WO1999031256A2 - Proteines cathepsine dc, adn codant pour ces proteines et utilisation de ces proteines dans l'etablissement de pronostics pour les cancers chez l'homme - Google Patents

Proteines cathepsine dc, adn codant pour ces proteines et utilisation de ces proteines dans l'etablissement de pronostics pour les cancers chez l'homme Download PDF

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WO1999031256A2
WO1999031256A2 PCT/US1998/026920 US9826920W WO9931256A2 WO 1999031256 A2 WO1999031256 A2 WO 1999031256A2 US 9826920 W US9826920 W US 9826920W WO 9931256 A2 WO9931256 A2 WO 9931256A2
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cathepsin
protein
proteins
polypeptides
nucleic acid
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PCT/US1998/026920
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WO1999031256A3 (fr
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Dirk Michael Anderson
Terry Y. Nakagawa
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Immunex Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention is directed to purified and isolated cathepsin DC proteins, the nucleic acids encoding such proteins, processes for production of recombinant forms of such proteins, antibodies directed against these proteins, affinity columns containing these proteins, and methods for using these proteins to determine and enhance the specificity of antibodies directed against cathepsin B protein in order to enhance the accuracy of human tumor prognosis.
  • Cancer is a broad medical term that encompasses a wide variety of specific types of human tumors. Examples of such tumors would include glioblastomas, astrocytomas, and colorectal carcinomas. Metastasis of tumors is the process describing the transmission and invasion of a human tumor to other sites in the body. Such a spread of tumor cells is usually associated with a poor prognosis for the patient.
  • metastasis may involve the action of various proteases including the urokinase form of plasminogen activator, cathepsin B, cathepsin D, and various metalloproteases (Duffy et al., Clin. Exp. Metastasis
  • cathepsin B protein in metastasis may be due in part to the involvement of cathepsin B protein in enhancing the invasion properties of tumor cells (Navab et al., Clin. Exp. Metastasis 15:121, 1997). This may further involve a role for cathepsin B protein in the angiogenic response in tumors (Keppler et al., Biochem. Cell Bio. 74:799, 1996).
  • Cathepsin B protein is a lysosomal protease that is a member of the cysteine, or thiol, family of proteases (Chapman et al., Am. J. Respir. Crit. Care Med. 150:S155, 1994).
  • cysteine-type cysteine proteases A number of amino acid residues have been shown to be conserved among members of the papain-type cysteine proteases including cysteine, histidine, and asparagine residues that reside within the active site of the enzyme (Id. and Coleman et al., U.S. Patent No. 5,637,462). Two additional glycine residues are also conserved in most cysteine proteases of the papain family (Fox et al., Prot. Engineering 8:53, 1995). The members of the papain family of cysteine proteases have a high conservation of active site sequences, but overall amino acid homologies in the range of 20- 60% (Chapman et al., 1994).
  • Cathepsin B protein is thought to be expressed constitutively in a wide variety of cell types (Id.). Other cathepsin proteins may demonstrate different tissue distribution patterns (Id.).
  • An increase in cathepsin B protein expression has been linked to tumor progression in a number of different human tumors. In human colorectal cancer, up-regulation of cathepsin B protein was correlated with tumor progression (Campo et al., Am. J. Path. 145:301, 1994). Increased cathepsin B protein expression was further correlated with the malignant progression of astrocytomas, especially in glioblastomas (Sivaparvathi et al., Clin. Exp. Metastasis 13:49, 1995). The highest expression of cathepsin B protein has been correlated with the most malignant tumors, particularly at the invasive edge of those tumors (Berquin and Sloane, in
  • Cathepsin B protein levels were studied in malignant and adjacent colorectal tissues (Adenis et al., Cancer Letters 96:267, 1995). The authors found a statistically significant increase of approximately 8-fold in cathepsin B protein levels in tumor tissue as compared to normal tissue. The correlation of increased cathepsin B protein expression with some types of malignant tumors allows cathepsin B protein expression to serve as a prognostic marker for the progression of certain types of cancers (Mikkelsen et al, J. Neurosurgery 83:285, 1995; Keppler and Sloane, E . Prot. 49:94, 1996; Berquin and Sloane, 1996; and Duffy, 1992).
  • cathepsin B protein in clinical specimens has been accomplished through the use of antisera generated against cathepsin B protein (Sivaparvathi et al., 1994; Mikkelsen et al., 1995; and Campo et al., 1994). These antisera were used as reagents in immunological staining techniques to determine the expression level and tissue distribution of cathepsin B protein. Therefore, antisera directed against cathepsin B protein constitute valuable reagents in the prognosis of human cancers in which cathepsin B protein expression is altered.
  • an antiserum directed against cathepsin B protein which is used for the prognosis of human cancer, is both sensitive enough to detect cathepsin B protein expression and specific for cathepsin B protein.
  • the variability of cathepsin B protein must be taken into account with regard to any allelic variations in amino acid sequence.
  • An antisera can either detect all allelic variants of cathepsin B protein or only detect a single cathepsin B amino acid sequence.
  • An antisera can either recognize epitopes unique to cathepsin B protein or epitopes conserved on other cathepsins, or even other proteins. Either polyclonal antibodies or monoclonal antibodies can be used as reagents in prognosis, but each type includes a set of inherent limitations.
  • Another potential problem is a lack of monospecificity of the antiserum for the protein (Goding, pp 42-45, 1986). That is, the antiserum can cross-react with other antigens, such as other cathepsins.
  • the use of a polyclonal antiserum can enhance the likelihood of detecting cross-reacting proteins.
  • the inability to discriminate the expression of cathepsin B protein from the expression of other proteins, such as other cathepsins can decrease the accuracy of a prognosis of the cancer in question.
  • an antiserum against cathepsin B protein in the prognosis of human cancers involves avoidance of the above mentioned problems.
  • an antiserum is both sensitive and specific for the detection of cathepsin B protein and variants thereof. Therefore, there exists a need in the art for methods to determine, as well as enhance, the specificity of antisera against cathepsin B protein to facilitate the accurate prognosis of human cancers.
  • the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation.
  • the investigator can rely upon a comparison of the unknown protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
  • Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Patent No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight.
  • the difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
  • composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein.
  • fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Patent No. 5,151,412), for determination of modification sites ⁇ e.g. phosphorylation), for generation of active biological compounds (T.D. Brock and M.T. Madigan, Biology of Microorganisms 300-301 (Prentice Hall, 6d ed. 1991)), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol. 50:309-316, 1980).
  • a peptide fingerprint of an unknown protein when obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W.J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyo et al., Electrophoresis 19:998-1005, 1998).
  • a variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), Multildent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id- frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance.
  • the programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining the number of fragmented peptides and their molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
  • peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J.K. Eng, et al., J. Am. Soc Mass Spec 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994); J.A. Taylor and R.S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075 (1997)).
  • polypeptides suitable for use in peptide fragmentation studies for use in molecular weight measurements, and for use in protein sequencing using tandem mass spectrometry.
  • This invention aids in fulfilling this need in the art by providing reagents and methods that facilitate the accurate prognosis of human cancers by determining and enhancing the specificity of antisera against human cathepsin B protein.
  • cathepsin DC and the interaction of this protein with anti-cathepsin B antibodies enables the use of this protein in methods that enhance the accuracy of the prognosis of human cancers.
  • the immunological cross-reactivity of anti-cathepsin B antibodies with cathepsin DC allows for both a more precise determination of the specificity of anti-cathepsin B antisera and the enhancement of this specificity.
  • the invention aids in fulfilling these various needs in the art by providing isolated cathepsin DC nucleic acids and polypeptides encoded by these nucleic acids.
  • Particular embodiments of the invention are directed to an isolated cathepsin DC nucleic acid molecule comprising the DNA sequence of SEQ ID NO: 1 and an isolated cathepsin DC nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, as well as nucleic acid molecules complementary to these sequences.
  • Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention, as well as nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising all or a portion of SEQ ID NO:l.
  • isolated nucleic acid molecules that are derived by in vitro mutagenesis of nucleic acid molecules comprising sequences of SEQ ID NO:l, that are degenerate from nucleic acid molecules comprising sequences of SEQ ID NO: l, and that are allelic variants of DNA of the invention.
  • the invention also encompasses a species homolog of cathepsin DC DNA.
  • the invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells stably or transiently transformed or transfected with these vectors.
  • the invention encompasses methods of using the nucleic acids noted above to identify nucleic acids encoding proteins having cathepsin DC activity, and to study cell signal transduction and the cathepsin DC system.
  • the invention also encompasses the use of sense or antisense oligonucleotides from the nucleic acid of SEQ ID NO: 1 to inhibit the expression of the polynucleotide encoded by the cathepsin DC gene.
  • the invention also encompasses isolated polypeptides and fragments thereof encoded by these nucleic acid molecules including soluble polypeptide portions of SEQ ID NO:2.
  • the invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium.
  • the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
  • the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell proliferation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses.
  • the invention includes assays utilizing these polypeptides to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using these polypeptides as therapeutic agents for the treatment of diseases mediated by cathepsin DC polypeptide counter-structure molecules. Further, methods of using these polypeptides in the design of inhibitors thereof are also an aspect of the invention.
  • the invention further provides a method for using these polypeptides as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein, as well as a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis.
  • the invention further encompasses methods for using the polypeptides of the invention as markers for determining the isoelectric point of an unknown protein, as well as controls for establishing the extent of fragmentation of a protein . Further encompassed by this invention are kits to aid in these determinations.
  • cathepsin DC nucleic acid sequences predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample nucleic acids and/or proteins.
  • the invention also encompasses isolated polypeptides encoded by these nucleic acid molecules, including isolated polypeptides having a molecular weight of approximately 40-45 kD as determined by SDS-PAGE and isolated polypeptides in non-glycosylated form.
  • Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, in addition the use of these antibodies to aid in purifying the cathepsin DC polypeptide.
  • the invention is further directed to methods for using cathepsin DC proteins to determine the specificity of antisera directed against cathepsin B protein and to enhance the specificity of antibodies directed against cathepsin B protein.
  • the invention encompasses methods for using the signal sequence of cathepsin DC protein to enhance the expression and secretion of heterologous proteins.
  • nucleic acid molecules encompassed in the invention includes the following nucleotide sequence:
  • the amino acid sequences ofthe polypeptides encoded by the nucleotide sequence ofthe invention includes: ARRGPG RP LLLVLLAGAAQGGLYFRRGQTCYRPLRGDG APLGRSTY PRPHEYLSPADLPKSWDWRNVDGVNYASITRNQHIPQYCGSCWAHASTSA MADRINIKRKGA PSTLLSVQNVIDCGNAGSCEGGNDLS ⁇ WDYAHQHGIP DETC NYQAKDQECDKFNQCGTCNEFKECHAIRNYTLWRVGDYGSLSGRE K MAEIYANGPISCGI ATERLANYTGGIYAEYQDTTYI HVVSVAG GI SDGTEYWIVRNSWGEPWGERGWLRIVT ⁇ TYKDGKGARYNLAIEEHCTFGD
  • a cDNA encoding human cathepsin DC proteins has been isolated and is disclosed in SEQ ID NO:l. This discovery of the cDNA encoding human cathepsin DC proteins enables construction of expression vectors comprising nucleic acid sequences encoding cathepsin DC proteins; host cells transfected or transformed with the expression vectors; biologically active human cathepsin DC proteins as isolated and purified proteins; and antibodies immunoreactive with cathepsin DC proteins.
  • nucleic acids of the invention enables the construction of expression vectors comprising nucleic acid sequences encoding polypeptides; host cells transfected or transformed with the expression vectors; isolated and purified biologically active polypeptides and fragments thereof; the use of the nucleic acids or oligonucleotides thereof as probes to identify nucleic acid encoding proteins having cathepsin activity, the use of single-stranded sense or antisense oligonucleotides from the nucleic acids to inhibit expression of polynucleotide encoded by the cathepsin DC gene; the use of such polypeptides and soluble fragments to facilitate cancer prognosis; the use of such polypeptides and fragmented peptides as molecular weight markers; the use of such polypeptides and fragmented peptides as controls for peptide fragmentation, and kits comprising these reagents; the use of such polypeptides and fragments thereof to generate antibodies; and the use of antibodies to pur
  • nucleotide sequence refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
  • the nucleic acid molecule has been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual,
  • sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.
  • Nucleic acid molecules of the invention include DNA in both single-stranded and double- stranded form, as well as the RNA complement thereof.
  • DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • Genomic DNA may be isolated by conventional techniques, e.g., using the cDNA of SEQ ID NO: 1 , or a suitable fragment thereof, as a probe.
  • the DNA molecules of the invention include full length genes as well as polynucleotides and fragments thereof.
  • the full length gene may include the N-terminal signal peptide.
  • Other embodiments include DNA encoding a soluble form, e.g., encoding the extracellular domain of the protein, either with or without the signal peptide.
  • the invention further encompasses isolated fragments and oligonucleotides derived from the nucleotide sequence of SEQ ID NO:l, including nucleotide sequence 1-909 of SEQ ID NO:l.
  • the invention also encompasses polypeptides encoded by these fragments and oligonucleotides
  • nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well.
  • the invention also encompasses DNA sequences encoding the mature cathepsin DC protein and comprise nucleotides 70-909 of SEQ ID NO:l.
  • the mature cathepsin DC protein does not contain amino acids 1-23 of SEQ ID NO:2, which are cleaved from cathepsin DC upon secretion from the cell. Therefore, the mature cathepsin DC protein comprises amino acids 24- 303 of SEQ ID NO:2.
  • a particularly preferred nucleotide sequence of the invention is SEQ ID NO:l, as set forth above.
  • the sequences of amino acids encoded by the DNA of SEQ ID NO: 1 is shown in SEQ ID NO:2. This sequence identifies the cathepsin DC polynucleotide as a member of the cathepsin family.
  • Cathepsin DC protein (SEQ ID NO:2) is a new member of the cysteine, or thiol, family of cysteine proteases.
  • the cDNA for cathepsin DC was identified in a library generated from
  • RNA from human bone marrow-derived dendritic cells By sequence homology, it can be assigned to the papain family of cysteine proteases. It is most closely related to a cathepsin B- like cathepsin protein described in the marine worm, Urechis caupo, with 60.2% amino acid identity. It exhibits 30.4% amino acid identity with human cathepsin C protein, 25.9% amino acid identity with human cathepsin B protein, 25.5% amino acid identity with human cathepsin
  • Cathepsin DC protein retains a high conservation of active site amino acid residues, including the cysteine, histidine, and asparagine residues that reside within the active site of the enzyme (Id. and Coleman et al, 1997). Cathepsin DC protein also conserves the two glycine residues that are conserved in most cysteine proteases of the papain family (Fox et al., 1995). This is consistent with the finding that the members of the papain family of cysteine proteases have high conservation of active site amino acid residues, but overall amino acid homologies in the range of 20-60% (Chapman et al., 1994).
  • the full-length cathepsin DC protein is encoded by nucleotides 1-909 of SEQ ID NO:l.
  • the predicted cysteine protease active site of cathepsin DC is encoded by nucleotides 766-824 of SEQ ID NO:l, and the predicted signal peptide sequence is encoded by nucleotides 1-69 of SEQ ID NO:l.
  • Cathepsin DC DNA was detected as an EST in the public databases; however, numerous errors were found in these ESTs, including insertions, deletions, and substitutions. No ESTs containing the cathepsin DC 5' coding sequences that encompass the initiator methionine were found in these databases.
  • Cathepsin DC appears to be the same as cathepsin Z, recently reported by Santamaria et al., J. Biol. Chem. 273:16816-16823 (1998). Northern blot analysis demonstrated that this cathepsin is widely expressed in human tissues, as well as in some tumor cell lines and primary tumors.
  • cathepsin B and cathepsin DC proteins The relationship between cathepsin B and cathepsin DC proteins has been analyzed using polyclonal antisera raised against purified human cathepsin B. Surprisingly, the anti-cathepsin B antiserum was capable of strong binding to cathepsin DC protein. This cross-reactivity occurred in spite of the limited amino acid identity between cathepsin B and cathepsin DC proteins. The antisera did not cross-react with purified human cathepsin D or recombinant human cathepsin L. The cross-reactivity of anti-cathepsin B antisera with cathepsin DC demonstrated the lack of specificity of this antisera for cathepsin B protein.
  • a DNA sequence can vary from that shown in SEQ ID NO:l, and still encode a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.
  • In vitro mutagenesis would include numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
  • the invention thus provides isolated DNA sequences encoding polypeptides of the invention, selected from: (a) DNA comprising the nucleotide sequence of SEQ ID NO:l; (b) DNA encoding the polypeptides of SEQ ID NO:2; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b), (c), or (d) and which encode polypeptides of the invention.
  • Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the native cathepsin DC nucleotide sequences disclosed herein under conditions of moderate or severe stringency, and which encode cathepsin DC proteins.
  • conditions of moderate stringency can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA.
  • the basic conditions are set forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed.
  • the invention further provides isolated DNA sequences encoding cathepsin DC proteins, selected from: (a) DNA derived from the coding region of a native mammalian cathepsin DC gene; (b) cDNA comprising the nucleotide sequences 1-909 or 70-909 of SEQ ID NO:l; (c) DNA capable of hybridization to a DNA of (a) under conditions of moderate stringency and which encodes cathepsin DC proteins; and (d) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b) or (c) and which encodes cathepsin DC proteins.
  • Cathepsin DC proteins encoded by such DNA equivalent sequences are encompassed by the invention.
  • DNA that is equivalent to the DNA sequence of SEQ ID NO: 1 will hybridize under moderately stringent conditions to the double-stranded native DNA sequence that encode proteins comprising amino acid sequences of 1-303 of SEQ ID NO:2.
  • Examples of cathepsin DC proteins encoded by such DNA include, but are not limited to, cathepsin DC protein fragments and cathepsin DC proteins comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described above.
  • Cathepsin DC proteins encoded by DNA derived from other mammalian species, wherein the DNA will hybridize to the complement of the cDNA of SEQ ID NO: 1 are also encompassed.
  • DNA encoding polypeptide fragments and polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described below.
  • nucleic acid molecules of the invention also comprise nucleotide sequences that are at least 80% identical to a native sequence. Also contemplated are embodiments in which a nucleic acid molecule comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence.
  • the percent identity may be determined by visual inspection and mathematical calculation.
  • the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. ⁇ Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non- identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6145, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of
  • the invention also provides isolated nucleic acids useful in the production of polypeptides.
  • polypeptides may be prepared by any of a number of conventional techniques.
  • a DNA sequence encoding a cathepsin DC polypeptide, or desired fragment thereof may be subcloned into an expression vector for production of the polypeptide or fragment.
  • the DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide.
  • the desired fragment may be chemically synthesized using known techniques.
  • DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels.
  • oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion.
  • Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.
  • PCR polymerase chain reaction
  • Oligonucleotides that define the desired termini of the DNA fragment are employed as 5' and 3' primers.
  • the oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into an expression vector.
  • PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to
  • the invention encompasses polypeptides and fragments thereof in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.
  • polypeptides and Fragments Thereof include full length proteins encoded by the nucleic acid sequences set forth above.
  • Particularly preferred polypeptides comprise the amino acid sequence of SEQ ID NO:2 with particularly preferred fragments comprising amino acids 1-303 or 24-303 of SEQ ID NO:2, or encoded by nucleotides 766-824 of SEQ ID NO:l.
  • the polypeptide of SEQ ID NO:2 includes an N-terminal hydrophobic region that functions as a signal peptide.
  • Computer analysis predicts that the signal peptide corresponds to residues 1 to 23 of SEQ ID NO:2. Cleavage of the signal peptide thus would yield a mature protein comprising amino acids 24 through 303 of SEQ ID NO:2.
  • the skilled artisan will recognize that the above-described boundaries of such regions of the polypeptide are approximate, and that the boundaries of the signal peptide (which may be predicted by using computer programs available for that purpose) may differ from those described above.
  • polypeptides of the invention may be membrane bound or they may be secreted and thus soluble. Soluble polypeptides are capable of being secreted from the cells in which they are expressed. In general, soluble polypeptides may be identified (and distinguished from non- soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the protein.
  • Soluble polypeptides thus include, but are not limited to, polypeptides comprising amino acids 24 to 303 of SEQ ID NO:2.
  • soluble forms are advantageous for certain applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Further, soluble polypeptides are generally more suitable for intravenous administration.
  • the invention also provides polypeptides and fragments of the extracellular domain that retain a desired biological activity. Particular embodiments are directed to polypeptide fragments that retain the cathepsin DC proteolytic activity or the ability to bind cathepsin DC counter-structures. Such a fragment may be a soluble polypeptide, as described above. In another embodiment, the polypeptides and fragments advantageously include regions that are conserved in the cathepsin family as described above.
  • polypeptide fragments comprising at least 20, or at least 30, contiguous amino acids of the sequence of SEQ ID NO:2. Fragments derived from the cytoplasmic domain find use in studies of signal transduction, and in regulating cellular processes associated with transduction of biological signals. Polypeptide fragments also may be employed as immunogens, in generating antibodies.
  • Naturally occurring variants as well as derived variants of the polypeptides and fragments are provided herein.
  • Cathepsin DC protein variant as referred to herein means a polypeptide substantially homologous to native cathepsin DC proteins, but which has an amino acid sequence different from that of native cathepsin DC proteins (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions.
  • the variant amino acid sequence preferably lo is at least 80% identical to a native cathepsin DC amino acid sequence, most preferably at least 90% identical.
  • the percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. ⁇ Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman ⁇ Adv. Appl. Math 2:482, 1981).
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical
  • Variants also include other embodiments in which a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the preferred polypeptide or fragment thereof.
  • Percent identity may be determined as above.
  • the percent identity of two protein sequences can be determined by comparing sequence information using the GAP computer program, based on the algorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps.
  • Other programs used by one skilled in the art of sequence comparison may also be used.
  • variants of the invention include, for example, those that result from alternate mRNA splicing events or from proteolytic cleavage.
  • Alternate splicing of mRNA may, for example, yield a truncated but biologically active protein, such as a naturally occurring soluble form of the protein.
  • Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the protein (generally from 1-5 terminal amino acids). Proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein.
  • the invention provides isolated and purified, or homogeneous, cathepsin DC proteins, both recombinant and non-recombinant.
  • Variants and derivatives of native cathepsin DC proteins can be obtained by mutations of nucleotide sequences coding for native cathepsin DC proteins. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion.
  • Exemplary methods of making the alterations set forth above are disclosed by Walder et al. ⁇ Gene 42:133, 1986); Bauer et al. ⁇ Gene 37:73, 1985); Craik
  • polypeptides that may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives may be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide.
  • the conjugate can comprise a signal or leader polypeptide sequence (e.g. the ⁇ -factor leader of Saccharomyces) at the N-terminus of a cathepsin DC protein.
  • the signal or leader peptide co-translationally or post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.
  • Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein, as discussed in more detail below.
  • fusion proteins include covalent or aggregative conjugates of the polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C- terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. Further, fusion proteins can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., BiolTechnology 6:1204, 1988.
  • FLAG ® peptide is the FLAG ® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
  • a murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG ® peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912, hereby incorporated by reference.
  • the 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG ® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.
  • variant polypeptides that retain the native biological activity or the substantial equivalent thereof.
  • variants that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Patent No. 5,512.457 and as set forth below.
  • Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions.
  • Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.
  • a given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics.
  • conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another.
  • Other conservative substitutions e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • the DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide.
  • the invention further includes polypeptides of the invention with or without associated native-pattern glycosylation.
  • Polypeptides expressed in yeast or mammalian expression systems e.g., COS-1 or COS-7 cells
  • yeast or mammalian expression systems e.g., COS-1 or COS-7 cells
  • Expression of polypeptides of the invention in bacterial expression systems, such as E. coli. provides non-glycosylated molecules.
  • a given preparation may include multiple differentially glycosylated species of the protein. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase.
  • glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).
  • similar DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are encompassed by the invention.
  • N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems.
  • N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.
  • nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain.
  • the Ser or Thr can by replaced with another amino acid, such as Ala.
  • Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846, hereby incorporated by reference.
  • sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation.
  • EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein.
  • KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg- Arg, Arg-Lys, and Lys- Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys- Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.
  • oligomers or fusion proteins that contain cathepsin DC polypeptides.
  • Such oligomers may be in the form of covalently-linked or non-covalently-linked multimers, including dimers, trimers. or higher oligomers.
  • preferred polypeptides are soluble and thus these oligomers may comprise soluble polypeptides.
  • the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites.
  • One embodiment of the invention is directed to oligomers comprising multiple polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides.
  • Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization.
  • Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.
  • an oligomer is prepared using polypeptides derived from immunoglobulins.
  • Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. ⁇ Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992).
  • One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody.
  • a gene fusion encoding the polypeptide/Fc fusion protein is inserted into an appropriate expression vector.
  • Polypeptide/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules.
  • Fc polypeptide as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides comprise an Fc polypeptide derived from a human IgGl antibody.
  • Fc polypeptide is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgGl antibody.
  • Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., ⁇ EMBO J. 13:3992-4001, 1994) incorporated herein by reference.
  • amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala.
  • the mutein exhibits reduced affinity for Fc receptors.
  • fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.
  • the Fc polypeptide preferably is fused to the C-terminus of a soluble cathepsin DC protein (comprising only the extracellular domain).
  • a gene fusion encoding the cathepsin DC prote ⁇ r.Fc fusion protein is inserted into an appropriate expression vector.
  • Cathepsin DC protein:Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc proteins, yielding divalent cathepsin DC proteins.
  • the polypeptides of the invention may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four cathepsin DC extracellular regions.
  • the oligomer is a fusion protein comprising multiple polypeptides, with or without peptide linkers (spacer peptides).
  • suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby incorporated by reference.
  • a DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique.
  • a chemically synthesized oligonucleotide encoding the linker may be ligated between the sequences.
  • a fusion protein comprises from two to four soluble cathepsin DC polypeptides, separated by peptide linkers. Leucine-Zippers
  • Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins.
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • the zipper domain (also referred to herein as an oligomerizing, or oligomer-forming, domain) comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids.
  • Examples of zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA-binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989).
  • Two nuclear transforming proteins, fos and jun also exhibit zipper domains, as does the gene product of the murine proto-oncogene.
  • c-myc (Landschulz et al., Science 240:1759, 1988).
  • the products of the nuclear oncogenes ⁇ -? andywn comprise zipper domains that preferentially form heterodimer (O'Shea et al., Science 245:646,
  • the zipper domain is necessary for biological activity (DNA binding) in these proteins.
  • the fusogenic proteins of several different viruses including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS
  • Zipper domains fold as short, parallel coiled coils (O'Shea et al., Science 254:539; 1991).
  • the general architecture of the parallel coiled coil has been well characterized, with a "knobs- into-holes" packing as proposed by Crick in 1953 ⁇ Acta Crystallogr. 6:689).
  • the dimer formed by a zipper domain is stabilized by the heptad repeat, designated ⁇ abcdefg) n according to the notation of McLachlan and Stewart (J. Mol. Biol. 98:293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine. which line up on the same face of a helix.
  • leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al. ⁇ FEBS Letters 344:191, 1994), hereby incorporated by reference.
  • SPD lung surfactant protein D
  • the use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al. ⁇ Semin. Immunol. 6:267-278, 1994).
  • Recombinant fusion proteins comprising a soluble polypeptide fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomer that forms is recovered from the culture supernatant.
  • leucine zipper moieties preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, 1994) and in U.S. Patent 5,716,805, hereby incorporated by reference in their entirety.
  • This lung SPD-derived leucine zipper peptide comprises the amino acid sequence Pro Asp Val Ala Ser Leu Arg Gin Gin Val Glu Ala Leu Gin Gly Gin Val Gin His Leu Gin Ala Ala Phe Ser Gin Tyr.
  • a leucine zipper that promotes trimerization is a peptide comprising the amino acid sequence Arg Met Lys Gin He Glu Asp Lys He Glu Glu He Leu Ser Lys He Tyr His He Glu Asn Glu He Ala Arg He Lys Lys Leu He Gly Glu Arg, as described in U.S. Patent 5,716,805.
  • an N-terminal Asp residue is added; in another, the peptide lacks the N-terminal Arg residue.
  • Fragments of the foregoing zipper peptides that retain the property of promoting oligomerization may be employed as well.
  • Examples of such fragments include, but are not limited to, peptides lacking one or two of the N-terminal or C-terminal residues presented in the foregoing amino acid sequences.
  • Leucine zippers may be derived from naturally occurring leucine zipper peptides, e.g., via conservative substitution(s) in the native amino acid sequence, wherein the peptide's ability to promote oligomerization is retained.
  • Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric cathepsin DC polypeptides.
  • synthetic peptides that promote oligomerization may be employed.
  • leucine residues in a leucine zipper moiety are replaced by isoleucine residues.
  • Such peptides comprising isoleucine may be referred to as isoleucine zippers, but are encompassed by the term "leucine zippers" as employed herein.
  • polypeptides and fragments of the invention may be accomplished by any suitable technique, including but not limited to the following:
  • the present invention also provides recombinant cloning and expression vectors containing DNA, as well as host cell containing the recombinant vectors.
  • Expression vectors comprising DNA may be used to prepare the polypeptides or fragments of the invention encoded by the DNA.
  • a method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture.
  • the skilled artisan will recognize that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble form that is secreted from the host cell.
  • the vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence.
  • a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence.
  • An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
  • a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell.
  • a protein preparation may include a mixture of protein molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
  • Suitable host cells for expression of polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory
  • Prokaryotes include gram-negative or gram-positive organisms. Suitable prokaryotic host cells for transformation include, for example, E. coli. Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • a polypeptide may include an N- terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide.
  • Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
  • useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • An appropriate promoter and a DNA sequence are inserted into the pBR322 vector.
  • Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA).
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 257:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4051, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
  • ⁇ -lactamase penicillinase
  • lactose promoter system Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 257:544, 1979
  • tryptophan (trp) promoter system Goeddel et al., Nucl. Acids Res. 8:40
  • a particularly useful prokaryotic host cell expression system employs a phage ⁇ P L promoter and a cI857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the ⁇ P L promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).
  • Cathepsin DC DNA may be cloned in-frame into the multiple cloning site of an ordinary bacterial expression vector.
  • the vector would contain an inducible promoter upstream of the cloning site, such that addition of an inducer leads to high-level production of the recombinant protein at a time of the investigator's choosing.
  • expression levels may be boosted by incorporation of codons encoding a fusion partner (such as hexahistidine) between the promoter and the gene of interest.
  • the resulting "expression plasmid" may be propagated in a variety of strains of E. coli.
  • the bacterial cells are propagated in growth medium until reaching a pre-determined optical density. Expression of the recombinant protein is then induced, e.g. by addition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activates expression of proteins from plasmids containing a lac operator/promoter. After induction (typically for 1-4 hours), the cells are harvested by pelleting in a centrifuge, e.g. at 5,000 x G for 20 minutes at 4°C.
  • IPTG isopropyl-b-D-thiogalactopyranoside
  • the pelleted cells may be resuspended in ten volumes of 50 mM Tris-HCl (pH 8)/l M NaCl and then passed two or three times through a French press. Most highly-expressed recombinant proteins form insoluble aggregates known as inclusion bodies. Inclusion bodies can be purified away from the soluble proteins by pelleting in a centrifuge at 5,000 x G for 20 minutes, 4°C. The inclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/l% Triton X-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/ 0.1 M DTT.
  • the protein of interest will, in most cases, be the most abundant protein in the resulting clarified supernatant.
  • This protein may be "refolded" into the active conformation by dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCL/5 mM Zn(OAc) : /l mM GSSG/0.1 mM GSH.
  • purification can be carried out by a variety of chromatographic methods such as ion exchange or gel filtration. In some protocols, initial purification may be carried out before refolding.
  • hexahistidine-tagged fusion proteins may be partially purified on immobilized Nickel.
  • polypeptides may be expressed in yeast host cells, preferably from the Saccharomyces genus ⁇ e.g., S. cerevisiae).
  • yeast host cells preferably from the Saccharomyces genus ⁇ e.g., S. cerevisiae.
  • yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replicating sequence
  • Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2013, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
  • enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase.
  • suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657.
  • Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2614, 1982) and Beier et al.
  • Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp r gene and origin of replication) into the above-described yeast vectors.
  • the yeast ⁇ -factor leader sequence may be employed to direct secretion of the polypeptide.
  • the ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984.
  • Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
  • a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.
  • the Hinnen et al. protocol selects for Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil.
  • Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a "rich" medium.
  • a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
  • Mammalian or insect host cell culture systems also may be employed to express recombinant polypeptides.
  • Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, BiolTechnology 6:47 (1988).
  • Established cell lines of mammalian origin also may be employed.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:115, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. ⁇ EMBO J. 10: 2821, 1991).
  • COS-7 line of monkey kidney cells ATCC CRL 1651
  • L cells C127 cells
  • 3T3 cells ATCC CCL 163
  • Chinese hamster ovary (CHO) cells HeLa cells
  • BHK ATCC CRL 104708
  • CV1/EBNA cell line ATCC CRL 104708
  • DHFR dihydrofolate reductase
  • a plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media.
  • selectable markers include cDNAs conferring resistance to antibiotics, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes.
  • Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included. Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp.
  • EASE expression augmenting sequence element
  • DHFR has been shown to improve transfectabihty of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology, 1990).
  • Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al.. Biotechniques 22:150-161, 1997, and p2A5I described by Morris et al., Animal Cell Technology, 1997, pp. 529-534.
  • a useful high expression vector, pCAVNOT has been described by Mosley et al., Cell 59:335-348, 1989.
  • Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg ⁇ Mol. Cell. Biol. 5:280, 1983).
  • a useful system for stable high level expression of mammalian cDNAs in C 127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. ⁇ Mol. Immunol. 25:935, 1986).
  • a useful high expression vector, PMLSV N1/N4 described by Cosman et al., Nature 572:768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982, incorporated by reference herein.
  • the vectors can be derived from retroviruses.
  • FLAG ® can also be used.
  • FLAG ® technology is centered on the fusion of a low molecular weight (lkD), hydrophilic, FLAG ® marker peptide to the N-terminus of a recombinant protein expressed by pFLAG ® expression vectors.
  • pDC311 is another specialized vector used for expressing proteins in CHO cells.
  • pDC311 is characterized by a bicistronic sequence containing the gene of interest and a dihydrofolate reductase (DHFR) gene with an internal ribosome binding site for DHFR translation, an expression augmenting sequence element (EASE), the human CMV promoter, a tripartite leader sequence, and a polyadenylation site.
  • DHFR dihydrofolate reductase
  • the native signal peptide may be replaced by a heterologous signal peptide or leader sequence, if desired.
  • the choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced.
  • heterologous signal peptides that are fimctional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.
  • IL-7 interleukin-7
  • the expression of recombinant cathepsin DC proteins has been accomplished utilizing a mammalian expression vector.
  • a mammalian expression vector See Example 1.
  • the expression of cathepsin DC proteins in mammalian cells employed a vector containing a heterologous cytomegalovirus immediate early promoter/enhancer to drive expression of the cathepsin DC proteins cDNA. Transformation of this vector into CV-1/EBNA cells resulted in high level expression and secretion of cathepsin DC protein. It is understood of course that many different vectors and techniques can be used for the expression and purification of cathepsin DC proteins and that this embodiment in no way limits the scope of the invention.
  • the invention also includes methods of isolating and purifying the polypeptides and fragments thereof.
  • cathepsin DC proteins refers to a genus of proteins that further encompasses proteins having the amino acid sequence 1-303 of SEQ ID NO:2, as well as those proteins having a high degree of similarity (at least 90% identity) with such amino acid sequences and which proteins are biologically active.
  • cathepsin DC proteins refers to the biologically active gene products of the nucleotides 1-909 of SEQ ID NO:l.
  • the isolated and purified cathepsin DC protein according to the invention has a molecular weight of approximately 40-45 kD. It is understood that the molecular weight of cathepsin DC proteins can be varied by fusing additional peptide sequences to both the amino and carboxyl terminal ends of cathepsin DC proteins. Fusions of additional peptide sequences at the amino and carboxyl terminal ends of cathepsin DC proteins can be used to enhance expression of cathepsin DC proteins or aid in the purification of the protein.
  • isolated and purified means that the cathepsin DC proteins are essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source.
  • substantially purified refers to a mixture that contains cathepsin DC proteins and is essentially free of association with other proteins or polypeptides, but for the presence of known proteins that can be removed using a specific antibody, and which substantially purified cathepsin DC proteins retain biological activity.
  • purified cathepsin DC proteins refers to either the “isolated and purified” form of cathepsin DC proteins or the “substantially purified” form of cathepsin DC proteins, as both are described herein.
  • isolated polypeptides or fragments thereof encompassed by this invention are polypeptides or fragments that are not in an environment identical to an environment in which it or they can be found in nature.
  • cathepsin DC protein can be produced by recombinant expression systems as described above or purified from naturally occurring cells.
  • Cathepsin DC proteins can be substantially purified, as indicated by a single protein band upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • the purification of recombinant polypeptides or fragments can be accomplished using fusions of polypeptides or fragments of the invention to another polypeptide to aid in the purification of polypeptides or fragments of the invention.
  • fusion partners can include the poly-His or other antigenic identification peptides described above as well as the Fc moieties described previously.
  • the recombinant polypeptide or fragment can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps.
  • the culture medium first can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the concentrate can be applied to a purification matrix such as a gel filtration medium.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a chromatofocusmg step can be employed.
  • a hydrophobic interaction chromatography step can be employed.
  • Suitable matrices can be phenyl or octyl moieties bound to resins.
  • affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal- chelating columns.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
  • RP-HPLC media e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups
  • an affinity column comprising a polypeptide-binding protein of the invention, such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides.
  • polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention.
  • polypeptide-binding proteins such as the anti-polypeptide antibodies of the invention or other proteins that may interact with the polypeptide of the invention, can be bound to a solid phase support such as a column chromatography matrix or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface.
  • Adherence of polypeptide-binding proteins of the invention to a solid phase contacting surface can be accomplished by any means, for example, magnetic microspheres can be coated with these polypeptide-binding proteins and held in the incubation vessel through a magnetic field. Suspensions of cell mixtures are contacted with the solid phase that has such polypeptide-binding proteins thereon.
  • Cells having polypeptides of the invention on their surface bind to the fixed polypeptide-binding protein and unbound cells then are washed away.
  • This affinity-binding method is useful for purifying, screening, or separating such polypeptide-expressing cells from solution.
  • Methods of releasing positively selected cells from the solid phase are known in the art and encompass, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-injurious to the cells and are preferably directed to cleaving the cell-surface binding partner.
  • mixtures of cells suspected of containing polypeptide-expressing cells of the invention first can be incubated with a biotinylated polypeptide-binding protein of the invention. Incubation periods are typically at least one hour in duration to ensure sufficient binding to polypeptides of the invention.
  • the resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides the binding of the polypeptide-binding cells to the beads.
  • avidin-coated beads is known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239 (1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.
  • cathepsin DC protein-binding proteins are anti- cathepsin DC protein antibodies, and other proteins that are capable of high-affinity binding of cathepsin DC proteins.
  • a preferred cathepsin DC protein-binding protein is an anti-cathepsin DC protein monoclonal antibody.
  • the desired degree of purity depends on the intended use of the protein.
  • a relatively high degree of purity is desired when the polypeptide is to be administered in vivo, for example.
  • the polypeptides are purified such that no protein bands corresponding to other proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE, due to differential glycosylation, differential post- translational processing, and the like.
  • the polypeptide of the invention is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-
  • the protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.
  • Recombinant protein produced in bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • Transformed yeast host cells are preferably employed to express cathepsin DC proteins as secreted proteins in order to simplify purification.
  • Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296:11 ⁇ , 1984).
  • Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
  • the purified polypeptides of the invention may be tested for the ability to bind cathepsin DC counterstructures in any suitable assay, such as a conventional binding assay.
  • the polypeptide may be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like).
  • a detectable reagent e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like.
  • the labeled polypeptide is contacted with cells expressing cathepsin DC counterstructures.
  • the cells then are washed to remove unbound labeled polypeptide, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
  • a binding assay procedure is as follows.
  • a recombinant expression vector containing cathepsin DC counterstructure cDNA is constructed, wherein the cathepsin DC counterstructure comprises an N-terminal cytoplasmic domain, a transmembrane region, and a C- terminal extracellular domain.
  • CVl-EBNA-1 cells in 10 cm 2 dishes are transfected with the recombinant expression vector.
  • CV-l/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen- 1 driven from the CMV immediate-early enhancer/promoter.
  • CV1-EBNA- 1 was derived from the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as described by McMahan et al. ⁇ EMBO J. 10:2821, 1991).
  • the transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4 x 10 4 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37°C with various concentrations of, for example, a soluble polypeptide/Fc fusion protein made as set forth above.
  • BM-NFDM binding medium
  • RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2
  • Cells then are washed and incubated with a constant saturating concentration of a 125 I-mouse anti- human IgG in binding medium, with gentle agitation for 1 hour at 37°C. After extensive washing, cells are released via trypsinization.
  • the mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA.
  • the antibody is radioiodinated using the standard chloramine-T method.
  • the antibody will bind to the Fc portion of any polypeptide/Fc protein that has bound to the cells.
  • nonspecific binding of 125 I-antibody is assayed in the absence of the Fc fusion protein, as well as in the presence of the Fc fusion protein and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody.
  • Cell-bound 125 I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. NY. Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.
  • Another type of suitable binding assay is a competitive binding assay.
  • biological activity of a variant may be determined by assaying for the variant's ability to compete with the native protein for binding to cathepsin DC counterstructures.
  • ком ⁇ онентs that may be employed in competitive binding assays include radiolabeled cathepsin DC and intact cells expressing cathepsin DC (endogenous or recombinant) on the cell surface.
  • a radiolabeled soluble cathepsin DC fragment can be used to compete with a soluble cathepsin DC variant for binding to cell surface cathepsin DC counterstructures.
  • a soluble cathepsin DC counterstructure/Fc fusion protein bound to a solid phase through the interaction of Protein A or Protein G (on the solid phase) with the Fc moiety.
  • Chromatography columns that contain Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, NJ.
  • Another type of competitive binding assay utilizes radiolabeled soluble cathepsin DC counterstructures, such as a soluble cathepsin DC counterstructure/Fc fusion protein, and intact cells expressing cathepsin DC.
  • Qualitative results can be obtained by competitive autoradiographic plate binding assays, while Scatchard plots (Scatchard, Ann. N. Y. Acad. Sci. 51:660, 1949) may be utilized to generate quantitative results.
  • nucleic acids of the invention including DNA, and oligonucleotides thereof can be used:
  • _ as probes to identify nucleic acid encoding proteins having cathepsin activity; _ as single-stranded sense or antisense oligonucleotides, to inhibit expression of polypeptide encoded by the cathepsin DC gene; _ to help detect defective genes in an individual; and
  • nucleic acids of the invention are useful as probes or primers.
  • Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence.
  • a DNA fragment comprises at least 30, or at least 60, contiguous nucleotides of a DNA sequence.
  • probes based on the (human) DNA sequence of SEQ ID NO:l may be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques.
  • sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
  • antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
  • Antisense or sense oligonucleotides comprise a fragment of DNA (SEQ ID NO:l). Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides.
  • Stein and Cohen ⁇ Cancer Res. 48:2659, 1988 and van der Krol et al. ⁇ BioTechniques 6:958, 1988).
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block or inhibit protein expression by one of several means, including enhanced degradation of the mRNA by RNAseH, inhibition of splicing, premature termination of transcription or translation, or by other means.
  • the antisense oligonucleotides thus may be used to block expression of proteins.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo ⁇ i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
  • sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
  • intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO 4 - mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein- Barr virus.
  • Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo.
  • Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5 A, DCT5B, and DCT5C (see PCT Application US 90/02656).
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • Cathepsin DC proteins are introduced into the intracellular environment by well-known means, such as by encasing the protein in liposomes or coupling it to a monoclonal antibody targeted to a specific cell type.
  • Uses include, but are not limited to, the following:
  • the polypeptides of the invention find use as a protein purification reagents.
  • the polypeptides may be attached to a solid support material and used to purify cathepsin DC counterstructure proteins by affinity chromatography.
  • a polypeptide in any form described herein that is capable of binding cathepsin DC counterstructures
  • chromatography columns conta ⁇ fifr ⁇ g > functional groups that will react with functional groups on amino acid side chains of proteins are available (Pharmacia Biotech, Inc., Piscataway, NJ).
  • a polypeptide/Fc protein is attached to Protein A- or Protein G-containing chromatography columns through interaction with the Fc moiety.
  • the polypeptide also finds use in purifying or identifying cells that express cathepsin DC counterstructures on the cell surface.
  • Polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate.
  • a solid phase such as a column chromatography matrix or a similar suitable substrate.
  • magnetic microspheres can be coated with the polypeptides and held in an incubation vessel through a magnetic field.
  • Suspensions of cell mixtures containing cathepsin DC counterstructure expressing cells are contacted with the solid phase having the polypeptides thereon.
  • Cells expressing cathepsin DC counterstructures on the cell surface bind to the fixed polypeptides, and unbound cells then are washed away.
  • polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for cathepsin DC counterstructure expression. After incubation, unbound labeled matter is removed and the presence or absence of the detectable moiety on the cells is determined.
  • mixtures of cells suspected of containing cathepsin DC counterstructure cells are incubated with biotinylated polypeptides. Incubation periods are typically at least one hour in duration to ensure sufficient binding.
  • the resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads.
  • Procedures for using avidin-coated beads are known (see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods.
  • cathepsin DC proteins can be used to determine the specificity of antisera against cathepsin B protein.
  • Cathepsin B and cathepsin DC proteins can be resolved in separate lanes of a denaturing 12% polyacrylamide gel by electrophoresis under conventional conditions, such as those described in Laemmli, U.K. ⁇ Nature 227:680, 1970). Both cathepsin B and cathepsin DC proteins can be resolved in duplicate lanes of the gel.
  • the proteins can be transferred from the gel to a nitrocellulose membrane under conventional conditions, such as those described in Towbin, H. et al. ⁇ Proc Natl. Acad. Sci.
  • the membrane can then be cut so as to generate two membranes, such that both cathepsin B and cathepsin DC proteins are present on both membranes.
  • An antiserum generated against cathepsin B protein can be incubated with one of the nitrocellulose membranes under conventional conditions for immunoblot analysis, such as those described in Harper and Murphy ⁇ Anal. Biochem. 193:59, 1991).
  • a labeled secondary antibody is used to detect binding of the antiserum to specific proteins under conventional conditions.
  • the duplicate nitrocellulose membrane is incubated only with the labeled secondary antibody.
  • a lack of interaction of the secondary antibody with cathepsin B and DC proteins allows an assessment of the specificity of the primary antiserum.
  • Interaction of the antiserum with cathepsin B protein indicates the ability of the antiserum to recognize cathepsin B protein.
  • Cross-reaction of the antiserum with cathepsin DC proteins indicates the lack of specificity of the antiserum for cathepsin B.
  • cathepsin DC proteins can be used to create an affinity column.
  • Isolated and purified cathepsin DC proteins can be attached to a cyanogen bromide-activated agarose beads (Pharmacia Biotech) by conventional means.
  • the beads can be rinsed to remove unbound cathepsin DC proteins.
  • a chromatography column can then be filled with the cathepsin DC protein-agarose beads. It is understood of course that many different techniques can be used for the generation of a cathepsin DC protein affinity column and that this embodiment in no way limits the scope of the invention.
  • a cathepsin DC protein affinity column can be used to enhance the specificity of an antiserum generated against cathepsin B protein.
  • An antiserum generated against cathepsin B protein is contacted with a cathepsin DC protein affinity column and the antibodies that interact with cathepsin DC proteins are allowed to bind under conventional conditions.
  • the unbound antiserum can be removed from the column.
  • the unbound antiserum having been depleted for antibodies that interact with cathepsin DC proteins, has enhanced specificity for cathepsin B protein.
  • cathepsin B protein can be used to determine the expression level of cathepsin B protein in biopsies of human tumor tissues using conventional immunochemical detection methods, wherein the level of cathepsin B protein expression serves as a prognostic marker for tumor progression.
  • cathepsin B protein Due to enhanced specificity in the detection of cathepsin B protein, the depletion of antibodies that cross-react with cathepsin DC proteins results in enhanced accuracy in the prognosis of the progression of these tumors. It is understood of course that many different antibodies and immunological techniques can be used for the detection of cathepsin B protein in tumor tissues using antisera depleted for anti-cathepsin DC antibodies and that this embodiment in no way limits the scope of the invention.
  • Polypeptides also find use in measuring the biological activity of cathepsin DC counterstructure protein in terms of their binding affinity.
  • the polypeptides thus may be employed by those conducting "quality assurance” studies, e.g., to monitor shelf life and stability of protein under different conditions.
  • the polypeptides may be employed in a binding affinity study to measure the biological activity of a cathepsin DC counterstructure protein that has been stored at different temperatures, or produced in different cell types.
  • the proteins also may be used to determine whether biological activity is retained after modification of a cathepsin DC counterstructure protein (e.g., chemical modification, truncation, mutation, etc.).
  • the binding affinity of the modified cathepsin DC counterstructure protein is compared to that of an unmodified cathepsin DC counterstructure protein to detect any adverse impact of the modifications on biological activity of cathepsin DC counterstructures.
  • the biological activity of a cathepsin DC counterstructure protein thus can be ascertained before it is used in a research study, for example.
  • the polypeptides also find use as carriers for delivering agents attached thereto to cells bearing cathepsin DC counterstructures.
  • the polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express cathepsin DC counterstructures on the cell surface) in in vitro or in vivo procedures.
  • Detectable (diagnostic) and therapeutic agents that may be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application.
  • Radionuclides suitable for diagnostic use include, but are not limited to, 123 1, 131 1, 99m Tc, m In, and 76 Br.
  • Radionuclides suitable for therapeutic use are 131 1, 211 At, 77 Br, 186 Re, 188 Re, 2I2 Pb, 21 Bi, 109 Pd, M Cu, and 67 Cu.
  • Such agents may be attached to the polypeptide by any suitable conventional procedure.
  • the polypeptide comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example.
  • the protein or agent may be derivatized to generate or attach a desired reactive functional group.
  • the derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling proteins are known. Radionuclide metals may be attached to polypeptides by using a suitable bifunctional chelating agent, for example.
  • Conjugates comprising polypeptides and a suitable diagnostic or therapeutic agent (preferably covalently linked) are thus prepared.
  • the conjugates are administered or otherwise employed in an amount appropriate for the particular application.
  • Polypeptides of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of the polypeptides. These polypeptides may be administered to a mammal afflicted with such a disorder.
  • the polypeptides may also be employed in inhibiting a biological activity of cathepsin DC counterstructures, in in vitro or in vivo procedures.
  • a purified polypeptide may be used to inhibit binding of cathepsin DC counterstructures to endogenous cell surface cathepsin DC counterstructures. Biological effects that result from the binding of cathepsin DC counterstructure to endogenous receptors thus are inhibited.
  • Cathepsin DC may be administered to a mammal to treat a cathepsin DC counterstructure-mediated disorder.
  • cathepsin DC counterstructure-mediated disorders include conditions caused (directly or indirectly) or exacerbated by cathepsin DC counterstructures.
  • compositions of the present invention may contain a polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, and biologically active fragments.
  • the composition comprises a soluble polypeptide or an oligomer comprising soluble cathepsin DC polypeptides.
  • compositions comprising an effective amount of a polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein.
  • the polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers.
  • Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980,
  • compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • PEG polyethylene glycol
  • metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc.
  • liposomes such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc.
  • Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.
  • compositions of the invention can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation.
  • parenteral includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g., at a site of disease or injury. Sustained release from implants is also contemplated.
  • suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
  • Compositions comprising nucleic acids in physiologically acceptable formulations are also contemplated. DNA may be formulated for injection, for example. Research Reagents
  • polypeptide of the present invention is as a research tool for studying the biological effects that result from inhibiting cathepsin DC counterstructure/cathepsin DC interactions on different cell types.
  • Polypeptides also may be employed in in vitro assays for detecting cathepsin DC counterstructure or cathepsin DC or the interactions thereof.
  • Cathepsin DC proteins can also be used as a reagent to identify (a) any protein that cathepsin DC protein regulates, and (b) other proteins with which it might interact. Cathepsin DC proteins could be used by coupling recombinant protein to an affinity matrix, or by using them as a bait in the 2-hybrid system. In addition, cathepsin DC proteins according to the invention are useful for the structure- based design of an cathepsin DC protein inhibitor.
  • Such a design would comprise the steps of determining the three-dimensional structure of such the cathepsin DC protein, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predictive reactive site, and determining the inhibiting activity of the molecule.
  • the purified cathepsin DC proteins according to the invention will facilitate the discovery of inhibitors of cathepsin DC proteins.
  • the use of a purified cathepsin DC proteins in the screening of potential inhibitors thereof is important and can eliminate or reduce the possibility of interfering reactions with contaminants.
  • cathepsin DC proteins can also be used for structure-based design of cathepsin DC protein-inhibitors. Such structure-based design is also known as "rational drug design.”
  • the cathepsin DC proteins can be three-dimensionally analyzed by, for example. X-ray crystallography, nuclear magnetic resonance or homo logy modeling, all of which are well-known methods.
  • the use of cathepsin DC protein structural information in molecular modeling software systems to assist in inhibitor design and inhibitor-cathepsin DC protein interaction is also encompassed by the invention.
  • Such computer-assisted modeling and drug design can utilize information such as chemical conformational analysis, electrostatic potential of the molecules, protein folding, etc.
  • a particular method of the invention comprises analyzing the three dimensional structure of cathepsin DC proteins for likely binding sites of substrates, synthesizing a new molecule that incorporates a predictive reactive site, and assaying the new molecule as described above.
  • Cathepsin DC DNA, cathepsin DC proteins, and antibodies against cathepsin DC proteins can be used as reagents in a variety of research protocols. A sample of such research protocols are given in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold
  • these reagents can serve as markers for cell specific or tissue specific expression of RNA or proteins.
  • these reagents can be used to investigate constituitive and transient expression of cathepsin DC RNA or proteins.
  • Cathepsin DC DNA can be used to determine the chromosomal location of cathepsin DC DNA and to map genes in relation to this chromosomal location.
  • Cathepsin DC DNA can also be used to examine genetic heterogeneity and heredity through the use of techniques such as genetic fingerprinting, as well as to identify risks associated with genetic disorders.
  • Cathepsin DC DNA can be further used to identify additional genes related to cathepsin DC DNA and to establish evolutionary trees based on the comparison of sequences.
  • Cathepsin DC DNA and proteins can be used to select for those genes or proteins that are homologous to cathepsin DC DNA or proteins, through positive screening procedures such as Southern blotting and immunoblotting and through negative screening procedures such as subtraction.
  • Cathepsin DC proteinase can be used as a reagent in analyses with other proteinases to compare the substrate specificity and activity of the proteinases.
  • Chimeric proteinases can be generated by swapping fragments of cathepsin DC proteinase with other proteinases. Such chimeric proteinases can be analyzed with respect to altered activity and specificity.
  • the proteinase activity of cathepsin DC can be used as a detergent additive for the removal of stains having a protein component, similar to the use of proteases described in U.S. Patent No. 5,599,400 and U.S. Patent No. 5,650,315.
  • the detergent composition can contain other known detergent constituents, such as surfactants, foam enhancers, fillers, enzyme stabilizers, chlorine bleach scavengers, other proteolytic enzymes, bacteriocides, dyes, perfumes, diluents, solvents, and other conventional ingredients.
  • the detergent composition preferably contains between .001% to 10% cathepsin DC proteinase.
  • cathepsin DC proteinase can be included in a detergent composition or can be combined with other constituents at the time of use as an additive.
  • the detergent additive can be formulated as a liquid, powder, granulate, slurry, or other conventional form of a detergent additive.
  • the leader sequence of cathepsin DC can be used to generate expression vectors for the expression an secretion of heterologous proteins.
  • heterologous refers to a sequence of nucleotides or amino acids that is unrelated to the nucleotide or amino acid sequence of mature cathepsin DC.
  • the nucleic acid sequence encoding the signal peptide of this protein can be fused to a nucleic acid sequence encoding a heterologous protein using conventional molecular techniques, such as those found in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol.
  • Sequences enhancing expression and secretion are preferably an amino acid sequence comprising amino acids 1-23 of cathepsin DC protein. Also preferred are amino acids 1-24, 1-25, 1-26, 1-27, and 1-28, the additional amino acids of which may enhance cleavage of the fusion protein.
  • polypeptides of the present invention can be subjected to fragmentation into smaller peptides by chemical and enzymatic means, and the peptide fragments so produced can be used in the analysis of other proteins or polypeptides.
  • peptide fragments can be used as peptide molecular weight markers, peptide isoelectric point markers, or in the analysis of the degree of peptide fragmentation.
  • the invention also includes these polypeptides and peptide fragments, as well as kits to aid in the determination of the apparent molecular weight and isoelectric point of an unknown protein and kits to assess the degree of fragmentation of an unknown protein.
  • chemical fragmentation is a preferred embodiment, and includes the use of cyanogen bromide to cleave under neutral or acidic conditions such that specific cleavage occurs at methionine residues (E. Gross, Methods in Enz. 11 :238-255, 1967). This can further include additional steps, such as a carboxymethylation step to convert cysteine residues to an unreactive species.
  • Enzymatic fragmentation is another preferred embodiment, and includes the use of a protease such as Asparaginylendo-peptidase, Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcus aureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C under conventional conditions to result in cleavage at specific amino acid residues.
  • Asparaginylendo-peptidase can cleave specifically on the carboxyl side of the asparagine residues present within the polypeptides of the invention.
  • Arginylendo-peptidase can cleave specifically on the carboxyl side of the arginine residues present within these polypeptides.
  • Achromobacter protease I can cleave specifically on the carboxyl side of the lysine residues present within the polypeptides (Sakiyama and Nakat, U.S. Patent No. 5,248,599; T. Masaki et al., Biochim. Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta 660:51-55,
  • Trypsin can cleave specifically on the carboxyl side of the arginine and lysine residues present within polypeptides of the invention. Enzymatic fragmentation may also occur with a protease that cleaves at multiple amino acid residues. For example, Staphlococcus aureus V8 protease can cleave specifically on the carboxyl side of the aspartic and glutamic acid residues present within polypeptides (D. W. Cleveland, J. Biol. Chem. 5:1102-1106, 1977).
  • Endoproteinase Asp-N can cleave specifically on the amino side of the asparagine residues present within polypeptides.
  • Endoproteinase Lys-C can cleave specifically on the carboxyl side of the lysine residues present within polypeptides of the invention.
  • Other enzymatic and chemical treatments can likewise be used to specifically fragment these polypeptides into a unique set of specific peptides.
  • the peptides and fragments of the polypeptides of the invention can also be produced by conventional recombinant processes and synthetic processes well known in the art.
  • the polypeptides and peptide fragments encompassed by invention can have variable molecular weights, depending upon the host cell in which they are expressed. Glycosylation of polypeptides and peptide fragments of the invention in various cell types can result in variations of the molecular weight of these pieces, depending upon the extent of modification. The size of these pieces can be most heterogeneous with fragments of polypeptide derived from the extracellular portion of the polypeptide.
  • Consistent polypeptides and peptide fragments can be obtained by using polypeptides derived entirely from the transmembrane and cytoplasmic regions, pretreating with N-glycanase to remove glycosylation, or expressing the polypeptides in bacterial hosts.
  • the molecular weight of these polypeptides can also be varied by fusing additional peptide sequences to both the amino and carboxyl terminal ends of polypeptides of the invention. Fusions of additional peptide sequences at the amino and carboxyl terminal ends of polypeptides of the invention can be used to enhance expression of these polypeptides or aid in the purification of the protein.
  • fusions of additional peptide sequences at the amino and carboxyl terminal ends of polypeptides of the invention will alter some, but usually not all, of the fragmented peptides of the polypeptides generated by enzymatic or chemical treatment.
  • mutations can be introduced into polypeptides of the invention using routine and known techniques of molecular biology.
  • a mutation can be designed so as to eliminate a site of proteolytic cleavage by a specific enzyme or a site of cleavage by a specific chemically induced fragmentation procedure. The elimination of the site will alter the peptide fingerprint of polypeptides of the invention upon fragmentation with the specific enzyme or chemical procedure.
  • the polypeptides and the resultant fragmented peptides can be analyzed by methods including sedimentation, electrophoresis, chromatography, and mass spectrometry to determine their molecular weights. Because the unique amino acid sequence of each piece specifies a molecular weight, these pieces can thereafter serve as molecular weight markers using such analysis techniques to assist in the determination of the molecular weight of an unknown protein, polypeptides or fragments thereof.
  • the molecular weight markers of the invention serve particularly well as molecular weight markers for the estimation of the apparent molecular weight of proteins that have similar apparent molecular weights and, consequently, allow increased accuracy in the determination of apparent molecular weight of proteins.
  • fragmented peptide molecular weight markers those markers are preferably at least 10 amino acids in size. More preferably, these fragmented peptide molecular weight markers are between 10 and 100 amino acids in size. Even more preferable are fragmented peptide molecular weight markers between 10 and 50 amino acids in size and especially between 10 and 35 amino acids in size. Most preferable are fragmented peptide molecular weight markers between 10 and 20 amino acids in size.
  • a particularly preferred embodiment is denaturing polyacrylamide gel electrophoresis (U. K. Laemmli, Nature 227:680-685, 1970).
  • the method uses two separate lanes of a gel containing sodium dodecyl sulfate and a concentration of acrylamide between 6-20%.
  • the ability to simultaneously resolve the marker and the sample under identical conditions allows for increased accuracy. It is understood, of course, that many different techniques can be used for the determination of the molecular weight of an unknown protein using polypeptides of the invention, and that this embodiment in no way limits the scope of the invention.
  • Each unglycosylated polypeptide or fragment thereof has a pi that is intrinsically determined by its unique amino acid sequence (which pi can be estimated by the skilled artisan using any of the computer programs designed to predict pi values currently available, calculated using any well-known amino acid pKa table, or measured empirically). Therefore these polypeptides and fragments thereof can serve as specific markers to assist in the determination of the isoelectric point of an unknown protein, polypeptide, or fragmented peptide using techniques such as isoelectric focusing. These polypeptide or fragmented peptide markers serve particularly well for the estimation of apparent isoelectric points of unknown proteins that have apparent isoelectric points close to that of the polypeptide or fragmented peptide markers of the invention.
  • the technique of isoelectric focusing can be further combined with other techniques such as gel electrophoresis to simultaneously separate a protein on the basis of molecular weight and charge.
  • Other techniques such as gel electrophoresis to simultaneously separate a protein on the basis of molecular weight and charge.
  • gel electrophoresis to simultaneously separate a protein on the basis of molecular weight and charge.
  • the ability to simultaneously resolve these polypeptide or fragmented peptide markers and the unknown protein under identical conditions allows for increased accuracy in the determination of the apparent isoelectric point of the unknown protein. This is of particular interest in techniques, such as two dimensional electrophoresis (T.D. Brock and M.T. Madigan,
  • these polypeptides and fragmented peptides thereof can assist in the determination of both the isoelectric point and molecular weight of an unknown protein or fragmented peptide.
  • Polypeptides and fragmented peptides can be visualized using two different methods that allow a discrimination between the unknown protein and the molecular weight markers.
  • the polypeptide and fragmented peptide molecular weight markers of the invention can be visualized using antibodies generated against these markers and conventional immunoblotting techniques. This detection is performed under conventional conditions that do not result in the detection of the unknown protein. It is understood that it may not be possible to generate antibodies against all polypeptide fragments of the invention, since small peptides may not contain immunogenic epitopes. It is further understood that not all antibodies will work in this assay; however, those antibodies which are able to bind polypeptides and fragments of the invention can be readily determined using conventional techniques.
  • the unknown protein is also visualized by using a conventional staining procedure.
  • the molar excess of unknown protein to polypeptide or fragmented peptide molecular weight markers of the invention is such that the conventional staining procedure predominantly detects the unknown protein.
  • the level of these polypeptide or fragmented peptide molecular weight markers is such as to allow little or no detection of these markers by the conventional staining method.
  • the preferred molar excess of unknown protein to polypeptide molecular weight markers of the invention is between 2 and 100,000 fold. More preferably, the preferred molar excess of unknown protein to these polypeptide molecular weight markers is between 10 and 10,000 fold and especially between 100 and 1,000 fold.
  • the analysis of the progressive fragmentation of the polypeptides of the invention into specific peptides can be used as a control for the extent of cleavage of an unknown protein.
  • cleavage of the same amount of polypeptide and unknown protein under identical conditions can allow for a direct comparison of the extent of fragmentation.
  • Conditions that result in the complete fragmentation of the polypeptide can also result in complete fragmentation of the unknown protein.
  • the fragmentation of the polypeptide of SEQ ID NO:2 with cyanogen bromide generates a unique set of fragmented peptide molecular weight markers with molecular weights.
  • the distribution of methionine residues determines the number of amino acids in each peptide and the unique amino acid composition of each peptide determines its molecular weight.
  • the use of these polypeptide molecular weight markers allows increased accuracy in the determination of apparent molecular weight of proteins that have apparent molecular weights close to that of cathepsin DC.
  • fragments are used, there is increased accuracy in determining molecular weight over the range of the molecular weights of the fragment.
  • kits that are encompassed by the invention
  • the constituents of such kits can be varied, but typically contain the polypeptide and fragmented peptide molecular weight markers.
  • such kits can contain the polypeptides wherein a site necessary for fragmentation has been removed.
  • the kits can contain reagents for the specific cleavage of the polypeptide and the unknown protein by chemical or enzymatic cleavage. Kits can further contain antibodies directed against polypeptides or fragments thereof of the invention.
  • a polypeptide or peptide fingerprint can be entered into or compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W.J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyo et al., Electrophoresis 19:998-1005, 1998).
  • a variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), Multildent (Internet site: www.expasy.ch sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/ prot-id-frag.html).
  • These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance.
  • the programs compare observed molecular weights to predicted peptide molecular weights derived from sequence databases to assist in determining the identity of the unknown protein.
  • a polypeptide or peptide digest can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J.K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399
  • Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding). Thus, the polypeptides, fragments, variants, fusion proteins, etc., as set forth above may be employed as immunogens in producing antibodies immunoreactive therewith.
  • cathepsin DC proteins and peptides based on the amino acid sequence of cathepsin DC proteins, can be utilized to prepare antibodies that specifically bind to cathepsin DC proteins.
  • the term "antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2, and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind cathepsin DC proteins with a K, of greater than or equal to about 10 7 M "1 . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N. Y Acad.
  • Polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual , Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well- known in the art.
  • cathepsin DC proteins In general, purified cathepsin DC proteins, or a peptide based on the amino acid sequence of cathepsin DC proteins that is appropriately conjugated, is administered to the host animal typically through parenteral injection.
  • the immunogenicity of cathepsin DC proteins can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to cathepsin DC proteins.
  • an adjuvant for example, Freund's complete or incomplete adjuvant.
  • Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Patent Nos. 4,376.110 and 4,486,530.
  • Antigen-binding fragments of such antibodies which may be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab') 2 fragments.
  • Monoclonal antibodies can be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980. Briefly, the host animals, such as mice are injected intraperitoneally at least once, and preferably at least twice at about 3 week intervals with isolated and purified cathepsin DC proteins or conjugated cathepsin DC proteins, optionally in the presence of adjuvant.
  • mice are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of cathepsin DC proteins or conjugated cathepsin DC proteins. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG).
  • Fusion is plated out into plates containing media that allows for the selective growth of the fused cells.
  • the fused cells can then be allowed to grow for approximately eight days.
  • Supematants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as, I25 I-cathepsin DC proteins is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supematants are subsequently purified over a Protein A column (Pharmacia).
  • the monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 5:1-9 (1990), which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).
  • the monoclonal antibodies of the present invention include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. ⁇ Nature 552:323, 1988), Liu et al. ⁇ PNAS 84:3439, 1987), Larrick et al.
  • the antibodies are specific for the polypeptides of the present invention, and do not cross-react with other proteins. Screening procedures by which such antibodies may be identified are well known, and may involve immunoaffinity chromatography, for example.
  • Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques.
  • One method for producing such a hybridoma cell line comprises immunizing an animal with a polypeptide; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds the polypeptide.
  • the monoclonal antibodies may be recovered by conventional techniques.
  • Antibodies that are immunoreactive with the polypeptides of the invention are provided herein. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody (as opposed to non-specific binding).
  • the polypeptides, fragments, variants, fusion proteins, etc. as set forth above may be employed as "immunogens" in producing antibodies immunoreactive therewith.
  • the polypeptides, fragment, variants, fusion proteins, etc. contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous).
  • Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland
  • epitopes Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any of the methods known in the art. Thus, one aspect of the present invention relates to the antigenic epitopes of the polypeptides of the invention. Such epitopes are useful for raising antibodies, in particular monoclonal antibodies, as described in more detail below.
  • epitopes from the polypeptides of the invention can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supematants from cultured hybridomas.
  • Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
  • both polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).
  • the antibodies of the invention can be used in assays to detect the presence of the polypeptides or fragments of the invention, either in vitro or in vivo.
  • the antibodies also may be employed in purifying polypeptides or fragments of the invention by immunoaffinity chromatography.
  • the antibodies against cathepsin DC proteins can be used to detect the presence of cathepsin DC proteins in a sample using established assay protocols. Further, the antibodies of the invention can be used therapeutically to bind to cathepsin DC proteins and inhibit its activity in vivo.
  • an affinity column is generated using antibodies against cathepsin DC protein.
  • Monoclonal antibodies directed against cathepsin DC protein are covalently attached to cyanogen bromide-activated agarose beads (Pharmacia Biotech).
  • the cathepsin DC protein-agarose beads are used in an affinity column as a step in the purification of cathepsin DC protein.
  • Those antibodies that additionally can block binding of the polypeptides of the invention to a cathepsin DC counterstructure may be used to inhibit a biological activity that results from such binding.
  • Such blocking antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of cathepsin DC to certain cells expressing the cathepsin DC counterstructure.
  • blocking antibodies may be identified in assays for the ability to inhibit a biological effect that results from binding of cathepsin DC counterstructure to target cells.
  • Antibodies may be assayed for the ability to inhibit cathepsin DC counterstructure-mediated lysis, for example.
  • Those antibodies that additionally can block interaction and/or cleavage of cathepsin DC proteolytic substrates may be used to inhibit a biological activity that results from such interaction and/or cleavage.
  • Such blocking antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit proteolytic activity of cathepsin DC.
  • Such an antibody may be employed in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by the entity that generated the antibody. Disorders caused or exacerbated (directly or indirectly) by the interaction of cathepsin DC counterstructures with cell surface (binding partner) receptor or by cathepsin DC proteolkytic activity thus may be treated.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting a cathepsin DC counterstructure-mediated biological activity. Monoclonal antibodies are generally preferred for use in such therapeutic methods.
  • an antigen-binding antibody fragment is employed.
  • Antibodies may be screened for agonistic ⁇ i.e., ligand-mimicking) properties. Such antibodies, upon binding to cell surface cathepsin DC, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when cathepsin DC counterstructures binds to cell surface cathepsin DC.
  • Compositions comprising an antibody that is directed against catkepsin DC, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above for compositions containing cathepsin DC proteins.
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to the antibody. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures.
  • an antiserum that lacks specificity for cathepsin B protein in the prognosis of human cancer progression leads to a reduction in the accuracy of such a prognosis.
  • an enhanced specificity of the antisera towards cathepsin B protein in order to increase accuracy in the prognosis of human cancers can be obtained through the removal from the antisera of those antibodies that cross-react with cathepsin DC protein (c.f. Goding, pp 43 and 290-291, 1986).
  • cathepsin DC protein with antibodies to cathepsin B protein.
  • Purified protein can be resolved on a polyacrylamide gel by electrophoresis under conventional conditions, such as those described in Laemmli, U.K. ⁇ Nature 227:680, 1970), hereby incorporated by reference. Proteins resolved on the gel can be transferred to a nitrocellulose membrane under conventional conditions, such as those described in Towbin, H. et al. ⁇ Proc Natl. Acad. Sci. USA 76:4350, 1979), hereby incorporated by reference.
  • An antisera raised against human cathepsin B protein can be incubated with the nitrocellulose membrane to detect cathepsin DC by conventional immunoblotting methods, such as those described in Harper and Murphy ⁇ Anal. Biochem. 193:59, 1991) and Goding (1986), hereby incorporated by reference. It is understood of course that many different antibodies and immunological techniques can be used for the detection of cathepsin DC proteins and that this embodiment in no way limits the scope of the invention. The references cited herein are incorporated by reference herein in their entirety.
  • a vector for expressing cathepsin DC was constructed by cloning a BamHI restriction enzyme fragment encoding cathepsin DC protein into a pDC409 mammalian expression vector.
  • BamHI restriction enzyme cuts after nucleotides 30 and 1077 of SEQ ID NO: 1.
  • the ⁇ DC409 mammalian expression vector described in Giri et al. ⁇ EMBO J. 13:2822, 1994) was derived from pDC406 (McMahon et al., EMBO J. 10:2821, 1991), but has a single Rg/II restriction site.
  • the pDC409 mammalian expression vector was cut with Bglil and ligated to the cathepsin DC BamHI restriction enzyme fragment using conventional techniques.
  • CV-1/EBNA cells were transfected by the DEAE/Dextran method with 2 ⁇ g of the cathepsin DC expression vector. The cells were grown to allow the production of soluble cathepsin DC in the cell supernatant.
  • the membrane was incubated with primary antibody (1:100 dilution of rabbit anti-human cathepsin B from Vital Products, St Louis, MO) for one hour. Blots were subsequently washed extensively with IX phosphate buffered saline (with and without 0.5% NP40). A secondary antibody (horseradish peroxidase conjugated donkey anti-rabbit serum from Amersham) was used at 1 : 1000 and the washing steps were repeated. A luminescent detection method was used to visualize the detected proteins (Harper and Murphy, 1991) on Kodak X-OMAT AR film.
  • the polyclonal antiserum generated against purified human cathepsin B (from liver) reacted strongly against roughly equivalent amounts of purified cathepsin B protein (human and bovine) and recombinant cathepsin DC protein. No reactivity was observed against purified human cathepsin D protein and recombinant cathepsin L protein. No signal was observed when a duplicate blot was probed with secondary antibody alone.

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Abstract

L'invention concerne de nouveaux polypeptides cathepsine DC isolés et purifiés, les acide nucléiques codant pour ces polypeptides, les protéinases ainsi codées, des techniques permettant de produire des formes recombinantes de ces polypeptides, les anticorps produits contre ces polypeptides, des peptides fragmentés dérivés de ces polypeptides, ainsi que les utilisations de ces diverses formes.
PCT/US1998/026920 1997-12-18 1998-12-18 Proteines cathepsine dc, adn codant pour ces proteines et utilisation de ces proteines dans l'etablissement de pronostics pour les cancers chez l'homme WO1999031256A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1336847A1 (fr) * 2002-02-14 2003-08-20 Biofrontera Pharmaceuticals AG Inhibiteurs de la Cathepsine Y pour le dévelopment de médicaments pour le traitement de la douleur
WO2004077053A1 (fr) * 2003-02-25 2004-09-10 Biofrontera Pharmaceuticals Gmbh Cathepsine y pour le traitement de la douleur
WO2005065693A2 (fr) * 2003-12-30 2005-07-21 Aventis Pharmaceuticals Inc. Utilisation d'inhibiteurs de la cathepsine z dans le traitement de la polyarthrite rhumatoide et d'autres maladies auto-immunes
US6974672B2 (en) 2002-03-19 2005-12-13 Amgen Inc. Gene amplification in cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039194A1 (fr) * 1995-06-06 1996-12-12 Athena Neurosciences, Inc. Nouvelle cathepsine et procedes et compositions d'inhibition de cathepsine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039194A1 (fr) * 1995-06-06 1996-12-12 Athena Neurosciences, Inc. Nouvelle cathepsine et procedes et compositions d'inhibition de cathepsine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
N[GLER D AND MÉNARD R: "Human cathepsin X: a novel cysteine protease of the papain family with a very short proregion and unique insertions" FEBS LETTERS., vol. 434, 1998, pages 135-139, XP002101279 AMSTERDAM NL *
SANTAMARIA I ET AL: "Cathepsin Z, a novel human cysteine proteinase with a short propeptide domain and a unique chromosomal location" JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 273, no. 27, 3 July 1998 (1998-07-03), pages 16816-16823, XP002101278 MD US cited in the application *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1336847A1 (fr) * 2002-02-14 2003-08-20 Biofrontera Pharmaceuticals AG Inhibiteurs de la Cathepsine Y pour le dévelopment de médicaments pour le traitement de la douleur
US6974672B2 (en) 2002-03-19 2005-12-13 Amgen Inc. Gene amplification in cancer
WO2004077053A1 (fr) * 2003-02-25 2004-09-10 Biofrontera Pharmaceuticals Gmbh Cathepsine y pour le traitement de la douleur
WO2005065693A2 (fr) * 2003-12-30 2005-07-21 Aventis Pharmaceuticals Inc. Utilisation d'inhibiteurs de la cathepsine z dans le traitement de la polyarthrite rhumatoide et d'autres maladies auto-immunes
WO2005065693A3 (fr) * 2003-12-30 2005-11-10 Aventis Pharma Inc Utilisation d'inhibiteurs de la cathepsine z dans le traitement de la polyarthrite rhumatoide et d'autres maladies auto-immunes

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