease M protein is a mature polypeptide which comprises amino acids 17-244 of SEQ ID NO: 2 or amino acids 22-244.
  • the isolated Protease M protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 and possesses a Protease M bioactivity in vitro.
  • the protein is at least 70 %, preferably at least 80 %, even more preferably at least 90% .
  • a Protease M protein of the present invention is at least about 95 % homologous to the amino acid sequence of SEQ ID NO: 2.
  • a Protease M protein of the invention can be incorporated into a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.
  • the invention provides a fusion protein comprising a Protease M polypeptide operatively linked to a non-Protease M polypeptide.
  • the Protease M proteins of the invention can be used to prepare anti-Protease M antibodies.
  • the invention provides an antigenic peptide of Protease M comprising at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and encompassing an epitope of Protease M such that an antibody raised against the peptide forms a specific immune complex with Protease M.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • the invention further provides an antibody that specifically binds Protease M.
  • the antibody is monoclonal.
  • the antibody is coupled to a detectable label.
  • the antibody is incorporated into a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.
  • Yet another aspect of the invention pertains to transgenic non-human animals in which a Protease M gene has been introduced or altered.
  • the genome of the nonhuman animal has been altered by introduction of a nucleic acid molecule of the invention encoding Protease M as a transgene.
  • an endogenous Protease M gene within the genome of the nonhuman animal has been altered, e.g., functionally disrupted, by homologous recombination.
  • Another aspect of the invention pertains to methods for detecting the presence or absence of Protease M in a biological sample.
  • the method involves contacting a biological sample (e.g., a tissue sample) with an agent capable of detecting Protease M protein or nucleic acid such that the presence of Protease M is detected in the biological sample.
  • the agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to Protease M mRNA or a labeled or labelable antibody capable of binding to a Protease M protein.
  • the invention further provides methods for detecting carcinomas or for staging a carcinoma based on detecting the presence, or absence, or amount of Protease M protein or nucleic acid in a test sample relative to a control sample.
  • the method involves contacting a cell or other sample from a subject with an agent capable of detecting Protease M protein or nucleic acid, determining the amount of Protease M protein or nucleic expressed in the sample, comparing the amount of Protease M protein or nucleic acid expressed in the sample to a control and forming a diagnosis and/or prognosis based on the amount of Protease M protein or nucleic acid expressed in the test sample as compared to the control sample.
  • the sample is mammary or ovarian tissue.
  • one such diagnostic method involves contacting the mRNA of a test cell with a nucleic acid probe containing a sequence antisense to (i.e. complementary to the sense strand of) a segment of the nucleic acid sequence shown in SEQ ID No: 1.
  • Kits for detecting Protease M in a biological sample are also within the scope of the invention.
  • the Protease M protein of the invention can be used therapeutically.
  • the present invention can be used to modulate the Protease M bioactivity associated with a cell (e.g., in the cell, secreted by the cell or in the extracellular milieu surrounding the cell).
  • the invention provides a method for modulating the Protease M serine protease activity associated with a cell by contacting the cell with an agent that modulates Protease M serine protease activity.
  • an agent can be, for example, a Protease M protein agonist or antagonist or a nucleic acid encoding a Protease M agonist or antagonist that has been introduced into the cell.
  • Protease M activity is stimulated in tumor cells, such as metastatic mammary tumor cells, in which endogenous Protease M expression is low or absent.
  • the invention provides a method for inhibiting the Protease M activity associated with a cell by contacting the cell with an agent that inhibits Protease M serine protease activity.
  • an agent can be, for example, an antisense Protease M nucleic acid molecule or an anti- Protease M antibody, or Protease M antagonist, or inhibitor.
  • the methods of the invention for modulating Protease M activity can be applied in vitro (e.g., to cells in culture) or in vivo, wherein an agent that modulates Protease M serine protease activity is administered to the subject.
  • the invention provides a method for inhibiting development or progression of cancer in a cell comprising contacting a cell with an agent which modulates the amount of or activity of Protease M in or around the tumor cell.
  • Drug screening methods for identifying modulators of Protease M expression or Protease M serine protease activity are also encompassed by the invention.
  • the modulator stimulates Protease M expression or activity, i.e., is an agonist or potentiator.
  • the modulator inhibits Protease M expression or activity, i.e., is an antagonist or inhibitor.
  • Figure 1 shows the identification of Protease M (1G3) by Differential Display (DD) gel and northern blot
  • DD gel 21 PT and 21 MT-1 RNA was reverse transcribed with and OPA1 primers in the presence of 35sdATP, mn on a ⁇ % acrylamide sequencing gel, and exposed to x-ray film for 18 hours. The portion of the gel surrounding the differentially displayed 0.28kb band is shown.
  • B. Northern Blot: lOmg of total cell RNA was northern blotted and probed with 32 P-labeled PCR-amplified 0.28kb band from the DD gel shown in (A).
  • Figure 2 shows Protease M cDNA.
  • the cDNA sequence and putative protein coding sequence of the longest clone from the 76N library is shown.
  • the postulated pre-pro N- terminal amino acids are underlined.
  • the predicted cleavage sites of pre and pro amino acids after ala ⁇ " and lys 21 respectively are indicated by arrows.
  • the potential n-linked glycosylation site at amino acids 134-136 and asp ⁇ l a t the bottom of the binding cleft are boxed.
  • the residues of the catalytic triad his" 2 , asp ⁇ ", and ser ⁇ ) are circled.
  • the actual polyadenylation signal at nucleotide 1 ,490 and an alternative polyadenylation signal at nucleotide 1 ,095 are underlined.
  • Figure 3 shows an alignment of Protease M with closely related members of the serine protease family.
  • the GCG pileup and pretty plot programs were used to align Protease M with closely related human serine proteases: They are from top to bottom: glandular kallikrein-hk2 (accession number SP
  • Figure 4 shows protease M mRNA expression in mammary and prostate cell lines (A.) lOmg of total mammary cell RNA was run on an agarose/ formaldehyde gel, blotted and hybridized to 32 P-labeled Protease M probe and exposed to x-ray film for 20 hours (B). lOmg of total prostate cell RNA was blotted and hybridized (as in A) and exposed to x-ray film for 20 hours.
  • Figure 5 shows Protease M mRNA expression in ovarian tissue. lOmg of total cell RNA isolated from ovarian tissue was blotted and hybridized to
  • Protease M probe (as in Figure 4) and exposed to x-ray film for 5 days.
  • Figure 6 shows Protease M mRNA expression in human tissue.
  • Figure 7 shows the expression of Protease M protein in mammary cell lines and insect cells infected with recombinant Protease M .
  • 50mg of total cell lysate from mammary cell lines, uninfected insect cells (SF9) or insect cells infected with 4.5ml recombinant Protease M baculovirus (SF9/1G3(1)) or 22.5ml recombinant baculovirus (SF9/Protease M(2)) was run on a 12% polyacryamide/SDS gel, transferred to a PDVF membrane, and reacted with Protease M polyclonal anti-peptide antibody as the primary antibody and horseradish peroxidase conjugated anti rabbit IgG secondary antibody. Bands were detected with ECL detection system.
  • Protease M was isolated by differential display (Liang L and Pardee AB.
  • Protease M is a novel member of the serine protease family which is most homologous to trypsin and members of the kallikrein family. Protease M is downregulated in metastatic breast cancer lines, but strongly expressed at the mRNA level in some primary breast cancer cell lines and in ovarian cancer tissues and tumor cell lines.
  • Protease M was originally identified as being differentially expressed in a primary ductal breast carcinoma (21PT) as compared to a pleural metastasis (21MT-1) derived from the same patient.
  • a full-length cDNA was subsequently isolated using the partial cDNA as a hybridization probe to screen a cDNA library prepared from a normal breast epithelial cell (76N).
  • the nucleotide sequence of the isolated human Protease M cDNA, and the predicted amino acid sequence of the human Protease M protein, are shown in SEQ ID NOs: 1 and 2, respectively.
  • the full length cDNA clone isolated is 1526 nucleotides in length and comprises 2456 base pairs of 5' nontranslated sequence, 732 base pairs of coding sequence, and 549 base pairs of 3' nontranslated sequence.
  • the predicted Protease M protein is 244 amino acids.
  • the NH2 terminus comprises 13 consecutive hydrophobic amino acids (leu4-alal°) ; wn ich is a predicted signal sequence.
  • the residues glu ⁇ -glu ⁇ - glul9- a sn 0-lys 2 l resemble a pro-polypeptide with a potential trypsin cleavage site after lys 2 * .
  • Glandular kallikrein 2 ( Schedlich LJ, et al. (1987) DNA 6:429-437; Riegman PH, et al. (1991) Mol. Cell Endocrinol. 76:181-190) has 44% exact matches and 48% match with conservative changes. Trypsin I ( Emi M, et al. (1986) Gene 41 :305-310) has 43% exact matches and 49% match with conservative changes. Both glandular kallikrein 1 (Fukushima D, et al. (1985) Biochemistry 24:8037-8043., Baker A, Shine J.
  • Structural features important for serine protease activity such as the catalytic triad (his6 2 asn!06 ser 197) 5 C y S t e ine bridges (Cys ⁇ -Cys ⁇ ; Cys ⁇ - ys ⁇ 3 ; C ys 138_ Cys 203. Cys 168 -Cys 182 ; and Cys 193 -Cys 218 ), and residues lining the binding cleft are almost perfectly conserved between Protease M and other members of the kallikrein family.
  • the Asp residue at position 191 predicts that Protease M has a trypsin-like cleavage pattern.
  • Protease M and trypsin lack the kallikrein loop at amino acid residues 109-119, which is important for kallikrein specificity.
  • Protease M mRNA has a distinct expression pattern that distinguishes it from other serine proteases.
  • a 1.7-1.8 kb message was found to be normal brain, kidney, and pancreas tissue, but not in heart, placenta, lung, liver, or skeletal muscle. The message detected in the pancreas was only about 1.2 kb.
  • Expression studies further indicate that expression of the Protease M gene is downregulated in metastatic breast cancer cell lines and is upregulated in primary breast cancer cell lines and ovarian cancer tissues and tumor cell lines.
  • the Protease M gene was localized by FISH analysis to chromosome 19ql3.4.
  • the three kallikrein genes also map to chromosome 19ql3.2-ql3.4, while trypsinogen 1 maps to chromosome 7.
  • the size of the detected Protease M protein is approximately 36 kD rather than the predicted size of 27 kD. This size discrepancy could be accounted for by glycosylation at asn* 3
  • the expression of Protease M is regulated both at the transcriptional and translational level.
  • certain aspects of the present invention relate to nucleic acids encoding Protease M proteins, the Protease M proteins themselves, antibodies immunoreactive with Protease M proteins, and preparations of such compositions.
  • the present invention provides diagnostic/prognostic assays and therapeutic reagents for detecting and treating disorders involving, for example, aberrant expression of Protease M or Protease M homologs.
  • drug discovery assays are provided for identifying agents which can modulate the biological function of Protease M proteins, such as by altering the binding of Protease M molecules to proteins, including substrates. Such agents can be useful therapeutically to alter the growth and/or differentiation of a cell.
  • Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.
  • polypeptides referred to herein as having an activity of a Protease M protein are defined as polypeptides which include an amino acid sequence corresponding (e.g., identical or homologous) to all or a portion of the amino acid sequences of a Protease M protein shown in SEQ ID No:2 and which mimic or antagonize all or a portion of the biological/biochemical activities of a naturally occurring Protease M protein.
  • biological activity include serine protease activity and/or the ability to compete with a bioactivity of a naturally occurring Protease M.
  • a Protease M molecule of the present invention is capable of modulating the proliferation or metastasis of a cell, either in vitro or in vivo.
  • a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally-occurring form of a Protease M protein.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding one of the subject Protease M polypeptides with a second amino acid sequence defining a domain (e.g. polypeptide portion) foreign to and not substantially homologous with any domain of one of the Protease M proteins.
  • a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • a fusion protein can be represented by the general formula X-Protease M-Y, wherein
  • Protease M represents a portion of the protein which is derived from a Protease M protein, and X and Y are, independently, absent or represent amino acid sequences which are not related to a Protease M sequence in an organism.
  • genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual.
  • Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
  • DNA sequence encoding a Protease M polypeptide may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individuals of the same species, which are called alleles.
  • allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • gene refers to a nucleic acid comprising an open reading frame encoding a Protease M polypeptide of the present invention, including both exon and (optionally) intron sequences.
  • recombinant gene refers to nucleic acid encoding a Protease M polypeptide and comprising Protease M-encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal Protease M gene or from an unrelated chromosomal gene.
  • An exemplary recombinant gene encoding the subject Protease M polypeptide is represented in the appended Sequence Listing.
  • the term "intron” refers to a DNA sequence present in a given Protease M gene which is not translated into protein and is generally found between exons.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40 percent identity, though preferably less than 25 percent identity, with one of the Protease M sequences of the present invention.
  • Protease M polypeptide which function in a limited capacity as one of either a Protease M agonist (mimetic) or a Protease M antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein.
  • a Protease M agonist mimetic
  • a Protease M antagonist e.g., a Protease M antagonist
  • Homologs of each of the subject Protease M protein can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the Protease M polypeptide from which it was derived.
  • antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to a Protease M substrate or other Protease M associated protein.
  • agonistic forms of the protein may be generated which are constitutively active, or have an altered K cat or K m for protease reactions.
  • the Protease M protein and homologs thereof provided by the subject invention may be either positive or negative regulators of protease activity.
  • isolated as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding a subject Protease M polypeptide preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the Protease M gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • modulation is meant to refer to the upregulation or downregulation of a response.
  • an "Protease M-related" protein refers to the Protease M proteins described herein, and other human homologs of those Protease M sequences, as well as orthologs and paralogs (homologs) of the Protease M proteins in other species.
  • the term “ortholog” refers to genes or proteins which are homologs via speciation, e.g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species.
  • paralog refers to genes or proteins which are homologs via gene duplication, e.g., duplicated variants of a gene within a genome. See also, Fritch, WM (1970) Syst Zool 19:99-113.
  • non-human animals refer to rodents, non- human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • Preferred non- human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • transgenic amphibians such as members of the Xenopus genus
  • transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal.
  • tissue-specific chimeric animal indicates that one of the recombinant Protease M genes is present and/or expressed or disrupted in some tissues but not others.
  • one aspect of the invention pertains to isolated nucleic acids comprising nucleotide sequences encoding Protease M polypeptides, and/or equivalents of such nucleic acids.
  • nucleic acid refers to DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA).
  • Nucleic acids may be double stranded or single stranded and the term is meant to include a nucleic acid which is complementary (i.e., can specifically hybridize to) a nucleic acid of the present invention (e.g., an antisense molecule).
  • nucleic acid as used herein is intended to include fragments as equivalents.
  • equivalent is understood to include nucleotide sequences encoding functionally equivalent Protease M polypeptides or functionally equivalent peptides having a bioactivity of a Protease M protein such as described herein.
  • Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the Protease M cDNA sequences shown in SEQ ID No:l due to the degeneracy of the genetic code. Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27°C below the melting temperature (T m ) of the DNA duplex formed in about 1M salt) to the nucleotide sequence represented in SEQ ID No:l . In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequence shown in SEQ ID No: 1.
  • the term “specifically hybridizes” refers to the ability of the probe/primer of the invention to hybridize to at least 15 consecutive nucleotides of a Protease M gene, such as a Protease M sequence designated in SEQ ID No: 1, or a sequence complementary thereto, or naturally occurring mutants thereof, such that it has less than 15%, preferably less than 10%, and more preferably less than 5% background hybridization to a cellular nucleic acid (e.g., mRNA or genomic DNA) encoding a protein other than a Protease M protein, as defined herein.
  • a cellular nucleic acid e.g., mRNA or genomic DNA
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of hepatic, pancreatic, neuronal or hematopoietic origin.
  • tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of hepatic, pancreatic, neuronal or hematopoietic origin.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but can cause at least low level expression in other tissues as well.
  • the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • "Transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a Protease M polypeptide or, where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the Protease M protein is disrupted.
  • a "transgenic animal” is any animal, preferably a non- human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • transgenic animal In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of a Protease M protein.
  • transgenic animals in which the recombinant Protease M gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more Protease M genes is caused by human intervention, including both recombination and antisense techniques.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of a recombinant Protease M gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell- type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of naturally- occurring forms of Protease M genes.
  • transgene means a nucleic acid sequence (encoding a Protease M polypeptide, or an antisense transcript thereto), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or. is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode Protease M or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify Protease M-encoding nucleic acid.
  • a Protease M nucleic acid or a portion thereof can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a human Protease M cDNA can be isolated from a cell line, (e.g., a normal mammary epithelial cell line) or from a cDNA library, using all or portion of SEQ ID NO: 1 as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1.
  • mRNA can be isolated from normal mammary epithelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO: 1.
  • primers suitable for amplification of a Protease M nucleic acid are provided in the appended Examples.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to Protease M nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.
  • the sequence of SEQ ID NO: 1 corresponds to the human Protease M cDNA.
  • This cDNA comprises sequences encoding the Protease M protein (i.e., "the coding region", from nucleotides 246 to 977), as well as 5' untranslated sequences (nucleotides 1 to 245) and 3' untranslated sequences (nucleotides 978 to 1526).
  • the nucleic acid molecule may comprise only the coding region of SEQ ID NO: 1 (e.g., nucleotides 246 to 977), for example a fragment encoding a biologically active portion of Protease M.
  • the Protease M nucleic acid of the present invention encodes the polypeptide shown in SEQ ID No:2.
  • a Protease M nucleic acid encodes a biologically active portion of Protease M.
  • a Protease M nucleic acid encodes a mature form of Protease M in which a hydrophobic, amino-terminal signal sequence (encompassing approximately amino acids 1-16) is absent.
  • a mature form of Protease M preferably comprises about amino acid residues 22 to 244 (i.e., Protease M which has been cleaved at a trypsin site).
  • a nucleic acid of the present invention encodes a protein in which amino acid residue 22 is the N-terminal residue of the mature protein
  • more than one native isoform differing in the length of the N-terminal sequence may exist for Protease M. Consequently, the skilled artisan will appreciate that some flexibility exists in the N-terminus of the mature form of Protease M lacking a signal sequence.
  • Additional nucleic acid fragments encoding biologically active portions of Protease M can be prepared by isolating a portion of SEQ ID NO: 1 , expressing the encoded portion of Protease M protein or peptide (e.g., by recombinant expression in vitro) and assessing the bioactivity of the encoded portion of Protease M protein or peptide.
  • an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60 % homologous to each other typically remain hybridized to each other.
  • the conditions are such that at least sequences at least 65 %, more preferably at least 70 %, and even more preferably at least 75 % homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the nucleic acid encodes a natural human Protease M.
  • the nucleic acid molecule encodes a murine homologue of human Protease M.
  • a Protease M nucleic acid of the present invention comprises the sequence shown in SEQ ID NO: 1 or a fragment thereof. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of Protease M may exist within a population (e.g., the human population). Such genetic polymorphism in the Protease M gene may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1 -5 % variance in the nucleotide sequence of a gene.
  • nucleic acid molecules encoding Protease M proteins from other species and thus which have a nucleotide sequence which differs from the human sequence of SEQ ID NO: 1, are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and nonhuman homologues of the human Protease M cDNA of the invention can be isolated based on their homology to the human Protease M nucleic acid disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • nucleic acid molecules encoding Protease M proteins that contain changes in amino acid residues that are not essential for Protease M activity , e.g., residues that are not conserved or only semi-conserved among members of the chymotrypsin family of serine proteases.
  • Protease M proteins differ in amino acid sequence from SEQ ID NO: 2 yet retain Protease M bioactivity.
  • the invention further encompasses nucleic acid molecules that differ from SEQ ID NO:l (and portions thereof) due to degeneracy of the genetic code.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 and exhibits serine protease activity in vitro.
  • the protein encoded by the nucleic acid molecule is at least 70 % homologous to SEQ ID NO: 2, more preferably at least 80 % homologous to SEQ ID NO: 2, even more preferably at least 90 % homologous to SEQ ID NO: 2.
  • a Protease M nucleic acid of the present invention is at least about 95 % homologous to SEQ ID NO: 2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein).
  • the amino acid residues at corresponding amino acid positions are then compared.
  • a position in one sequence e.g., SEQ ID NO: 2
  • amino acid residues are then compared.
  • a position in one sequence e.g., SEQ ID NO: 2
  • a mutant form of Protease M occupied by the same amino acid residue as the corresponding position in the other sequence
  • the molecules are homologous at that position (i.e., as used herein amino acid "homology" is equivalent to amino acid "identity").
  • An isolated nucleic acid molecule encoding a Protease M protein homologous to the protein of SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein, as detailed below.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire Protease M coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding Protease M.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the entire coding region of SEQ ID NO: 1 comprises nucleotides 246 to 977).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding Protease M.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • an oligonucleotide which is antisense to only a portion of the coding or noncoding region of Protease M mRNA.
  • the antisense oligonucleotide may be complementary to the region surrounding the translation start site of Protease M mRNA.
  • An antisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • a ribozyme having specificity for a Protease M-encoding nucleic acid can be designed based upon the nucleotide sequence of a Protease M cDNA disclosed herein (i.e., SEQ ID NO: 1).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a Protease M-encoding mRNA.
  • Protease M mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Barrel, D. and Szostak, J.W. (1993) Science 261 : 1411-1418.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., Protease M proteins, mutant forms of Protease M, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of Protease M in prokaryotic or eukaryotic cells.
  • Protease M can be expressed in bacterial cells such as E. coli or insect cells (using baculovirus expression vectors) as described in detain in the appended Examples.
  • Other possible host cells include yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • the coding sequence of the mature form of Protease M (i.e., encompassing amino acids 22-244) is cloned into a pGEX-2t expression vector to create a vector encoding a fusion protein which was solubilized from bacteria and purified on glutathionine agarose beads by standard methods (Smith DB, and Johnson. 1988.
  • E. coli expression vectors examples include pTrc (Amann et ah, (1988) Gene 69:301-315) and pET 1 Id (Studier et a , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 1 19-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the Protease M expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Protease M can be expressed in insect cells using baculovirus expression vectors as described herein.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to Protease M mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to recombinant host cells into which a recombinant expression vector of the invention has been introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell may be any prokaryotic or eukaryotic cell.
  • Protease M protein may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding protease M or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) Protease M protein.
  • the invention further provides methods for producing Protease M protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding Protease M has been introduced) in a suitable medium until Protease M is produced.
  • the method further comprises isolating Protease M from the medium or the host cell.
  • Another aspect of the invention pertains to isolated Protease M proteins, and biologically active portions thereof, as well as peptide fragments suitable as immunogens to raise anti-Protease M antibodies.
  • the invention provides an isolated preparation of Protease M, or a biologically active portion thereof.
  • An "isolated" protein is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the Protease M protein has an amino acid sequence shown in SEQ ID NO: 2.
  • the Protease M protein is substantially homologous to SEQ ID NO: 2 and retains the functional activity of the protein of SEQ ID NO: 2 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the Protease M protein is a protein which comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 and possesses a Protease M bioactivity in vitro.
  • the protein is at least 70 % homologous to SEQ ID NO: 2, more preferably at least 80 % homologous to SEQ ID NO: 2, even more preferably at least 90 % homologous to SEQ ID NO: 2.
  • a Protease M polypeptide is at least about 95 % homologous to SEQ ID NO: 2.
  • An isolated Protease M protein may comprise the entire amino acid sequence of SEQ ID NO: 2 (i.e., amino acids 1-244) or a biologically active portion thereof.
  • a biologically active portion of Protease M can comprise a mature form of Protease M in which a hydrophobic, amino-terminal signal sequence is absent, or which has been cleaved at a trypsin site.
  • such a mature form of Protease M comprises about amino acids 17-244 of SEQ ID NO: 2 and in another embodiment comprises about amino acids 22-244.
  • the term "about" amino acids 17- 244 or 22-244 is intended to indicate that there is some flexibility in the amino-terminal residue, as discussed further in subsection I above.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for serine protease activity as described in detail above.
  • Protease M proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the Protease M protein is expressed in the host cell. The Protease M protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a Protease M protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native Protease M protein can be isolated from cells (e.g., cultured human mammary epithelial cells), for example using an anti-Protease M antibody (discussed further below).
  • a Protease M protein is encoded by a nucleic acid of SEQ ID No: 1.
  • a Protease M protein is encoded by a nucleic acid at least about 60%, preferably about 70%, or more preferably about 80% homologous to the nucleic acid of SEQ ID No: 1.
  • a Protease M protein is encoded by a nucleic acid at least about 90% and preferably about 95% homologous to the nucleic acid of SEQ ID No: 1.
  • a Protease M polypeptide is encoded by a nucleic acid which hybridizes to the nucleic acid of SEQ ID No:l under stringent conditions.
  • a Protease M "fusion protein” comprises a Protease M polypeptide operatively linked to a non-Protease M polypeptide.
  • An "Protease M polypeptide” refers to a polypeptide having an amino acid sequence corresponding to Protease M
  • a “non-Protease M polypeptide” refers to a polypeptide having an amino acid sequence corresponding to another protein.
  • the term "operatively linked” is intended to indicate that the Protease M polypeptide and the non-Protease M polypeptide are fused in-frame to each other.
  • the non-Protease M polypeptide may be fused to the N-terminus or C-terminus of the Protease M polypeptide.
  • the fusion protein is a GST-Protease M fusion protein in which the Protease M sequences are fused to the C-terminus of the GST sequences (see Example 3).
  • Such fusion proteins can facilitate the purification of recombinant Protease M.
  • the fusion protein is a Protease M protein containing a heterologous signal sequence at its N-terminus.
  • the native Protease M signal sequence i.e., about amino acids 1-16
  • a Protease M fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a Protease M- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the Protease M protein.
  • an isolated Protease M protein, or fragment thereof, can be used as an immunogen to generate antibodies that bind Protease M using standard techniques for polyclonal and monoclonal antibody preparation.
  • the Protease M immunogen comprises an epitope unique to Protease M.
  • the full-length Protease M protein can be used or, alternatively, the invention provides antigenic peptide fragments of Protease M for use as immunogens.
  • the antigenic peptide of Protease M comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and encompasses an epitope of Protease M such that an antibody raised against the peptide forms a specific immune complex with Protease M.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of Protease M that are located on the surface of the protein, e.g., hydrophilic regions. Exemplary immunogens are described in more detail in the appended examples.
  • a Protease M immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed Protease M protein or a chemically synthesized Protease M peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic Protease M preparation induces a polyclonal anti-Protease M antibody response.
  • Modification of the structure of the subject Protease M polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications.
  • Such modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the Protease M polypeptides described in more detail herein.
  • Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
  • Protease M homolog (e.g. functional in the sense that the resulting polypeptide mimics or antagonizes the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
  • Amino acid residues of Protease M that are strongly conserved among members of the chymotrypsin family of serine proteases are predicted to be essential to the bioactivity of Protease M and thus are not likely to be amenable to alteration.
  • the catalytic residues of a serine protease are Ser ⁇ , His ⁇ 7 and Asp ⁇ 2 (chymotrypsin numbering system). These three residues form a hydrogen bonding system often referred to as the catalytic triad, or the charge relay system (Powers and Harper, supra).
  • the catalytic triad of serine proteases is conserved in Protease M (i.e.
  • Protease M contains twelve cysteine residues. Ten of these are conserved in the two kallikreins, PSA and human trypsin and would be expected to form the following disulfide bridges: (Cys 28 -Cys 157 ). (Cys 47 -Cys 63 ), (Cys 138 -Cys 203 ), (Cys 168 -Cys 182 ), and (Cys 193 -Cys 21 8 ).
  • the other two cysteines (Cys 131 and Cys 23 1) are not found in the kallikreins, PSA and human trypsin, but are found in similar positions in bovine trypsin and would be expected to form a disulfide bond.
  • This invention further contemplates a method for generating sets of combinatorial mutants of the subject Protease M proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that have a Protease M activity.
  • the purpose of screening such combinatorial libraries is to generate, for example, novel Protease M homologs which can act as either agonists or antagonists, or alternatively, possess novel activities all together.
  • Protease M homologs can be engineered by the present method to provide selective, constitutive activation of enzymatic activity.
  • combinatorially-derived homologs can be generated to have an increased potency relative to a naturally occurring form of the protein.
  • Protease M homologs can be generated by the present combinatorial approach to selectively inhibit (antagonize) a Protease M activity.
  • mutagenesis can provide Protease M homologs which are able to prevent serine protease activity, e.g. the homologs can be dominant negative mutants.
  • a dominant negative mutant of a Protease M protein is mutated at one or more residues of its catalytic site and/or specificity subsites.
  • the amino acid sequences for a population of Protease M homologs or other related proteins are aligned, preferably to promote the highest homology possible.
  • a population of variants can include, for example, Protease M homologs from one or more species.
  • Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
  • the variegated library of Protease M variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential Protease M sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of Protease M sequences therein.
  • a library of coding sequence fragments can be provided for a Protease M clone in order to generate a variegated population of Protease M fragments for screening and subsequent selection of bioactive fragments.
  • a variety of techniques are known in the art for generating such libraries, including chemical synthesis.
  • a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of a Protease M coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of Protease M homologs.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • the library of Protease M variants is expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages Ml 3, fd., and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:125-134; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
  • RPAS recombinant phage antibody system
  • Catalog number 27-9400-01 can be easily modified for use in expressing and screening Protease M combinatorial libraries by panning on glutathione immobilized substrate/GST fusion proteins to enrich for Protease M homologs which retain an ability to bind a substrate or regulatory protein.
  • Each of these Protease M homologs can subsequently be screened for further biological activities in order to differentiate agonists and antagonists.
  • homologs isolated from the combinatorial library can be tested for their enzymatic activity directly, or for their effect on cellular proliferation relative to the wild-type form of the protein.
  • the invention also provides for reduction of the Protease M proteins to generate mimetics, e.g. peptide or non-peptide agents, which are able to disrupt a biological activity of a Protease M polypeptide of the present invention, e.g. as catalytic inhibitor or an inhibitor of protein-protein interactions.
  • mimetics e.g. peptide or non-peptide agents
  • Such mutagenic techniques as described above are also useful to map the determinants of the Protease M proteins which participate in protein-protein interactions.
  • the critical residues of a subject Protease M polypeptide which are involved proteolytic cleavage can be used to generate Protease M-derived peptidomimetics which competitively inhibit binding of the authentic Protease M protein with that moiety.
  • peptidomimetic compounds By employing, for example, scanning mutagenesis to map the amino acid residues of a protein which is involved in binding other proteins, peptidomimetic compounds can be generated which mimic those residues which facilitate the interaction. Such mimetics may then be used to interfere with the normal function of a Protease M protein.
  • non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as Protease M.
  • the invention provides polyclonal and monoclonal antibodies that bind Protease M.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of Protease M.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular Protease M protein with which it immunoreacts.
  • Polyclonal Protease M antibodies can be prepared as described above by immunizing a suitable subject with a Protease M immunogen, as described in more detail in the appended Examples.
  • the anti-Protease M antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized Protease M.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against Protease M can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J Immunol 127:539- 46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31 ; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds Protease M.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • PEG polyethylene glycol
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind Protease M, e.g., using a standard ELISA assay.
  • a monoclonal anti-Protease M antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with Protease M to thereby isolate immunoglobulin library members that bind Protease M. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
  • An anti-Protease M antibody (e.g., monoclonal antibody) can be used to isolate Protease M by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-Protease M antibody can facilitate the purification of natural Protease M from cells and of recombinantly produced Protease M expressed in host cells.
  • an anti-Protease M antibody can be used to detect Protease M protein (e.g., in a cellular lysate or cell supernatant). Detection may be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes i nc i l l T ⁇ -3 luminol; and examples of suitable radioactive material include I, I, S or H.
  • Another aspect of the invention features transgenic non-human animals which express a heterologous Protease M gene of the present invention, or which have had one or more genomic Protease M genes disrupted in at least one of the tissue or cell- types of the animal.
  • the invention features an animal model for proliferative disorders, which animal has one or more Protease M allele which is mis- expressed.
  • a mouse can be bred which has one or more Protease M alleles deleted or otherwise rendered inactive.
  • Such a mouse model can then be used to study disorders arising from mis-expressed Protease M genes, as well as for evaluating potential therapies for similar disorders.
  • transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express an exogenous Protease M protein in one or more cells in the animal.
  • a Protease M transgene can encode the wild- type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
  • the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.
  • such mosaic expression of a Protease M protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of Protease M expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
  • temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences. Genetic techniques which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are known to those skilled in the art.
  • target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject Protease M proteins.
  • excision of a target sequence which interferes with the expression of a recombinant Protease M gene can be designed to activate expression of that gene.
  • This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the Protease M gene from the promoter element or an internal stop codon.
  • the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
  • cre/loxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
  • loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation expression of a recombinant Protease M protein can be regulated via control of recombinase expression.
  • cre/loxP recombinase system to regulate expression of a recombinant Protease M protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and a recombinant Protease M gene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., a Protease M gene and recombinase gene.
  • One advantage derived from initially constructing transgenic animals containing a Protease M transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal.
  • a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern.
  • prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the Protease M transgene.
  • Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.
  • a Protease M transgene could remain silent into adulthood until "turned on” by the introduction of the trans-activator.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell.
  • the zygote is the best target for micro-injection.
  • the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution.
  • the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
  • Retroviral infection can also be used to introduce Protease M transgenes into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
  • the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
  • Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
  • Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
  • Protease M knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert recombinase target sequences flanking portions of an endogenous Protease M gene, such that tissue specific and/or temporal control of inactivation of a Protease M allele can be controlled as above.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the protein or antibody and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier As used herein the term
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylene
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL * M (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a Protease M protein or anti-Protease M antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a Protease M protein or anti-Protease M antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the Protease M protein of the invention exhibits serine protease activity. Accordingly, Protease M is useful as a serine protease, either in vitro or in vivo.
  • the isolated nucleic acid molecules of the invention can be used to express Protease M protein (e.g., via a recombinant expression vector in a host cell), to detect Protease M mRNA (e.g., in a biological sample) and to modulate Protease M activity, as discussed and further below.
  • the anti-Protease M antibodies of the invention can be used to detect and isolate Protease M protein and modulate Protease M activity, also discussed further below.
  • the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation.
  • the methods can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding a Protease -protein, or (ii) the mis-expression of the Protease gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a Protease M gene, (ii) an addition of one or more nucleotides to a Protease M gene, (iii) a substitution of one or more nucleotides of a Protease M gene, (iv) a gross chromosomal rearrangement of a Protease M gene, (v) a gross alteration in the level of a messenger RNA transcript of a Protease M gene, (vii) aberrant modification of a Protease M gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a Protease M gene, (viii) a non-wild type level of a Protease M- protein, (ix) allelic loss of a Protease M gene, and (x) inappropriate post-translational modification of
  • the present invention provides a large number of assay techniques for detecting lesions in a Protease M gene, and importantly, provides the ability to discern between different molecular causes underlying Protease -dependent aberrant cell growth, proliferation and/or differentiation.
  • a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a Protease M gene, such as represented by any of SEQ ID Nos: 1 and 3, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject Protease M genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is contacted with nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • one aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, if mutations have arisen in one or more Protease M of the sample cells.
  • the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant cell proliferation and/or metastasis.
  • the method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by an alteration affecting the integrity of a gene encoding a Protease M.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a Protease M-gene, (ii) an addition of one or more nucleotides to a Protease M- gene, (iii) a substitution of one or more nucleotides of a Protease -gene, and (iv) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a Protease M-gene.
  • the present invention provides a large number of assay techniques for detecting lesions in Protease M genes, and importantly, provides the ability to discern between different molecular causes underlying Protease M- dependent aberrant cell growth and/or metastasis.
  • detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to a Protease M gene under conditions such that hybridization and amplification of the Protease M-gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874- 1878), transcriptional amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P.M.
  • mutations in a Protease M gene from a sample cell are identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No.
  • 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the Protease M gene and detect mutations by comparing the sequence of the sample Protease Mwith the corresponding wild-type (control) sequence.
  • Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463).
  • any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including by sequencing by mass spectrometry (see, for example PCT publication WO 94/16101 ; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159).
  • sequencing by mass spectrometry see, for example PCT publication WO 94/16101 ; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159.
  • the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-tract or the like, e.g., where only one nucleic acid is detected, can be carried out.
  • protection from cleavage agents can be used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers, et al. (1985) Science 230: 1242).
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • mismatched bases in RNA/RNA or RNA/DNA heteroduplexes Myers, et al. (1985) Science 230: 1242).
  • mismatch cleavage starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type Protease M sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DN A hybrids treated with S 1 nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in Protease cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on a Protease M sequence e.g., a wild-type Protease M sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in Protease M genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control Protease M nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGG ⁇ ) (Myers et al (1985) Nature 313:495).
  • DGG ⁇ denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele speicific oligonucleotide hybridization techniques may be used to test one mutation per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238.
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a Protease M-gene, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject Protease M-genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • Such oligonucleotide probes can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, neoplastic or hyperplastic disorders (e.g. aberrant cell growth).
  • nucleotide probes can be generated from the subject Protease M gene which facilitate histological screening of intact tissue and tissue samples for the presence (or absence) of Protease M-encoding transcripts. Similar to the diagnostic uses of anti-Protease M antibodies, the use of probes directed to Protease M messages, or to genomic Protease M sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, neoplastic or hyperplastic disorders (e.g. unwanted cell growth) or abnormal differentiation of tissue.
  • the oligonucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a Protease M protein. For instance, variation in polypeptide synthesis can be differentiated from a mutation in a coding sequence. Diagnostic procedures may be performed on any "biological sample” including, for example, cells, body fluids, or in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Antibodies directed against wild type or mutant Protease M proteins, which are discussed, above, may also be used in disease diagnostics and prognostics.
  • Such diagnostic methods may be used to detect abnormalities in the level of Protease M protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of Protease M protein.
  • Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant
  • Protease M protein relative to the normal Protease M protein.
  • Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to one of skill in the art, including but not limited to western blot analysis.
  • western blot analysis For a detailed explanation of methods for carrying out western blot analysis, see Sambrook et al, 1989, supra, at Chapter 18.
  • the protein detection and isolation methods employed herein may also be such as those described in Harlow and Lane, for example, (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety.
  • probe or labelable is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of Protease M proteins.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection may also be accomplished using any of a variety of other methods.
  • Antibodies may be labeled with radioactivity, fluorescent compounds (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine), chemiluminescent compounds (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester), bioluminescent compounds (e.g., luciferin, luciferase and aequorin).
  • fluorescent compounds e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine
  • chemiluminescent compounds e.g., luminol, isoluminol, theromatic
  • detection of Protease M is based on the detection a Protease M bioactivity, such as enzymatic activity.
  • a Protease M bioactivity such as enzymatic activity.
  • serine protease substrate cleavage may be measured in a sample.
  • substrates include, gelatin, casein, or n-a-benzoyl-L-arginine ethyl ester (BAEE).
  • the invention provides a diagnostic method comprising: (i) contacting a tumor sample from a subject with an agent capable of detecting Protease M protein or mRNA; (2) determining the amount of Protease M protein or mRNA expressed in the tumor sample; (3) comparing the amount of Protease M protein or mRNA expressed in the tumor sample to a control sample; and (4)forming a diagnosis based on the amount of Protease M protein or mRNA expressed in the tumor sample as compared to the control sample.
  • the biological sample is a tumor sample.
  • the tumor sample may comprise tumor tissue or a suspension of tumor cells.
  • a tissue section for example, a freeze-dried or fresh frozen section of tumor tissue removed from a patient, can be used as the tumor sample.
  • the tumor sample may comprise a biological fluid obtained from a tumor-bearing subject.
  • Protease M contains a signal sequence and thus is likely to be detectable in biological fluids.
  • tumor samples can be stored at temperatures below -20°C to prevent degradation until the detection method is to be performed.
  • Preferred tumor samples in which Protease M mRNA or protein is to be detected are mammary tumor samples and/or ovarian tumor samples.
  • Primary malignancy of the tumor cell sample can be diagnosed based on an increase in the level of expression of Protease M mRNA or protein in the tumor sample as compared to the control.
  • the control is from normal cells or a primary tumor and the tumor sample is a suspected metastatic tumor sample. Acquisition of the metastatic phenotype by the suspected metastatic tumor sample can be diagnosed based on a decrease in the level of, or absence of, Protease M mRNA or protein in the tumor sample compared to the control.
  • the detection method of the invention can be used to detect Protease M mRNA or protein in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of Protease M mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of Protease M protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • Protease M protein can be detected in vivo in a subject by introducing into the subject a labeled anti-Protease M antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g. , in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a Protease M gene.
  • the kit can comprise a labeled or labelable agent capable of detecting Protease M protein or mRNA in a biological sample; means for determining the amount of Protease M in the sample; and means for comparing the amount of Protease M in the sample with a standard.
  • the agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect Protease M mRNA or protein.
  • Another aspect of the invention pertains to methods of modulating
  • Protease M bioactivity associated with a cell e.g., for therapeutic purposes.
  • Protease M activity "associated with a cell” is intended to include Protease M activity within the cell, secreted by the cell and in the extracellular milieu surrounding the cell.
  • the modulatory method of the invention involves contacting the cell with an agent that modulates Protease M activity associated with the cell.
  • the agent stimulates Protease M serine protease activity.
  • Examples of such stimulatory agents include active Protease M protein agonists and a nucleic acid molecule encoding Protease M that has been introduced into the cell.
  • the agent inhibits the Protease M activity .
  • inhibitory agents include antisense Protease M nucleic acid molecules, anti-Protease M antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • Stimulation of Protease M bioactivity is desirable in situations in which Protease M is abnormally downregulated and/or in which increased Protease M activity is likely to have a beneficial effect.
  • One example of such a situation is in tumor cells, and in particular metastatic tumor cells.
  • acquisition of a metastatic phenotype by tumor cells is associated with downregulation of Protease M expression.
  • increasing the expression and/or activity of Protease M in or around the tumor cells is expected to inhibit the development or progression of the metastatic phenotype.
  • the invention provides a method for inhibiting development or progression of a tumorogeinc phenotype in a cell comprising contacting the tumor cell with an agent which elevates the amount of Protease M associated with the cell.
  • the agent that elevates Protease M in or around the tumor cell can be Protease M protein itself.
  • Protease M preferably in a pharmaceutically acceptable carrier, can be administered to a tumor-bearing subject by an appropriate route to inhibit the development or progression of a proliferative disorder.
  • Suitable routes of administration include intravenous, intramuscular or subcutaneous injection, injection directly into the tumor site or implantation of a device containing a slow-release formulation.
  • the Protease M preparation can also be incorporated into liposomes or other carrier vehicles to facilitate delivery to the tumor site.
  • a non-limiting dosage range is 0.001 to 100 mg/kg/day, with the most beneficial range to be determined by routine pharmacological methods.
  • Protease M protein or agonist Alternative to administration of Protease M protein or agonist itself, the development of or progression of cancer in a cell may be slowed by modifying them to express Protease M by introducing into the cells a nucleic acid encoding Protease M (e.g., via a recombinant expression vector).
  • Expression vectors suitable for gene therapy including retroviral and adenoviral vectors carrying appropriate regulatory elements, can be used to deliver the Protease M-encoding nucleic acid to the tumor cells.
  • the ability of Protease M protein or DNA to inhibit tumor progression and/or metastasis can be evaluated using in vivo and in vitro assays known in the art.
  • a suitable in vivo assay utilizes the mammary epithelial tumor cell line MDA-MB-435, which forms tumors at the site of orthotopic injection and metastasizes in nude mice (describe further in Price et al. (1990) Cancer Res. 50:717).
  • MDA-MB- 435 cells which do not express detectable Protease M mRNA, can be transfected with a Protease M expression vector and stable transfectants can be selected. These transfectants can then be injected into nude mice. At 10-weeks post-inoculation, the mice are sacrificed and their tumors are excised and weighed to determine the effect of Protease M expression on tumor progression and metastasis.
  • a suitable in vitro assay is tumor cell invasion through reconstituted basement membrane matrix (e.g., Matrigel) as described in Hendrix et al. (1987) Cancer Letters 38:137. The invasive ability of
  • Protease M-transfected MDA-MB-435 cells can be compared to untransfected MDA- MB-435 cells to determine the effect of Protease M expression on tumor invasiveness.
  • Protease M mRNA expression is markedly upregulated in certain primary tumor cells.
  • inhibiting the expression or activity of Protease M in cells may be useful for inhibiting or reducing carcinogenesis.
  • the present invention facilitates the development of assays which can be used to screen for drugs, including Protease M homologs, which are either agonists or antagonists of the normal cellular function of the subject Protease M polypeptides, or of their role in the pathogenesis of cellular differentiation and/or proliferation and disorders related thereto.
  • the assay evaluates the ability of a compound to modulate binding between a Protease M polypeptide and a molecule, be it protein or DNA, that interacts either upstream or downstream of the Protease M polypeptide in the TGFb signaling pathway.
  • a variety of assay formats will suffice and, in light of the present inventions, will be comprehended by a skilled artisan.
  • the compound of interest is contacted with Protease M and a molecule which interacts with Protease M (including both activators and repressors of its activity), such as a substrate.
  • Detection and quantification of complexes o ⁇ Protease Mwith it's binding protein provide a means for determining a compound's efficacy at inhibiting (or potentiating) complex formation between Protease Mand the Protease M-binding elements.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified Protease M polypeptide is added to a composition containing the Protease M- binding element, and the formation of a complex is quantitated in the absence of the test compound.
  • Complex formation between the Protease M polypeptide and a Protease M binding element may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled Protease M polypeptides, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled Protease M polypeptides
  • modulators of Protease M activity may be identified in a method wherein Protease M, a substrate for the serine protease, and a test substance are incubated under conditions suitable for the serine protease to cleave the substrate.
  • Cleavage of the substrate is then measured and the amount of cleavage of the substrate in the presence of the test substance is compared to the amount of cleavage of the substrate in the absence of the test substance.
  • the test substance can then be identified as a modulator of Protease M activity based on this comparison. For example, when the amount of cleavage of the substrate in the presence of the test substance is less than the amount of cleavage of the substrate in the absence of the test substance, the test substance can thereby be identified as a stimulator of the Protease M activity. Alternatively, when the amount of cleavage of the substrate in the presence of the test substance is greater than the amount of cleavage of the substrate in the absence of the test substance, the test substance can thereby be identified as an inhibitor of the Protease M activity.
  • Binding of Protease to a binding protein can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/Prote ⁇ e M (GST/Protease M) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates, e.g. an 35 S-labeled, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level oi Protease M- binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • Protease Mor its cognate binding protein can be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated Protease M molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with Protease but which do not interfere with binding of Protease M and a binding protein (BP) can be derivatized to the wells of the plate, and Protease M trapped in the wells by antibody conjugation.
  • preparations of a Protease M-binding protein and a test compound are incubated in the Protease M-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the Protease M binding element, or which are reactive with Protease M protein and compete with the binding element; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding element, either intrinsic or extrinsic activity.
  • the enzyme can be chemically conjugated or provided as a fusion protein with the Protease M-BP.
  • the Protease M-BP can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l- napthol.
  • a fusion protein comprising the polypeptide and glutathione-S- transferase can be provided, and complex formation quantitated by detecting the GST activity using 1 -chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
  • antibodies against the protein can be used.
  • the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the Protease M sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.
  • Cell based assays In addition to cell-free assays, such as described above, the readily available source of mammalian Protease M proteins provided by the present invention also facilitates the generation of cell-based assays for identifying small molecule agonists/antagonists and the like. For example, cells can be caused to overexpress a recombinant Protease M protein in the presence and absence of a test agent of interest, and the assay scored for modulation in Protease M bioactivity in the target cell mediated by the test agent. As with the cell-free assays, agents which produce a statistically significant change in Protease M-dependent responses (either inhibition or potentiation) can be identified.
  • the expression or activity of a Protease M is modulated in or cells and the effects of compounds of interest on the readout of interest (such as tumorigenesis or metastatic potential) are measured.
  • modulators of Protease M expression are identified in a method wherein a cell is contacted with a test substance and the expression of Protease M mRNA or protein in the cell is determined. The level of expression of Protease M mRNA or protein in the presence of the test substance is compared to the level of expression of Protease M mRNA or protein in the absence of the test substance. The test substance can then be identified as a modulator of Protease M expression based on this comparison.
  • the test substance when expression of Protease M mRNA or protein is greater in the presence of the test substance than in its absence, the test substance is identified as a stimulator of Protease M mRNA or protein expression.
  • the test substance when expression of Protease M mRNA or protein is less in the presence of the test substance than in its absence, the test substance is identified as an inhibitor of Protease M mRNA or protein expression.
  • the level of Protease M mRNA or protein expression in the cells can be determined by methods described above for detecting Protease M mRNA or protein.
  • the regulatory regions of a Protease M gene e.g., the 5' flanking promoter and enhancer regions, may be operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.
  • a detectable marker such as luciferase
  • the subject Protease M polypeptides can be used to generate a "two hybrid" assay (see, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins.
  • the method makes use of chimeric genes which express hybrid proteins.
  • a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for a Protease M polypeptide.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e.g., form a Protease M-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of the Protease M and sample proteins.
  • Normal human mammary epithelial cell strains (70N and 76N) were derived from reduction mammoplasties in this lab as described (Band V, Sager R. ( 1 989) Proc. Natl. Acad. Sci. USA86. I249-1253.). Primary (21PT, 21NT) and metastatic (21MT-1, 21MT-2) tumor lines were established in this lab from a single patient as described ( Band V and Sager R. ( 1 989) Proc. Natl. Acad. Sci. USA86.1249-1253; Band V, et al. (1990) Cancer Res. 50:735 1 -7357).
  • Human mammary epithelial tumor cell lines MCF-7, T47D, ZR75- 1 , BT549, MDA-MB- 157, MDA-MB-231 , MDA-MB- 435, MDA-MB436, MDA-MB-361, and BT-474 were obtained from American Tissue Culture Collection (Rockville, MD). Cells were grown in DFCI-1 media (Schachter M. (1980) Pharmacol. Rev. 31: 1 -1 7) and harvested at approximately 70% confluence for RNA isolation and when near confluent for DNA isolation.
  • CF3 HPV immortalized
  • CF91 SV40 immortalized
  • MLC SV40 immortalized
  • HOSE 10/11, 16, and 21 The primary human ovarian surface epithelial cell cultures (HOSE 10/11, 16, and 21) were established from the ovarian surface epithelium as described previously (Tsao SW, Mok SC, Fey E, et al. (1995) Exp. Cell Res. 218:499-507).
  • Immortalized ovarian surface epithelial cells (HOSE6.3E6E7) was obtained by infecting the HOSE cells with a replication-defective retrovirus construct, LXSN16E6E7 as described (Tsao SW, et al. (1995). Exp. Cell Res. 218:499-507).
  • the eight ovarian carcinoma cell lines used for this comparative study include DOV13, OVCA420, OVCA429, OVCA432, and OVCA433, which were established in the laboratory of Gynecologic Oncology; CAOV3 and SKOV3 which were purchased from ATCC (Rockville, MD); and OVCA3 which was obtained from the National Cancer Institute (Frederick, MD).
  • Ovarian tumors were obtained from consenting patients at the Brigham and Women's hospital in Boston as described previously (Mok SC, et al. (1992) Cancer Res. 52:51 19-5122). These include six borderline ovarian tumors (354A, 373 A, 395A, 405A, 466A, and 469A); twenty stage III/IV high grade invasive ovarian adenocarcinomas from the primary ovarian site; two metastatic adenocarcinoma from colon primary tumors (327A, 339A) and three normal ovaries (366N, 379N, and 465N).
  • Total cell RNAs (50mg) from 21PT and 21MT-1 were treated with DNasel ( Worthington DPRF) in the presence of Rnasin ribonuclease inhibitor
  • RNAs were reverse transcribed using the 3' -anchored primer T12MG (where M is a mixture of A, G, or C).
  • the resultant cDNAs were then PCR amplified in the presence of 35S-dATP using T12MG and the arbitrary primer OPA1 (CAGGCCCTTC) and run side by side on a 6% sequencing gel. Differentially displayed bands were recovered from the dried gel, reamplified by PCR, 2 P-labeled by the oligo method (Feinberg AP and Vogelstein B. (1983) Anal. Biochem. 132:6-13) and used as a probe on Northern strips prepared with 21PT and 21MT-1 total RNA to confirm the result obtained by differential display.
  • the reamplified band from differential display was cloned into the TA cloning vector PCRII (Invitrogen) and sequenced on both strands using T7 and SP6 primers.
  • cDNA libraries from 21PT and 76N cells constructed in Lambda Zap II (Stratagene, San Diego, CA) were screened using the cloned PCR product as a probe and several cDNA clones were isolated and sequenced on both strands.
  • the longest cDNA clone (from the 76N library) was sequenced on both strands using an ABI automated sequencer Model 373A by the Dana Farber Molecular Biology Core Facility.
  • Oligonucleotides used for sequencing were synthesized by the Dana Farber Molecular Biology Core facility or by Amitof, Inc. (Cambridge, MA). The predicted protein coding region and non-translated regions were determined and formatted using the GCG Publish program. The predicted protein sequence was compared to protein databases using the Blast algorithm (Altschul SF, (1990) J. Mol. Biol. 215:403-410). Protein alignment with related proteins performed on GCG using the Pileup, Distances, and Prettyplot programs.
  • Total cell RNA was isolated by the guanidinium isothiocyanate/cesium chloride method and analyzed on Northern blots as previously described (Sambrook j, (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.). 36B4 (Masiakowski P, Breathnach R, Bloch J, et al. (1982) Nucl. Acids Res. 10:7895-7903), a ribosomal protein whose message is constant under a variety of conditions, was used to normalize the blots. Total cellular DNA was isolated and analyzed on Southern blots as described (Sambrook J, (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.). Densitometric analysis of autoradiographs was performed with an imaging densitomer (Biorad GS-700) using the Molecular Analyst software.
  • GKNNLRQRESSQEQS 87 (0.5mg) was emulsified with an equal volume of Freund's adjuvant and injected into 3 to 9 month old New Zealand white rabbits. Boosts were done 2 and 6 weeks later. The animals were bled and serum was collected and stored at -20° C. Peptide and antibody production was done at Research Genetics, Huntsville, AL.
  • Whole cell lysates were prepared by sonicating 10 7 cells/ml for 20, 30 second pulses in a Sonicator Ultrasonic Processor in mammalian lysis buffer (4mMNaHCO3 5 lOOmM NaF, 20mMKH 2 PO 45 2mM Sodium orthovanadate, 5mM EDTA, 5mM disoflurophoshate, 2mM PMSF, 2 mg/ ml leupeptin, 2mg/ml aprotinin, pH 7.2). Lysates were clarified by spinning at 14,000 x g for 30 minutes in a microfuge (Eppendorf).
  • the full length cDNA clone was PCR amplified using the sense 5' 26- mer oligonucleotide 5 'GGAATTCCGTTGGTGCATGGCGGACC 3 ' and the antisense 3' oligonucleotide 5 'GTCGGAATTCAGGGTCACTTGGCCTG 3 ' at 95°C, 1 minute, ⁇ O ⁇ C, 1 minute, 72"C, 1 minute for 30 cycles to yield a 0.7 kb product which contained the open reading frame without the hydrophobic n-terminal amino acids.
  • a full length cDNA clone was cut with EcoNl and BstXl to give a fragment which spanned nucleotides 233 to 1019. This fragment was incorporated into the baculovirus transfer vector pVL1392 (Pharmingen). Generation and amplification of recombinant baculovirus was as described (23,24).
  • Protease M Spodoptera Frugiperda (cell line sf9) was infected with amplified recombinant virus to obtain nearly 100% infection as gauged by enlarged cells. 96 hours post-infection, cells were harvested and lysed by sonication in mammalian lysis buffer followed by adjusting to 500mM NaCl and rocking for one hour at 4°C. All subsequent purifications were done at 4°C.
  • the lysate was adjusted to 125mM NaCl, loaded onto p- aminobenzamidine agarose (Sigma A7155), washed with loading buffer, and eluted with (25mMNaPO 4 , 0.02% NaN3, 500mM NaCl, lOmM benzamidine), pH 6.0.
  • the eluted fractions were loaded onto concanavalin A agarose (Sigma C8402) by rocking for 1 hour, washed with (25mMNaPO 45 0.02% NaN 3 , 500 mM NaCl), pH 6.0, and eluted in wash buffer containing 10% methyl-a-D-mannopyranoside (Sigma M6882).
  • Protease activity was also determined by monitoring the cleavage of n-a- benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500. Reactions were set up in (25mMNaPO 4 , lmM EDTA, and ImM BAEE), pH 7.5. Samples were added and the change in absorbance at 260nm was monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin was used as a positive control.
  • BAEE n-a- benzoyl-L-arginine ethyl ester
  • RNAs from 21 PT and 21 MT-1 cell lines were compared by differential display. Approximately 100 bands appeared in each lane of each primer pair tested, and on the average 2-3 bands were differentially expressed. One of the bands that was overexpressed in the 21 PT lane (with primer pair OPA 1/T12MG) (280 bp in Figure 1 A) was excised from the gel and PCR amplified. The resulting 280 bp PCR product was used to probe a northern blot ( Figure IB). Two bands were detected; a band of 1.7kb which was very high in 21 PT and barely detectable in 21 MT-1, and a band of approximately 1 kb which was equal in both lanes. This mixture of two clones was purified and the clone which hybridized only to the differentially expressed 1.7 kB message was recovered. Pro tease M: Sequence Identification
  • the 0.28 kb insert was used to screen a 76N cDNA library constructed in IZapII.
  • the longest clone isolated was sequenced in its entirety. This clone is 1,526 nt in length and contains 245bp of 5'nt sequences, 732 bp of coding sequences (coding for a postulated protein of 244 aa), and 549 bp of 3'nt sequences.
  • Figure 2 The presumptive protein coding region begins with an ATG codon, which is in a good Kozak consensus sequence (Kozak M. (1984) Nucleic Acids Research 12:857872) CGGCCATGA, and ends with a TGA translation stop codon.
  • the amino terminal portion of the postulated protein has 13 consecutive hydrophobic residues (leu ⁇ to ala ⁇ ) which is characteristic of a signal peptide followed by glu ⁇ -glu-gln-asn-lys 2 ⁇ which resembles a pro-peptide with a potential trypsin susceptible cleavage site after lys 2
  • a potential N-linked glycosylation site is found at asn ⁇ 4-tfr -thr ⁇ 3 ⁇ in the 3'nt region, the expected polyadenylation signal AATAAA is found 11 base pairs upstream of the poly A tail at 1,490 bp.
  • Another polyadenylation signal AATAAA was found at 1,095 bp.
  • the postulated protein sequence compared to proteins in the database using the blast program, was similar to other proteins of the serine protease family.
  • the postulated sequence was compared to the four most closely related proteins using the pileup program and distances program and displayed by the prettyplot program and was found to be novel. (Figure 3).
  • Figure 4A shows the results of northern blots of mammary cell lines and strains.
  • the two normal cells strains shown (76N and 70N) and another normal cell strain 8 IN (not shown) expressed the 1.7kb Protease M message at low levels.
  • Two primary tumor lines (21 PT and 21 NT) as well as one metastatic line from the same patient (21 MT-2) expressed high levels of message (approximately 20 to 100 fold higher than the normal strains). However, the most metastatic cell line from the same patient (21 MT-1) expressed low levels of RNA (see Figure 1A).
  • FIG. 4B shows northern blots of prostate cell lines.
  • the normal, immortalized cell strains CF3 and CF91 express moderate levels of Protease M mRNA while another normal immortalized strain, MLC expresses just trace amounts.
  • MLC expresses just trace amounts.
  • all three of the tumor cell lines examined failed to express any Protease M message.
  • RNA from a series of normal ovarian tissue and biopsies from primary tumors one of the two northern blots is shown ( Figure 5). While mRNA was not expressed in the three normal tissues examined, the six borderline ovarian tumor tissues, and the two metastatic tumors from colon primaries, it was expressed in the primary ovarian tumor tissue in sixteen of the twenty specimens examined.
  • a polyclonal antibody was produced in rabbits against a hydrophilic peptide which was not highly conserved among other serine proteases ( 73 GKHNLRQRESSQEQS 87 ).
  • the western blot ( Figure 7) shows that the antibody detects a protein of 37kd in total cell lysates of the normal mammary epithelial cell strain 8 IN, and in the primary tumor cell line 2 INT. No protein is detected in the metastatic breast cell line MDA-MB-435. In other western blots (not shown) the antibody detected a 37kd protein in the normal strains 70N and 76N, as well as the primary tumor cell line 21PT, but not in the metastatic cell lines T47D and MCF-7.
  • RNA levels for the serine protease are not always correlated with the protein levels.
  • the antipeptide polyclonal Protease M antibody has been used successfully in western blots but does not seem to work in cellular immunofluorescence studies in which the antibody gave a high background with MDA-MB-435 cells which do not express the Protease M message.
  • transfectants were produced expressing Protease M in the mammary tumor cell line MDA-MB-435. Transfectants were screened initially for protein production, and as shown below, the results demonstrated that only 5 of the 76 transfectants produced any protein and this was at low levels.
  • the expected 52kd GST/Protease M fusion protein was purified and yielded approximately 600 mg of fusion protein per 500 ml culture. We were able to cleave the fusion protein by incubation with thrombin but the Protease M fragment was degraded, even at limiting dilutions, while only the GST portion stayed intact. When we ran the fusion protein on western blots, we needed at least lmg to get a detectable signal. Up to lmg of GST/Protease M fusion protein was run on casein and gelatin zymograms (Heusen C, Dowdle EB. (1980) Anal. Biochem. 102: 196-202;
  • a pCMV/Neo/Protease M construct as well as neo-vector controls were transfected into MDA-MB435 cells (5 x 10" cells for each construct) by electroporation. Of the 10" cells which survived the electroporation, approximately 400 colonies (one in 2,500) survived the G418 selection. 80 colonies of protease transfected clones and 20 colonies of vector transfected clones were transferred to 24 well dishes when they were 2 to 3 mm in diameter. The protease transfected cells grew more slowly and had more enlarged, dying cells than the vector controls. Total cell lysates were prepared from the 76 protease transfectants when the cells were approximately 70% confluent.
  • Table 2 shows that Protease M RNA was found in clones expressing protein as well as the majority of those not expressing protein. Thus, in MDA-MB-435 cells there is either inefficient translation of the message, or the protein translated is extremely unstable.
  • Table 1 Shows the Expression of Protease M mRNA and protein in mammary cells
  • RNA values were obtained by running 1 Omg of total RNA on a northern blot, hybridizing to P-labeled Protease M probes and quantitating the resulting autoradiograms.
  • the most intense band was set equal to 100 and the other values normalized accordingly 2 protein values were obtained by running 5 Omg of total cell lysates on a western blot and probing with the Protease M antibody as described in methods.
  • the 37kd bands on the autoradiograms were quantitated, the most intense band was set equal to 100 and the other values normalized accordingly.
  • Trp Gly Lys Thr Ala Asp Gly Asp Phe Pro Asp Thr lie Gin Cys Ala 145 150 155 TAC ATC CAC CTG GTG TCC CGT GAG GAG TGT GAG CAT GCC TAC CCT GGC 767 Tyr lie His Leu Val Ser Arg Glu Glu Cys Glu His Ala Tyr Pro Gly 160 165 170
  • CAG ATC ACC CAG AAC ATG TTG TGT GCT GGG GAT GAG AAG TAC GGG AAG 815 Gin lie Thr Gin Asn Met Leu Cys Ala Gly Asp Glu Lys Tyr Gly Lys 175 180 185 190
  • Lys Thr lie Gin Ala Lys 240

Abstract

Molecules nucléotidiques isolées codant une nouvelle sérine protéase, la protéase M. Cette protéase est obtenue par régulation négative dans des cellules métastatiques de tumeurs épithéliales mammaires, ainsi que dans d'autres cellules tumorales, et elle est obtenue par régulation positive dans des cellules sénescentes. On décrit par ailleurs des molécules nucléotidiques anti-sens, des vecteurs d'expression recombinés contenant une molécule nucléotidique visée dans l'invention, des cellules hôtes dans lesquelles les vecteurs d'expression ont été introduits, et des animaux transgéniques non humains dans lesquels on a introduit ou dissocié un gène de protéase M. On décrit par ailleurs des protéines de protéase M isolées, des protéines fusionnées, des peptides antigéniques et des anticorps anti-protéase M, et par ailleurs, des dosages diagnostiques, des méthodes de criblage de médicaments ainsi que des procédés thérapeutiques faisant appel aux compositions spécifiées.
PCT/US1997/016175 1996-09-13 1997-09-11 Protease m, une nouvelle serine protease WO1998011238A2 (fr)

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WO2002003787A2 (fr) * 2000-07-06 2002-01-17 Deltagen, Inc. Souris transgéniques contenant des disruptions géniques ciblées
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