EP1307568A2 - Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci - Google Patents

Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci

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Publication number
EP1307568A2
EP1307568A2 EP01957300A EP01957300A EP1307568A2 EP 1307568 A2 EP1307568 A2 EP 1307568A2 EP 01957300 A EP01957300 A EP 01957300A EP 01957300 A EP01957300 A EP 01957300A EP 1307568 A2 EP1307568 A2 EP 1307568A2
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European Patent Office
Prior art keywords
polypeptide
seq
mpd
polypeptides
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01957300A
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German (de)
English (en)
Inventor
Robert F. Dubose
Steven R. Wiley
Roy A. Black
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Immunex Corp
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Immunex Corp
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Publication of EP1307568A2 publication Critical patent/EP1307568A2/fr
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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
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • ADAM family polypeptides are cell surface polypeptides that consist of pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like repeat, 'transmembrane and cytoplasmic domains.
  • the metalloproteinase domain is believed to be involved in protein processing functions such as release of growth factors, adhesion proteins, and inflammatory factors.
  • the disintegrin domain may play a role in integrin-mediated cell adhesion (cell to cell and cell to matrix) interactions, such as platelet aggregation, migration of tumor cells or neutrophils, and angiogenesis.
  • the invention further provides a method for identifying an agent that modulates an activity of a polypeptide as set forth herein, comprising contacting the agent with the polypeptide under conditions such that the agent and the polypeptide interact; and determining activity of the polypeptide in the presence of the agent compared to a control (e.g., the polypeptide in the absence of the agent) wherein a change in activity is indicative of an agent that modulates the polypeptide' s activity.
  • the activity of the polypeptide is selected from the group consisting of disintegrin activity, cell adhesion activity, angiogenic activity, metalloproteinase activity, and a combination thereof (e.g., metalloproteinase and disintegrin activity).
  • the agent is selected from the group consisting of an antibody, a small molecule, a peptide, and a peptidomimetic.
  • the invention provides a method of inhibiting the binding of an integrin to a ligand comprising contacting or administering to a cell or mammal that expresses the integrin an effective amount of a polypeptide having disintegrin activity, hi one embodiment, the mammal is afflicted with a condition selected from the group consisting of ocular disorders; malignant and metastatic conditions; inflammatory diseases; osteoporosis and other conditions mediated by accelerated bone reso ⁇ tion; restenosis; inappropriate platelet activation, recruitment, or aggregation; thrombosis; and a condition requiring tissue repair or wound healing.
  • Figure 1 shows polypeptide sequences of the invention in single letter amino acid code.
  • the metalloproteinases identified herein are candidate proteins contributing to the pathogenesis of, for example, inflammatory diseases and disorders. Accordingly, the metalloproteinase polypeptides provided herein can be used to treat inflammatory disorders and as a source for the development of inhibitors both in s ⁇ lico and in vitro. Inhibitors to the metalloproteinases provided herein can amerliorate diseases and disorders associated with excessive proteinase activity including, for example, inflammation.
  • the structure of most metalloproteinase-disintegrin polypeptides includes a signal domain, a metalloproteinase domain, a disintegrin domain, a transmembrane domain and a cytoplasmic domain.
  • the typical structural elements common to various members of the ADAM family of polypeptides include, in N-to-C order, a signal sequence, a prodomain, a metalloproteinase domain, a disintegrin domain, a cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain.
  • ADAM family of polypeptides There are 31 highly conserved cysteines in the disintegrin and cysteine rich region; almost all of the ADAM family of polypeptides have these 31 cysteines. The skilled artisan will recognize that the boundaries of these regions within the polypeptides are approximate and that the precise boundaries of such domains (which can be predicted by using computer programs available for that pmpose) can differ from member to member within the ADAM family of polypeptides.
  • the ADAM family of polypeptides is reasonably well conserved, with the human family members similar to each other and to ADAM family members from other species such as mouse, rat, and even Drosophila melanogaster and Caenorhabditis elegans (see, e.g., Yamamoto et al., Immunol.
  • ADAM family polypeptides exhibit integrin-binding activities via the disintegrin domain
  • modulation of disintegrin activity will modulate adhesion, e.g., the role of ADAMs 1 and 2 in sperm binding to egg and the role of ADAM-9 in interactions of glomerular and tubular epithelial cells with the basal laminae in renal tissue.
  • the degree to which individual members of the ADAM family of polypeptides and fragments and other derivatives of these polypeptides exhibit these activities can be determined by standard assay methods, such as inhibition of endothelial cell migration by disintegrin-Fc constructs, and the like.
  • Particularly suitable assays to detect or measure the binding between ADAM polypeptides and their binding partners are FACS analyses.
  • methods of treating or ameliorating these conditions comprise increasing the amount or activity of, for example, MPD polypeptides having disintegrin activity by providing such polypeptides or active fragments or fusion polypeptides thereof, or by providing agents that activate endogenous or exogenous MPD polypeptides.
  • MPD polypeptides having disintegrin activity include diagnostic reagents for inflammation, cancer, allergy, reproductive, neural disorders, and vascular diseases; research reagents for investigation of integrin polypeptides and fertilization processes, purification and processing of integrins and/or endothelial cells or T cells; or as a carrier/targeting polypeptide to deliver therapeutic agents to cells.
  • protein and polypeptide mean any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), and include natural proteins, synthetic or recombinant polypeptides and peptides as well as a recombinant molecule consisting of a hybrid with one portion, for example, comprising all or part of an MPD amino acid sequence and a second portion being encoded by all or part of a different nucleotide sequence.
  • the protein or polypeptide is substantially pure of other components from which it is normally present in nature.
  • the term "between about” or “from about” will be understood to include sequences between any such referenced residues of a sequence.
  • “from about 43 to 148” means 44 to 148, 45 to 148, 46 to 148, and so on; and 43 to 147, 43 to 146, and so on.
  • MPD polypeptides comprising sequences associated withi metalloproteinase activity include SEQ ED NO: 14 from about residue 1 to 622; SEQ D NO: 14 from about residue 84 to 622; SEQ ID NO: 18; SEQ ED NO:21 from about residue 1 to 701; SEQ ID NO:22; SEQ ID NO:24; SEQ ED NO:24 from about residue 1 to 277; SEQ ID NO:25 from about residue 1 to 332; SEQ ED NO:25 from about residue 1 to 627; SEQ ED NO:26 from about residue 1 to 215; SEQ ED NO:26 from about residue 118 to 215; and SEQ ED NO:26, termed herein "MPD metalloproteinase polypeptides.”
  • the MPD polypeptides have been shown to have a high degree of homology to members of the ADAM family and related metalloproteinase/disintegrin polypeptides and thus have a predicted function or activity of an ADAM polypeptide, a disintegrin polypeptide, or a metalloproteinase polypeptide. Accordingly, the invention provides an MPD polypeptide comprising a sequence selected from the group consisting of SEQ ED Nos:l to 26, and 27. In one embodiment, the MPD polypeptide has disintegrin activity, metalloproteinase activity, or a combination thereof.
  • Methods of determining whether a polypeptide of the invention has a desired disintegrin activity or metalloproteinase activity can be accomplished by assaying the polypeptide by any of the methods described herein below.
  • a number of conserved sequences have been identified in ADAM and matrix metalloproteinases (MMPs) including, for example, the HExGHxxGxxHD motif (SEQ ID N0:28).
  • MMPs matrix metalloproteinases
  • a potential conserved motif includes a LNIx(I/V)(A/V)LVGLE(V/l)WT motif (SEQ D NO:29).
  • the invention also provides polypeptides having homology to the ADAM (A Disintegrin And Metalloproteinase) family of proteins.
  • Such polypeptides include metalloproteinase domains.
  • the metalloproteinase domain of the polypeptides comprising SEQ D Nos:4, 10, 14, 21, 25, and 26 comprise a sequence from about amino acids residues 65 to 274 of SEQ ED NO:4; 24 to 235 of SEQ ID NO:10; 85 to 290 of SEQ ID NO:14; 202 to 411 of SEQ ED NO:21; 123 to 332 of SEQ ID NO:25; and 118 to 215 of SEQ ID NO:26.
  • N-terminal and C-terminal residues of the respective metalloproteinase domains are approximate and variations of about 1 to 10 amino acid(s) from either end of the domain will not depart from the scope of the present invention.
  • the addition of amino acids to either end of the domain may not change the molecule's activity.
  • the effects of any such modification can be assayed using the methods described herein.
  • Polypeptides comprising sequences as set forth in SEQ ED Nos: 2 to 5, 7 to 10, 14, 17 to 19, 21 to 22, and 24 to 27 may also have disintegrin activity in addition to metalloproteinase activity.
  • SEQ ED NO: 11 has a putative CGN-GEEC sequence at residues 43 to 57 and thus a polypeptide comprising SEQ ED NO: 11 is predicted to have disintegrin activity.
  • ADAM family of proteins are characterized as having a number of conserved cysteine residues in their disintegrin and cysteine-rich domains. For example, SEQ ED Nos:6, 8, 12, 13, 16, and 23, when aligned with a number of ADAM family members (e.g., ADAM9 (accession no. NP 003807, which is inco ⁇ orated herein by reference)), align with the conserved cysteine residues in the disintegrin domain of such ADAM family members.
  • the invention also provides polypeptides comprising a sequence as set forth in
  • Table 2 provides a summary of the relative domains and residues characterizing the domains of some of the polypeptides of the invention.
  • the relative domains and residues corresponding to such activity are estimates based on similar molecules and computer algorithms and accordingly may vary slightly depending upon a number of factors including, for example, the source of material, the cell type used, the expression system used, and the like. Such factors will
  • a polypeptide of the invention also encompasses an amino acid sequence that has a sufficient or a substantial degree of identity or similarity to a sequence set forth in Figure 1.
  • Substantially identical sequences can be identified by those of skill in the art as having structural domains and/or having biological activity in common with an MPD polypeptide. Methods of determining similarity or identity may employ computer algorithms such as, e.g., BLAST, FASTA, and the like.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 1800, usually about 50 to 200, more usually about 70 to 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFtT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987), and is similar to the method described by Higgins & Sha ⁇ , CABIOS 5:151 (1989).
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • HSPs high scoring sequence pairs
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the percent identity of two amino acid or 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.
  • 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 Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • substitutions, deletions or additions to a nucleic acid sequence, peptide, or polypeptide sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in a molecule having substantially the same biological activity (e.g., disintegrin and/or metalloproteinase activity).
  • an alteration that results in the substitution of an amino acid with a chemically similar amino acid is a conservatively modified variant.
  • Conservative substitution tables providing functionally similar amino acids are known in the art.
  • the following six groups each contain amino acids that are conservative substitutions for one another 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) (see, e.g., Creighton, Proteins (1984)).
  • polypeptide encoded by a first polynucleotide is immunologically cross reactive with the antibodies raised against the polypeptide encoded by a second polynucleotide.
  • Another indication that two polynucleotides are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions.
  • Polypeptides derived from the MPD polypeptides of the invention by any type of alteration are also encompassed by the invention. Therefore, the polypeptides provided by the invention include polypeptides characterized by amino acid sequences similar to those as set forth in Figure 1, but into which modifications are naturally provided or deliberately engineered.
  • a polypeptide that shares biological activities in common with a polypeptide comprising a sequence as set forth in SEQ D NO: 1-26, or 27 having disintegrin activity and/or metalloproteinase activity are encompassed by the invention.
  • the present invention encompasses the use of various forms of MPD disintegrin polypeptides or domains that retain at least one activity selected from the group consisting of integrin binding activity, inhibition of endothelial cell migration, and inhibition of angiogenesis.
  • a MPD disintegrin polypeptide/domain (MPDdis) is intended to encompass polypeptides comprising all or part of an MPD polypeptide of the invention having disintegrin activity.
  • an MPDdis contains all or part of an MPD disintegrin domain, with or without other domains (such as the cysteine-rich region), as well as related forms including, but not limited to: (a) fragments, (b) variants, (c) derivatives, (d) fusion polypeptides, and (e) multimeric forms (multimers).
  • the ability of these related forms to inhibit integrin binding, endothelial cell migration, and/or inhibition of angiogenesis may be determined in vitro or in vivo by using methods such as those exemplified below or by using other assays known in the art.
  • One of skill in the art can easily assay for activity using the methods described herein. Such methods measure, for example, metalloproteinase activity, disintegrin activity, or a biological activities exhibited by members of the ADAM family of polypeptides including, without limitation, cell adhesion.
  • anti-MPD antibodies which neutralize MPD activity (e.g, metalloproteinase activity and/or disintegrin activity) can be used to assay for similar polypeptides by contacting an anti-MPD antibody with a polypeptide of interest and determining if the activity associated with the polypeptide of interest is neutralized.
  • the cross-reactivity of an antibody that specifically binds to an MPD polypeptide of the invention with another polypeptide of interest is indicative that the polypeptide of interest shares structural characteristics (e.g., primary, secondary, or tertiary protein characteristics) with an MPD polypeptide of the invention.
  • Full-length polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated.
  • the amino acid sequences of full-length polypeptides can be obtained, for example, by translation of the complete open reading frame ("ORF") of a cDNA molecule.
  • ORF complete open reading frame
  • Several full-length polypeptides may be encoded by a single genetic locus if multiple mRNA forms are produced from that locus by alternative splicing or by the use of multiple translation initiation sites.
  • the "mature form" of a polypeptide refers to a polypeptide that has undergone post- translational processing steps, if any, such as, for example, cleavage of the signal sequence or proteolytic cleavage to remove a prodomain.
  • Multiple mature forms of a particular full-length polypeptide may be produced, for example, by imprecise cleavage of the signal sequence, or by differential regulation of proteases that cleave the polypeptide.
  • the mature form(s) of such polypeptide may be obtained by expression, in a suitable mammalian cell or other host cell, of a polynucleotide that encodes the full-length polypeptide.
  • the sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites.
  • the MPD polypeptides of the invention also include polypeptides that result from post-transcriptional or post-translational processing events such as alternate mRNA processing which can yield a truncated but biologically active polypeptide, for example, a naturally occurring soluble form of the polypeptide.
  • variations attributable to proteolysis such as 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 polypeptide (generally from 1-5 terminal amino acids).
  • a polypeptide of the invention may be prepared by culturing transformed or recombinant host cells under culture conditions suitable to express a polypeptide of the invention.
  • the resulting expressed polypeptide may then be purified from such culture using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • a polypeptide of the invention may also be expressed in a form that will facilitate purification.
  • it may be expressed as a fusion polypeptide, joined to, for example, maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ), and InVitrogen, respectively.
  • MBP maltose binding polypeptide
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • RP-HPLC reverse-phase high performance liquid chromatography
  • polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the invention as a "substantially purified polypeptide"; such purified polypeptides include antibodies that specifically bind to an MPD polypeptide, fragment, variant, and the like.
  • a polypeptide of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide encoding a polypeptide of the invention.
  • an affinity column such as a monoclonal antibody generated against polypeptides of the invention, to affinity-purify expressed polypeptides.
  • These 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.
  • proteins that bind a polypeptide of the invention can be bound to a solid phase support or a similar substrate suitable for identifying, separating, or purifying cells that express polypeptides of the invention on their surface.
  • Adherence of, for example, an anti-MPD antibody of the invention to a solid phase 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.
  • Anti-MPD antibodies bind cells having polypeptides of the invention on their surface (e.g., an extracellular domain of MPD). Unbound cells (e.g., cell lacking and MPD polypeptide) are washed away from the bound cells.
  • 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 are first incubated with a biotinylated binding polypeptide 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 cells to the beads.
  • avidin-coated beads is known in the art (see, Berenson, et al I. Cell. Biochem., 10D:239, 1986). Wash of unbound material and the release of the bound cells is performed using conventional methods.
  • a polypeptide of the invention may also be produced by known conventional chemical synthesis. Methods for constructing the polypeptides of the invention by synthetic means are known to those skilled in the art.
  • the synthetically-constructed polypeptide sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with a native polypeptides may possess biological properties in common therewith, including biological activity.
  • the synthesized polypeptides may be employed as biologically active or immunological substitutes for natural, purified polypeptides in screening of therapeutic compounds, and in immunological processes for the development of antibodies.
  • the desired degree of purity depends on the intended use of the polypeptide.
  • 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 polypeptide bands corresponding to other polypeptides 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 can 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 polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.
  • Species homologues of MPD polypeptides and polynucleotides encoding the polypeptides are also provided by the invention.
  • a "species homologue” is a polypeptide or polynucleotide with a different species of origin from that of a given polypeptide or polynucleotide, but with significant sequence similarity to the given polypeptide or polynucleotide.
  • Species homologues may be isolated and identified by making suitable probes or primers from polynucleotides encoding the polypeptides provided herein and screening a suitable nucleic acid source from the desired species.
  • homologues may be identified by screening a genome database containing sequences from one or more species utilizing a sequence (e.g., nucleic acid or amino acid sequence) of an MPD of the invention.
  • genome databases are readily available for a number of species (e.g., on the world wide web (www) at tigr.org/tdb; genetics.wisc.edu; stanford.edu/ ⁇ ball; hiv-web.lanl.gov; ncbi.nlm.nig.gov; ebi.ac.uk; and pasteur.fr/other/biology).
  • the invention also encompasses allelic variants of MPD polypeptides and nucleic acids encoding them that are naturally-occurring alternative forms of such polypeptides and polynucleotides in which differences in amino acid or nucleotide sequence are attributable to genetic polymo ⁇ hism.
  • Saturated BLAST is a package that performs ISS. Starting with a protein sequence, Saturated BLAST runs a BLAST search and identifies representative sequences for the next generation of searches. The procedure is run until convergence or until some predefined criteria are met. Saturated BLAST is available on the world wide web (www) at: bioinformatics.burnham-inst.org/xblast (see also, Li et al Bioinformatics 16(12): 1105, 2000).
  • Fragments of the MPD polypeptides of the invention are encompassed by the invention and may be in linear form or cyclized using known methods (see, e.g., H.U. Saragovi, et al, Bio/Technology 10, 773 (1992); and R. S. McDowell, et al, J. Amer. Chem. Soc. 114:9245 (1992), both of which are inco ⁇ orated by reference herein).
  • Peptide fragments of MPD polypeptides of the invention, and polynucleotides encoding such fragments include amino acid or nucleotide sequence lengths that are at least 25% (more preferably at least 50%, 60%, or 70%, and most preferably at least 80%) of the length of an MPD polypeptide or polynucleotide.
  • sequences will have at least 60% sequence identity (more preferably at least 70%-75%, 80%- 85%, 90%-95%, at least 97%-97.5%, or at least 99%, and most preferably at least 99.5%) with an MPD polypeptide or polynucleotide when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • polypeptides, peptide fragments, and polynucleotides encoding such fragments that contain or encode a segment preferably comprising at least 8 to 10, or more preferably at least 20, or still more preferably at least 30, or most preferably at least 40 contiguous amino acids.
  • Such polypeptides and fragments may also contain a segment that shares at least 70% (at least 75%, 80%-85%, 90%-95%, at least 97%-97.5%, or at least 99%, and most preferably at least 99.5%) with any such segment of, for example, any of the ADAM family polypeptides, when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • Visual inspection, mathematical calculation, or computer algorithms can determine the percent identity.
  • the invention also provides soluble forms of MPD polypeptides comprising certain fragments or domains of these polypeptides.
  • Soluble fragments having disintegrin activity are of particular interest. For example, an amino acid sequence beginning with a highly conserved CGN- GEEC sequence (as discussed above) but which lacks a transmembrane region (see, e.g., Table 2). Transmembrane regions can be identified using publicly available computer algorithms.
  • polypeptides comprising SEQ ED NO:6 beginning at an amino acid between and including residues 1 and 43 to 148; SEQ ED NO:8 beginning at an amino acid between and including residues 1 and 38 to 366; SEQ ED NO: 11; SEQ ED NO: 13; SEQ ID NO: 14 beginning at an amino acid between and including residues 1 and 84 to 622; SEQ ED NO: 14 beginning at an amino acid between and including residues 1 and 299 to 622; SEQ ED NO: 16; SEQ ED NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ED NO:24 beginning at an amino acid between and including residues 1 and 278 to 435; SEQ ED NO:25 from about residue 1 to 627; and SEQ ED NO:26 beginning at an amino acid between and including residues 1 and 224 to 383.
  • polypeptides can be secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of polypeptides of the invention can be identified in accordance with known techniques for determination of such domains from sequence information. For example, alignment of the polypeptide sequences of the invention with other members of the ADAM family of polypeptides having known domains will provide information regarding the domains of the polypeptides of the invention.
  • One of skill in the art will recognize that slight modifications in the range of sequences of a particular domain can be made without affecting the molecule's biological activity. Accordingly, changes in the identified sequences of 1, 2, 3, 4, or 5 to 10 amino acids in either direction of the particular domain are encompassed by the present invention.
  • a polypeptide may comprise various combinations of ADAM polypeptide domains, such as a metalloproteinase domain, a disintegrin domain, or a cytoplasmic domain.
  • ADAM polypeptide domains such as a metalloproteinase domain, a disintegrin domain, or a cytoplasmic domain.
  • polypeptides of the invention and polynucleotides include those comprising or encoding two or more copies of a domain such as the metalloproteinase domain, two or more copies of a domain such as the disintegrin domain, or at least one copy of each domain, and these domains may be presented in any order within such polypeptides.
  • the second polypeptide can be any polypeptide of interest having an activity or function independent of, or related to, the function of an MPD polypeptide.
  • the second polypeptide can be a domain of a related but distinct member of the ADAM family of polypeptides such as, for example, an extracellular, cytoplasmic, metalloprotease, or transmembrane domain of an ADAM polypeptide.
  • the term "operatively linked" is intended to indicate that the MPD sequence and the second polypeptide sequence are fused in-frame to each other.
  • the second polypeptide can be fused to the N-terminus or C-terminus of an MPD sequence as set forth in Figure 1.
  • the fusion polypeptide is a GST-MPD fusion polypeptide in which an MPD sequence is fused to the C-terminus of the GST sequences.
  • Such fusion polypeptides can facilitate the purification of recombinant MPD sequences.
  • the fusion polypeptide is an MPD sequence comprising a heterologous signal sequence at its N-terminus.
  • an MPD polypeptide in certain host cells (e.g., mammalian host cells), expression and/or secretion of an MPD polypeptide can be increased through use of a heterologous signal sequence.
  • an MPD polypeptide or fragment thereof may be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
  • fusion polypeptides can comprise, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al, Bio/Technology 6:1204, 1988.
  • FLAG ® peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant polypeptide.
  • 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 inco ⁇ orated by reference.
  • the 4E11 hybridoma cell line has been deposited with the ATCC under accession no. HB9259.
  • Monoclonal antibodies that bind the FLAG ® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.
  • oligomers or fusion polypeptides that comprise an MPD polypeptide.
  • Oligomers that can be used as fusion partners can be in the form of covalently linked or non-covalently-linked multimers, including dimers, trimers, or higher oligomers.
  • the oligomers maintain the binding ability or catalytic ability of the polypeptide components and provide therefor, bivalent, trivalent, and the like, binding or catalytic sites, an alternative embodiment 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 can 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.
  • a linker will be a peptide linker moiety.
  • the length of the linker moiety is chosen to optimize the biological activity of the polypeptide comprising an MPD sequence and can be determined empirically without undue experimentation.
  • the linker moiety should be long enough and flexible enough to allow an MPD polypeptide to freely interact with a substrate or ligand.
  • the preferred linker moiety is a peptide between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues.
  • Preferred linker moieties are — Gly-Gly-, GGGGS (SEQ ID NO:31), (GGGGS) n (SEQ ID NO:32), GKSSGSGSESKS (SEQ ED NO:33), GSTSGSGKSSEGKG (SEQ ID NO:34), GSTSGSGKSSEGSGSTKG (SEQ ED NO:35), GSTSGSGKPGSGEGSTKG (SEQ ID NO:36), or EGKSSGSGSESKEF (SEQ ID NO:37).
  • Linking moieties are described, for example, in Huston, J. S., et al, PNAS 85:5879 (1988), Whitlow, M., et al, Protein Engineering 6:989 (1993), and Newton, D.
  • Suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby inco ⁇ orated by reference.
  • a DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, a DNA sequences encoding an MPD polypeptide or fragment thereof, using any suitable conventional technique.
  • a chemically synthesized oligonucleotide encoding the linker can be ligated between the sequences.
  • a fusion polypeptide comprises from two to four soluble MPD polypeptides, separated by peptide linkers.
  • variants of an MPD polypeptide are constructed to include a membrane-spanning domain, they will form a Type I membrane polypeptide.
  • the membrane-spanning polypeptides can be fused with known extracellular receptor domain polypeptides, for which the ligand is also known.
  • Such fusion polypeptides can then be manipulated to control the intracellular signaling pathways triggered by the bound MPD polypeptide.
  • Polypeptides that span the cell membrane can also be fused with agonists or antagonists of cell-surface receptors, or cellular adhesion molecules to further modulate MPD intracellular effects.
  • interleukins can be situated between the preferred MPD polypeptide fragment and other fusion polypeptide domains.
  • the MPD polypeptides of the invention can also include a localization sequence to direct the polypeptide to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins.
  • a polynucleotide encoding a localization sequence, or signal sequence can be ligated or fused at the 5' terminus of a polynucleotide encoding an MPD polypeptide such that the signal peptide is located at the amino terminal end of the resulting fusion polynucleotide/polypeptide.
  • the signal peptide functions to transport a polypeptide across the endoplasmic reticulum.
  • the secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or the external environment.
  • Signal peptides include pre-pro peptides that contain a proteolytic enzyme recognition site.
  • the localization sequence can be a nuclear-, an endoplasmic reticulum-, a peroxisome-, or a mitochondrial-localization sequence, or a localized protein. Localization sequences can be targeting sequences that are described, for example, in "Protein Targeting", chapter 35 of Stryer, L., Biochemistry (4th ed.). W. H. Freeman, 1995.
  • Some important localization sequences include those targeting the nucleus (e.g., KKKRK (SEQ ED NO:38)), mitochondria (MLRTSSLFTPvRVQPSLFRNE RLQST (SEQ ID NO:39)), endoplasmic reticulum (KDEL (SEQ ED NO:40)), peroxisome (SKF), plasma membrane (CAAX (SEQ ED NO:41), CC, CXC, or CCXX (SEQ ED NO:42)), cytoplasmic side of plasma membrane (fusion to SNAP-25), or the Golgi apparatus (fusion to furin).
  • KKKRK SEQ ED NO:38
  • mitochondria MLRTSSLFTPvRVQPSLFRNE RLQST
  • KDEL SEQ ED NO:40
  • KF peroxisome
  • plasma membrane CAAX (SEQ ED NO:41), CC, CXC, or CCXX (SEQ ED NO:42)
  • a polypeptide of the invention or fragments thereof may be fused to carrier molecules such as immunoglobulins for a variety of pu ⁇ oses including increasing the valency of polypeptide binding sites.
  • carrier molecules such as immunoglobulins for a variety of pu ⁇ oses including increasing the valency of polypeptide binding sites.
  • fragments of the polypeptide may be fused through linker sequences,, to the Fc portion of an immunoglobulin.
  • linker sequences for a bivalent form of the polypeptide, such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • a polypeptide-IgM fusion would generate a decavalent form of the polypeptide of the invention
  • the invention provides a fusion polypeptide having an Fc polypeptide domain and an MPD polypeptide sequence SEQ ID NO:6 beginning at an amino acid between and including residues 1 and 43 to 148; SEQ ED NO: 8 beginning at an amino acid between and including residues 1 and 38 to 366; SEQ ID NO:ll; SEQ ED NO:13; SEQ ED NO: 14 beginning at an amino acid between and including residues 1 and 84 to 622; SEQ ED NO: 14 beginning at an amino acid between and including residues 1 and 299 to 622; SEQ ED NO: 16; SEQ ED NO:21 from about residue 1 to 701; SEQ ID NO:23; SEQ ED NO:24 beginning at an amino acid between and including residues 1 and 278 to 435; SEQ ED NO:25 from about residue 1 to 627; and SEQ ED NO:26 beginning at an amino amino acid between and including
  • 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.
  • an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion polypeptides 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.
  • One embodiment of the invention is directed to a dimer comprising two fusion polypeptides created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion polypeptide is inserted into an appropriate expression vector.
  • Polypeptide/Fc fusion polypeptides are expressed in host cells transformed or transfected with the recombinant expression vector or recombinant polynucleotide encoding the fusion polypeptide, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules.
  • One suitable Fc polypeptide described in PCT application WO 93/10151 (hereby inco ⁇ orated by reference), 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.
  • the 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.
  • the above-described fusion polypeptides comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Polypeptide A or Polypeptide G columns.
  • polypeptides of the invention can be substituted for the variable portion of an antibody heavy or light chain. If fusion polypeptides are made with both heavy and light chains of an antibody, it is possible to form an oligomer with as many as four MPD polypeptides or fragments thereof.
  • Leucine zipper domains are peptides that promote oligomerization (dimers and trimers) of the polypeptides in which they are found.
  • Leucine zippers were originally identified in several DNA-binding polypeptides (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different polypeptides.
  • the zipper domain comprises a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids.
  • a chimeric or fusion polypeptide of the invention can be produced by standard recombinant DNA techniques.
  • polynucleotide fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example, by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • the invention further includes polypeptides with or without associated native-pattern glycosylation.
  • Polypeptides expressed in yeast or mammalian expression systems e.g., COS-1 or CHO cells
  • yeast or mammalian expression systems e.g., COS-1 or CHO cells
  • expression of polypeptides of the invention in bacterial expression systems, such as E. coli provides non- glycosylated molecules.
  • a given preparation can include multiple differentially glycosylated species of the polypeptide. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase.
  • modifications in the polypeptide or polynucleotide can be made using known techniques. Modifications of interest in the polypeptide sequences may include the alteration, substitution, replacement, insertion, or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule, an alteration which may involve preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation. Techniques for such alteration, substitution, replacement, insertion, or deletion are known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).
  • N-glycosylation sites in a polypeptide' s 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. Appropriate substitutions, additions, or deletions to the 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 polypeptides include those described in U.S. Patent 5,071,972 and EP 276,846, hereby inco ⁇ orated by reference.
  • Additional variants within the scope of the invention include polypeptides that can be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives can 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.
  • Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein. Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the polypeptide.
  • the invention also provides polynucleotides encoding MPD polypeptides.
  • polynucleotide refers to a polymeric form of nucleotides of at least 10 bases in length.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide.
  • DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • the polynucleotides of the invention include full-length genes and cDNA molecules as well as a combination of fragments thereof.
  • the polynucleotides of the invention are preferentially derived from human sources, but the invention includes those derived from non- human species, as well.
  • isolated polynucleotide is meant a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant polynucleotide molecule, which is inco ⁇ orated into a vector, e.g., an expression vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • An MPD polynucleotide of the invention (1) encodes a polypeptide comprising a sequence as set forth in SEQ D NO: 1-26 or 27 or a fragment thereof; (2) has a sequence complementary to a (1); (3) polynucleotides that specifically hybridize to the polynucleotide of (1) under moderate to highly stringent conditions; and (4) polynucleotides of (1) - (3) wherein T can also be U (e.g., RNA sequences). Also encompassed by the invention are homologs of an MPD polynucleotide of the invention. These polynucleotides can be identified in several ways, including isolation of genomic or cDNA molecules from a suitable source, or computer searches of available sequence databases.
  • Oligonucleotides or polynucleotides corresponding to the amino acid sequences described herein can be used as probes or primers for the isolation of polynucleotide homologs or as query sequences for database searches. Degenerate oligonucleotide sequences can be obtained by "back-translation" from the amino acid sequences of the invention. The polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding an MPD polypeptide. Oligonucleotides that define the desired termini of a target polynucleotide molecule are employed as 5' and 3' primers.
  • PCR polymerase chain reaction
  • fragments of the polynucleotides of the invention are useful as probes and primers to identify or amplify related sequence or obtain full- length sequences of an MPD of the invention.
  • the oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector.
  • PCR techniques are known in the art (see, e.g., PCR Protocols: A Guide to Methods and Applications, Innis et. al., eds., Academic Press, Inc. (1990)).
  • the invention also includes polynucleotides and oligonucleotides that hybridize under reduced stringency conditions, more preferably moderately stringent conditions, and most preferably highly stringent conditions, to MPD polynucleotides.
  • the basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, F. M.
  • One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about 55 °C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 °C), and washing conditions of about 60 °C, in 0.5 x SSC, 0.1% SDS.
  • highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 °C, 0.2 x SSC, 0.1% SDS.
  • SSPE lxSSPE is 0.15M NaCl, 10 mM NaH 2 P0 4 , and 1.25 mM EDTA, pH 7.4
  • IxSSC 0.15M NaCl and 15 mM sodium citrate
  • wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al, 1989).
  • the hybrid length is assumed to be that of the hybridizing nucleic acid.
  • the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity.
  • each such hybridizing nucleic acid has a length that is at least 25% (more preferably at least 50%, 60%, or 70%, and most preferably at least 80%) of the length of a polynucleotide of the invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, or at least 99%, and most preferably at least 99.5%) with a polynucleotide of the invention to which it hybridizes.
  • Constantly modified variants applies to both polypeptide and polynucleotide.
  • conservatively modified variants refer to codons in the polynucleotide which encode identical or essentially identical amino acids. Because of the degeneracy of the genetic code, a large number of functionally identical polynucleotides encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such variations are “silent variations,” which are one species of conservatively modified variations.
  • Every polynucleotide sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a polynucleotide except AUG, which is ordinarily the only codon for methionine
  • AUG which is ordinarily the only codon for methionine
  • the invention also provides methodology for analysis of polynucleotides of the invention on "DNA chips" as described in Hacia et al., Nature Genetics, 14:441-447 (1996).
  • high-density arrays of oligonucleotides comprising a sequence encoding an MPD polypeptide, fragment, or a variant or mutant thereof are applied and immobilized to the chip and can be used to detect sequence variations in a population.
  • Polynucleotides in a test sample are hybridized to the immobilized oligonucleotides.
  • the hybridization profile of the target polynucleotide to the immobilized probe is quantitated and compared to a reference profile.
  • the resulting genetic information can be used in molecular diagnosis.
  • the density of oligonucleotides on DNA chips can be modified as needed.
  • the invention also provides genes corresponding to the polynucleotides disclosed herein.
  • Corresponding genes are the regions of the genome that are transcribed to produce the mRNAs from which cDNA molecules are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements.
  • the corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • polypeptides and fragments of the invention can be accomplished by any suitable technique, including but not limited to the following methods.
  • the isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19:4485 (1991); and Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985, and Supplements), in order to produce a polypeptide of the invention recombinantly.
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant polypeptides are also known and are exemplified in R. Kaufman, Methods in Enzymology 185:537 (1990).
  • operably linked means that an isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the polypeptide encoded by the polynucleotide is expressed by a host cell which has been transformed (transfected) with the vector or polynucleotide operably linked to the control sequence.
  • a sequence encoding an appropriate signal peptide can be inco ⁇ orated into expression vectors.
  • the choice of signal peptide or leader can depend on factors such as the type of host cells in which the recombinant polypeptide is to be produced.
  • heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin (IL)-7 (see, U.S. Patent 4,965,195); the signal sequence for IL-2 receptor (see, Cosman et al, Nature 312:768, 1984); the IL-4 receptor signal peptide (see, EP 367,566); the type I IL-1 receptor signal peptide (see, U.S.
  • Patent 4,968,607 and the type II IL-1 receptor signal peptide (see, EP 460,846).
  • 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 a polypeptide from the cell.
  • a polypeptide preparation can include a mixture of polypeptide molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
  • DHFR dihydrofolate reductase
  • a suitable strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216, 1980).
  • 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.
  • gene products can be obtained via homologous recombination, or "gene targeting" techniques. Such techniques employ the introduction of exogenous transcription control elements (such as the CMV promoter or the like) in a particular predetermined site on the genome, to induce expression of an endogenous gene encoding an MPD polypeptide of the invention.
  • exogenous transcription control elements such as the CMV promoter or the like
  • the location of integration into a host chromosome or genome can be easily determined by one of skill in the art, given the known location and sequence of the gene.
  • the invention also contemplates the introduction of exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product.
  • exogenous transcriptional control elements in conjunction with an amplifiable gene, to produce increased amounts of the gene product.
  • the practice of homologous recombination or gene targeting is explained by Schimke, et al. 'Amplification of Genes in Somatic Mammalian cells," Methods in Enzymology 151:85 (1987), and by Capecchi, et al. , “Tlie New Mouse Genetics: Altering the Genome by Gene Targeting," TIG 5:70 (1989).
  • Suitable host cells for expression of the polypeptide include eukaryotic and prokaryotic cells.
  • Mammalian host cells include, for example, 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, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see, McMahan et al EMBO J.
  • yeast in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides.
  • bacterial strains include, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the polypeptide may also be produced by operably linking a polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • a host cell that comprises an isolated polynucleotide of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell". Any method, which neutralizes MPD polypeptides or inhibits expression (either transcription or translation) of an MPD polynucleotide can be used to reduce the biological activities of MPD polypeptides.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a mRNA having an MPD polynucleotide sequence. Absolute complementarity, although preferred, is not required. Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to, and including, the AUG initiation codon, should work most efficiently at inhibiting translation.
  • Antisense nucleic acids are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
  • the oligonucleotides can be DNA, RNA, chimeric mixtures, derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, and the like.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci. U.S.A. 86:6553, 1989; Lemaitre et al, Proc. Natl. Acad. Sci. 84:648, 1987; PCT Publication No.
  • the antisense molecules are delivered to cells, which express a transcript having an MPD polynucleotide sequence in vivo by, for example, direct injection into the tissue or cell derivation site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • Preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • Tetrahymena-type ribozymes recognize sequences, which are four bases in length, while "hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena- type ribozymes.
  • ribozymes can be composed of modified oligonucleotides and delivered using using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol El promoter.
  • endogenous MPD expression can be reduced by targeting DNA sequences complementary to a regulatory region of the target gene (e.g., the target gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the target gene (see generally, Helene, Anticancer Drug Des., 6(6), 569, 1991; Helene, et al, Ann. N.Y. Acad. Sci., 660:27, 1992; and Maher, Bioassays 14(12), 807, 1992).
  • a regulatory region of the target gene e.g., the target gene promoter and/or enhancers
  • Endogenous gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination (see, e.g., Smithies, et al, Nature 317:230, 1985; Thomas and Capecchi, Cell 51, 503, 1987; Thompson, et al, Cell 5, 313, 1989; each of which is inco ⁇ orated by reference herein in its entirety).
  • targeted homologous recombination see, e.g., Smithies, et al, Nature 317:230, 1985; Thomas and Capecchi, Cell 51, 503, 1987; Thompson, et al, Cell 5, 313, 1989; each of which is inco ⁇ orated by reference herein in its entirety.
  • a mutant non-functional target gene flanked by DNA homologous to the endogenous target gene can be used, with or without a selectable marker and/or a negative selectable marker. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation
  • RNAi RNA interference
  • a “transgene” is a polynucleotide that comprises one or more selected sequences (e.g., encoding ribozymes that cleave MPD mRNA, encoding an antisense molecule to an MPD mRNA, encoding a mutant MPD sequence, and the like) to be expressed in a transgenic animal.
  • the polynucleotide is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal's genome at a location which differs from that of the natural gene.
  • a transgene may include one or more promoters and any other DNA sequences, such as introns, necessary for expression of the selected DNA, all operably linked to the selected DNA, and may include an enhancer sequence.
  • polypeptides, fragment, variants, fusion polypeptides, and the like contain antigenic determinants or epitopes that elicit the formation of antibodies.
  • 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 polypeptide folding.
  • Epitopes can be identified by any of the methods known in the art.
  • MPD polypeptides, MPD fragments and antibodies The influence of MPD polypeptides, MPD fragments and antibodies on intercellular communication, co-stimulation, integrin binding, endothelial cell migration, angiogenesis or immune cell activity can be assayed by contacting a cell or a group of cells with a polynucleotide, polypeptide, agonist or antagonist, to induce, enhance, suppress, or arrest cellular communication, costimulation, integrin binding, endothelial cell migration, angiogenesis or activity in the target cells.
  • Identification of MPD polypeptides, agonists or antagonists can be carried out via a variety of assays known to those skilled in the art.
  • the invention provides a method of detecting the ability of a test agent to affect the cell-cell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity of the test agent on a cell or culture.
  • the method comprises: (1) contacting a first group of target cells with a test agent including a polypeptide comprising an MPD sequence (e.g., SEQ ED NO: 1-27; or a soluble MPD disintegrin polypeptide), a ligand or receptor for an MPD polypeptide, or fragment thereof, under conditions appropriate to the particular assay being used; (2) measuring the net rate of cell-cell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity among the target cells; and (3) observing the net rate of cell-cell interaction, cell-matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity among control cells containing an MPD polypeptide ligand or fragments thereof, in the absence of a test agent, under otherwise identical conditions as the first group of cells.
  • a test agent including a polypeptide comprising an MPD sequence (e.g., SEQ ED NO:
  • the net rate of intercellular communication or co-stimulation in the control cells is compared to that of the cells treated with both an MPD molecule as well as a test agent.
  • the comparison will provide a difference in the net rate of cell-cell interaction, cell- matrix interaction, integrin-associated binding activity, endothelial cell migratory activity, or angiogenic activity indicative of an agent that modulates MPD activity.
  • the test agent can function as an effector by either activating or up-regulating, or by inhibiting or down-regulating cell-cell interaction, cell-matrix interaction, integrinassociated binding, endothelial cell migratory activity, or angiogenic activity.
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al, Proc. Natl. Acad. Sci. USA 77:6091, 1980; Weinberger et al, Eur. J. Immun. 11:405, 1981; Takai et al, J. Immunol. 137:3494, 1986; Takai et al, J. Immunol. 140:508, 1988.
  • Assays for thymocyte or splenocyte cytotoxicity include, without limitation, Current Protocols in Immunology, Coligan eds., Pub. Greene Publishing Associates and Wiley- Interscience (In vitro assays for Mouse Lymphocyte Function pp. 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al, Proc. Natl. Acad. Sci. USA 78:2488, 1981; Herrmann et al, J.Immunol. 128:1968, 1982; Handa et al, J.Immunol. 135:1564, 1985; Takai et al, J.Immunol.
  • Assays for T-cell-dependent IgG responses and isotype switching include, without limitation, those described in: Maliszewski, J. Immunol.
  • Dendritic cell-dependent assays (which will identify, among others, polypeptides expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al, J. Immunol. 134:536, 1995; Inaba et al, J. of Exp. Med. 173:549, 1991;
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cell. Biol. 15: 141, 1995; Keller et al, Mol. and Cell. Biol.
  • Assays for stem cell survival and differentiation include, without limitation, those described in:
  • Assays for tissue generation activity include, without limitation, those described in: Patent Publication No. WO95/16035 (bone, cartilage, tendon); Patent Publication No. WO95/05846 (nerve, neuronal); Patent Publication No. WO91/07491 (skin, endothelium).
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, and Rovee, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al, Endocrinol. 91:562, 1972; Ling et al, Nature 321:779, 1986; Vale et al, Nature 321:776, 1986; Mason et al, Nature 318:659, 1985; Forage et al, Proc. Natl. Acad. Sci. USA 83:3091, 1986.
  • a polynucleotide encoding a polypeptide having an MPD sequence provided by the invention can be used for numerous diagnostic or other useful pu ⁇ oses.
  • a polynucleotide of the invention e.g., a polynucleotide encoding SEQ ED NO: 1-26 or 27
  • Probes and Primers are Among the uses of the disclosed MPD polynucleotides, and combinations of fragments thereof, is the use of fragments as probes or primers. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60 contiguous nucleotides of a DNA sequence.
  • the basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al, 1989 and are described in detail above. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared.
  • a polynucleotide encoding a polypeptide having an MPD polypeptide sequence of the invention, and the disclosed fragments and combinations of these polynucleotides can be used to analyze abnormalities associated with the genes corresponding to MPD polypeptides. This enables one to distinguish conditions in which this marker is rearranged or deleted.
  • polynucleotides of the invention or a fragment thereof can be used as a positional marker to map other genes of unknown location.
  • the polynucleotide can be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, genes (e.g., an MPD-associated disorder) corresponding to the polynucleotides of the invention.
  • the polynucleotides and associated sequences disclosed herein permit the detection of defective genes, and the replacement thereof with normal genes.
  • Defective genes can be detected in in vitro diagnostic assays, and by comparison of the polynucleotide sequences disclosed herein with that of a gene derived from a subject suspected of harboring a defect in this gene or having an MPD- associated disorder.
  • MPD polypeptides and peptide fragments thereof include, but are not limited to, the following: delivery agents; therapeutic and research reagents; molecular weight and isoelectric focusing markers; controls for peptide fragmentation; identification of unknown polypeptides; and preparation of antibodies.
  • the MPD polypeptides (e.g., SEQ ID NO: 1-26 or 27) of the invention can be used as polypeptide purification reagents.
  • MPD polypeptides can be attached to a solid support material and used to purify its binding partners (e.g., an integrin molecule) by affinity chromatography.
  • a polypeptide is attached to a solid support by conventional procedures.
  • chromatography columns containing functional groups that will react with amino acid side chains of polypeptides are available (Pharmacia Biotech, Inc., Piscataway, NJ).
  • an MPD-Fc polypeptide is attached to Polypeptide A- or Polypeptide G-containing chromatography columns through interaction with the Fc moiety.
  • the polypeptide also finds use in purifying or identifying cells that express a binding partner 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 the binding partner expressing cells are contacted with the solid phase having the polypeptides thereon. Cells expressing the binding partner on the cell surface bind to the polypeptides on the solid phase, and unbound cells then are washed away.
  • the polypeptides can be conjugated to a detectable moiety, then incubated with cells to be tested for binding partner expression.
  • the polypeptides also find use as carriers for delivering agents attached thereto to cells bearing identified binding partners (e.g., an integrin).
  • the polypeptides thus can be used to deliver diagnostic or therapeutic agents to such cells in in vitro or in vivo procedures.
  • Detectable (diagnostic) and therapeutic agents that can 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 I, 131 1, 99m Tc, m In, and 76 Br.
  • radionuclides suitable for therapeutic use are 131 I, 211 At, 77 Br, 186 Re, 188 Re, 212 Pb, 12 Bi, 109 Pd, 64 Cu, and 67 Cu.
  • Such agents can 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 polypeptide or agent can be derivatized to generate or attach a desired reactive functional group.
  • the derivatization can involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to polypeptides (Pierce Chemical Company, Rockford, Illinois).
  • soluble MPD disintegrins that can be used to target cells expressing a binding partner for the MPD disintegrin moiety (e.g., an integrin).
  • Such soluble MPD disintegrins can be used to target reagents to cells expressing, for example, the disintegrin' s cognate integrin.
  • antibodies specific for an MPD polypeptide can be labeled with a diagnostic or therapeutic agent and used to target the diagnostic or therapeutic to cells expressing an MPD polypeptide.
  • MPD polypeptides and MPD fragments can be employed in modulating a biological activity of an ADAM polypeptide, particularly MPD polypeptide, in in vitro or in vivo procedures.
  • an ADAM polypeptide particularly MPD polypeptide
  • MPD polypeptide can be employed in modulating a biological activity of an ADAM polypeptide, particularly MPD polypeptide, in in vitro or in vivo procedures.
  • domains of MPD polypeptides that act as modulators of native ADAM polypeptide function, including native MPD activity, when expressed as fragments or as components of fusion polypeptides.
  • a substantially purified polypeptide domain of the invention can be used to inhibit binding of an MPD polypeptide to endogenous binding partners. Such use effectively would block MPD interactions and inhibit MPD activities.
  • a soluble form of an MPD binding partner e.g., a soluble integrin domain
  • a soluble form of an MPD binding partner is used to bind to, and competitively inhibit activation of the
  • Antibodies that bind to MPD polypeptides can inhibit MPD polypeptide activity and may act as antagonists.
  • antibodies that specifically bind to one or more epitopes of an MPD polypeptide, or epitope of conserved variants of MPD polypeptides, or fragments can be used to inhibit MPD activity.
  • specifically bind means that an antibody to an MPD polypeptide or fragment thereof will not cross-react with unrelated polypeptides. Preferably such an antibody will not cross-react with other members of the ADAM family.
  • the invention further encompasses the use of agonists of MPD activity to treat or ameliorate the symptoms of a disease for which increased disintegrin activity is beneficial.
  • the invention entails administering compositions comprising an MPD polynucleotide or fragment thereof or a polypeptide comprising an MPD amino acid sequence (e.g., SEQ D NO: 1-26 or 27) or fragment thereof.
  • the administering may be to cells in vitro, to cells ex vivo, to cells in vivo, and/or to a multicellular organism.
  • Preferred therapeutic forms include soluble forms of an MPD having disintegrin activity.
  • Such a soluble MPD disintegrin polypeptide will bind to its binding partner (e.g., an integrin) and stimulate a biological activity associated with the binding partner.
  • compositions comprise administering a polynucleotide encoding an MPD polypeptide for expression in a host organism for treatment of disease.
  • a polynucleotide encoding an MPD polypeptide for expression in a host organism for treatment of disease is particularly preferred in this regard.
  • the invention encompasses the administration of compounds found to increase the endogenous activity of polypeptides comprising an MPD amino acid sequence to cells and/or organisms.
  • One example of compounds that increase MPD polypeptide activity are antibodies that bind to MPD polypeptides, preferably monoclonal antibodies, and increase or stimulate MPD polypeptide activity by causing constitutive intracellular signaling (or "ligand mimicking"), or by preventing the binding of a native inhibitor of MPD polypeptide activity.
  • an MPD polypeptide, fragment, variant, antagonist, agonist, antibody, and binding partner of the invention can be useful for treating medical conditions and diseases associated with cell-cell and cell matrix interactions (e.g., integrin-mediated disorders), endothelial migration, angiogenesis, inflammation, cancer, allergy, reproductive, neurological and vascular conditions as described further herein.
  • the therapeutic molecule or molecules to be used will depend on the etiology of the condition to be treated and the biological pathways involved, and will consider that different variants, antagonists, and binding partners of MPD polypeptides may have similar or different effects.
  • an MPD polypeptide or fragment thereof may act as an antagonist of a protein processing function of metalloproteinases (e.g., from other members of the ADAM family of polypeptides) by interacting with an ADAM binding partner and preventing the activity of the metalloproteinase upon its substrate. Accordingly, an MPD may modulate protein processing, such as release of growth factors, adhesion proteins, and inflammatory factors.
  • the disclosed MPD polypeptides, fragments thereof, antibodies, compositions and combination therapies described herein are useful in medicines for treating bacterial, viral or protozoal infections, and complications resulting therefrom.
  • Cardiovascular disorders are treatable with the disclosed MPD polypeptides, fragments thereof, antibodies, pharmaceutical compositions or combination therapies, including aortic aneurysms; arteritis; vascular occlusion; complications of coronary by-pass surgery; ischemia/reperfusion injury; heart disease; heart failure; and myocardial infarction.
  • the MPD polypeptides, fragments thereof, antibodies, compositions and combination therapies of the invention can be used to treat chronic pain conditions, to treat various disorders of the endocrine system, conditions of the gastrointestinal system, disorders of the genitourinary system, and anemias and hematological disorders.
  • integrins e.g., o ⁇ 5 3 , GC v ⁇ s, ⁇ i, ⁇ 4 , cci, a 2 , a 3 , a , as, and ⁇ 6 integrins
  • methods for using MPD polypeptides, fragments thereof, antibodies, compositions or combination therapies to treat various hematologic and oncologic disorders include o ⁇ 5 3 , GC v ⁇ s, ⁇ i, ⁇ 4 , cci, a 2 , a 3 , a , as, and ⁇ 6 integrins
  • soluble MPD disintegrin domains can be used to treat various forms of cancer, including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer- associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia, and hypercalcemia by modulating integrin-associated interactions.
  • cancer including acute myelogenous leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer- associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia, and hypercalcemia by modulating integrin-associated interactions.
  • the MPD polypeptides, fragments thereof, compositions or combination therapies are useful for treating leukemia, including acute myelogenous leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia.
  • Other malignancies with invasive metastatic potential that can be treated with the MPD polypeptides, fragments, antibodies, compositions and combination therapies, include multiple myeloma, various lymphoproliferative disorders such as autoimmune lymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia, hairy cell leukemia, chronic lymphatic leukemia, peripheral T-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, Epstein-Barr virus- positive T cell lymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressive lymphoma, acute lymphatic leukemias, T gamma lymphoproliferative
  • a combination of at least one MPD polypeptide, fragment thereof, or antibody, and one or more anti-angiogenesis factors or other therapeutic agent(s) may be administered to the subject.
  • the additional therapeutic agent(s) may be administered prior to, concurrently with, or following the administration of the MPD polypeptide, fragment thereof, or antibody.
  • the use of more than one therapeutic agent is particularly advantageous when the subject that is being treated has a solid tumor.
  • the treatment further comprises treating the mammal with radiation. Radiation, including brachytherapy and teletherapy, may be administered prior to, concurrently with, or following the administration of the MPD polypeptide, fragment, antibody, or MPD binding partner and/or additional therapeutic agent(s).
  • the method includes the administration of, in addition to a MPD polypeptide, fragment thereof, or antibody, one or more therapeutics selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones and antihormones, antibodies, immunotherapeutics, radiotherapeutics, and biological response modifiers.
  • one or more therapeutics selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones and antihormones, antibodies, immunotherapeutics, radiotherapeutics, and biological response modifiers.
  • the method includes administration of, in addition to an MPD polypeptide, fragment thereof, or antibody, one or more therapeutic polypeptides, including soluble forms thereof, selected from the group consisting of Flt3 ligand (see, U.S. Patent No. 5,554,512), CD40 ligand (see, U.S. Patent No. 5,716,805), EL-2, EL-12, 4-1BB ligand (see, U.S. Patent No. 5,674,704), anti-4-lBB antibodies, TRAIL, TNF antagonists and TNF receptor antagonists including TNFR/Fc, Tek antagonists, TWEAK antagonists and TWEAK-R (see, U.S.
  • an MPD polypeptide, fragment thereof, or antibody of the invention is used as a component of, or in combination with, "metronomic therapy,” such as that described by Browder et al. and Klement et al. (Cancer Research 60:1878, 2000; J. Clin. Invest. 105(8):R15, 2000; see also Barinaga, Science 288:245, 2000).
  • This invention provides compounds, compositions, and methods for treating a subject, preferably a mammalian subject, and most preferably a human subject, who is suffering from a medical disorder and in particular, an MPD-associated disorder.
  • MPD-associated disorders include conditions caused (directly or indirectly) or exacerbated by binding between a polypeptide having an MPD sequence and its binding partner (e.g., an integrin).
  • the terms “illness,” “disease,” “disorder,” “medical condition,” “abnormal condition” and the like are used interchangeably with the term “medical disorder.”
  • the terms “treat”, “treating”, and “treatment” used herein include curative, preventative (e.g., prophylactic) and palliative or ameliorative treatment.
  • MPD polypeptides and fragments, MPD polynucleotides encoding an MPD polypeptide, and/or agonists or antagonists of the MPD polypeptide such as antibodies can be administered to the subject in need through known means.
  • compositions of the invention can contain a polypeptide in any form described herein, such as native polypeptides, variants, derivatives, oligomers, and biologically active fragments.
  • the composition comprises a soluble polypeptide or an oligomer comprising soluble MPD polypeptides (e.g., a soluble MPD disintegrin domain).
  • a therapeutically effective amount of a therapeutic agent of the invention is administered to a subject having a condition to be treated, preferably to treat or ameliorate diseases associated with the activity of an MPD polypeptide.
  • “Therapeutic agent” includes without limitation any MPD polypeptide, fragment, and variant; polynucleotide encoding an MPD polypeptide, fragment, and variant; agonists or antagonists of the an MPD polypeptide such as antibodies; an MPD polypeptide binding partner; complexes formed from an MPD polypeptide, fragment, variant, and binding partner, and the like.
  • the term "therapeutically effective amount” means the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method that is sufficient to show a meaningful subject benefit, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful subject benefit e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • administering a therapeutically effective amount of a therapeutic agent means that the subject is treated with said therapeutic agent in an amount and for a time sufficient to induce an improvement, and preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder.
  • An improvement is considered “sustained” if the subject exhibits the improvement on at least two occasions separated by one or more weeks.
  • the degree of improvement is determined based on signs or symptoms, and determinations may also employ questionnaires that are administered to the subject, such as quality-of-life questionnaires.
  • Various indicators that reflect the extent of the subject's illness may be assessed for determining whether the amount and time of the treatment is sufficient.
  • the baseline value for the chosen indicator or indicators is established by examination of the subject prior to administration of the first dose of the therapeutic agent. Preferably, the baseline examination is done within about 60 days of administering the first dose. If the therapeutic agent is being administered to treat acute symptoms, the first dose is administered as soon as practically possible after the injury has occurred. Improvement is induced by administering therapeutic agents such as an MPD polypeptide, fragment, antibody, or MPD binding partner until the subject manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, this degree of improvement is obtained by repeatedly administering this medicament over a period of at least a month or more, e.g., for one, two, or three months or longer, or indefinitely.
  • therapeutic agents such as an MPD polypeptide, fragment, antibody, or MPD binding partner
  • suitable dosages will vary, depending upon such factors as the nature and severity of the disorder to be treated, the subject's body weight, age, general condition, and prior illnesses and/or treatments, 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 such as standard dosing trials.
  • the therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the specific activity of the compound and can be readily determined by routine experimentation.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture, while minimizing tonicities. Such information can be used to more accurately determine useful doses in humans.
  • the attending physician will decide the amount of polypeptide of the invention with which to treat each individual subject. Initially, the attending physician will administer low doses of polypeptide of the invention and observe the subject's response. Larger doses of polypeptide of the invention may be administered until the optimal therapeutic effect is obtained for the subject, and at that point the dosage is not increased further.
  • the various pharmaceutical compositions used to practice the method of the invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 microgram to about 1 mg) of a polypeptide of the invention per kg body weight.
  • an MPD polypeptide, fragment, antibody, or MPD binding partner is administered one time per week to treat the various medical disorders disclosed herein.
  • polypeptide, fragment, antibody, or MPD binding partner is administered at least two times per week and in another embodiment at least three times per week.
  • the effective amount of an MPD polypeptide, fragment, antibody, or MPD binding partner per adult dose ranges from 1-20 mg/m 2 , and preferably is about 5-12 mg/m 2 .
  • a flat dose may be administered whose amount may range from 5-100 mg/dose.
  • Exemplary dose ranges for a flat dose to be administered by subcutaneous injection are 5-25 mg/dose, 25-50 mg/dose and 50-100 mg/dose.
  • the various indications described herein are treated by administering a preparation acceptable for injection containing an MPD polypeptide, fragment, antibody, or MPD binding partner at 25 mg/dose, or alternatively, containing 50 mg per dose.
  • the 25 mg or 50 mg dose may be administered repeatedly, particularly for chronic conditions.
  • the dose is appropriately adjusted in accord with standard medical practices.
  • an improvement in a subject's condition will be obtained by injecting a dose of about 25 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner one to three times per week over a period of at least three weeks, or a dose of 50 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner one or two times per week for at least three weeks (a treatment for longer periods may be necessary to induce the desired degree of improvement).
  • the regimen may be continued indefinitely, with adjustments being made to dose and frequency if such are deemed necessary by the subject's physician.
  • a suitable regimen involves the subcutaneous injection of 0.4 mg/kg, up to a maximum dose of 25 mg of an MPD polypeptide, fragment, antibody, or MPD binding partner, administered by subcutaneous injection one or more times per week.
  • an antibody against an MPD polypeptide is used as an MPD polypeptide antagonist, a preferred dose range is 0.1 to 20 mg/kg, and more preferably is 1-10 mg/kg.
  • Another preferred dose range for an anti-MPD polypeptide antibody is 0.75 to 7.5 mg/kg of body weight.
  • Humanized antibodies are preferred. Such antibodies may be injected or administered intravenously.
  • Compositions comprising an effective amount of an MPD polypeptide of the invention
  • compositions suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • 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, Mack Publishing Company, Easton, PA.
  • the polypeptide may undergo pegylation to assist in adso ⁇ tion or uptake.
  • compositions can be complexed with polyethylene glycol (PEG), metal ions, or inco ⁇ orated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, or inco ⁇ orated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • PEG polyethylene glycol
  • metal ions or inco ⁇ orated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, and the like, or inco ⁇ orated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are inco ⁇ orated herein by reference.
  • 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, so that the characteristics of the carrier will depend on the selected route of administration, i one preferred embodiment of the invention, sustained-release forms of an MPD polypeptide are used.
  • Sustained-release forms suitable for use in the disclosed methods include, but are not limited to, an MPD polypeptide that is encapsulated in a slowly-dissolving biocompatible polymer (such as the alginate microparticles described in U.S. Pat. No. 6,036,978), admixed with such a polymer (including topically applied hydrogels), and or encased in a biocompatible semi-permeable implant.
  • An MPD polypeptide of the invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other polypeptides.
  • pharmaceutical compositions of the invention may comprise a polypeptide of the invention in such multimeric or complexed form.
  • the pharmaceutical composition of the invention may be in the form of a complex of the polypeptide(s) of invention.
  • the invention further includes the administration of an MPD polypeptide, fragment, antibody, or MPD binding partner concurrently with one or more other drugs that are administered to the same subject in combination, each drug being administered according to a regimen suitable for that medicament.
  • Conscurrent administration encompasses simultaneous or sequential treatment with the components of the combination, as well as regimens in which the drugs are alternated, or wherein one component is administered long-term and the other(s) are administered intermittently.
  • Components may be administered in the same or in separate compositions, and by the same or different routes of administration.
  • components that may be included in the pharmaceutical composition of the invention are cytokines, lymphokines, or other hematopoietic factors such as: M-CSF, GM-CSF, TNF, IL-1, IL- 2, E -3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, I -10, IL-11, D -12, E -13, EL-14, IL-15, EL-17, E -18, EFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents that either enhance the activity of the polypeptide or compliment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with a polypeptide of the invention, or to minimize side effects.
  • an MPD polypeptide, fragment, antibody, or MPD binding partner of the invention may be included in formulations with a particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • drugs to be administered concurrently include but are not limited to antivirals, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, non- steroidal anti-inflammatories, pentoxifylline, thalidomide, and disease-modifying antirheumatic drugs (DMARDs) such as azathioprine, cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine and gold compounds such as oral gold, gold sodium thiomalate, and aurothioglucose.
  • DMARDs disease-modifying antirheumatic drugs
  • an MPD polypeptide, fragment, antibody, or MPD binding partner may be combined with a second MPD polypeptide, antibody against an MPD polypeptide, or an MPD polypeptide-derived peptide that acts as a competitive inhibitor of a native an MPD polypeptide.
  • Any efficacious route of administration may be used to therapeutically administer an MPD polypeptide, fragment, antibody, or MPD binding partner thereof, including those compositions comprising MPD polynucleotides.
  • Parenteral administration includes injection, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes by bolus injection or by continuous infusion.
  • Other routes include localized administration, e.g., at a site of disease or injury.
  • MPD polypeptide, fragment, antibody, or MPD binding partner may be delivered by implanting cells that express the polypeptide, for example, by implanting cells that express an MPD polypeptide, fragment, antibody, or MPD binding partner.
  • Cells may also be cultured ex vivo in the presence of polypeptides of the invention in order to proliferate or to produce a desired effect on or activity in such cells.
  • Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
  • the subject's own cells are induced to produce an MPD polypeptide, fragment, antibody, or MPD binding partner by transfection in vivo or ex vivo with a polynucleotide that encodes an MPD polypeptide, fragment, antibody, or MPD binding partner.
  • the polynucleotide can be introduced into the subject's cells, for example, by injecting naked DNA or liposome-encapsulated DNA that encodes an MPD polypeptide, fragment, antibody, or MPD binding partner, or by other means of transfection.
  • Polynucleotides of the invention may also be administered to subjects by other known methods for introduction of nucleic acids into a cell or organism (including, without limitation, in the form of viral vectors).
  • the polypeptide When a therapeutically effective amount of an MPD polypeptide, fragment thereof, antibody, or binding partner of the invention is administered orally, the polypeptide will typically be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% a polypeptide of the invention, and preferably from about 25 to 90% a polypeptide of the invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of a polypeptide of the invention, and preferably from about 1 to 50% a polypeptide of the invention.
  • an MPD polypeptide, fragment, antibody, or binding agent of the invention When a therapeutically effective amount of an MPD polypeptide, fragment, antibody, or binding agent of the invention is administered by intravenous, cutaneous or subcutaneous injection, the polypeptide will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable polypeptide solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to a polypeptide of the invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the duration of intravenous therapy using the pharmaceutical composition of the invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual subject.
  • the duration of each application of a polypeptide of the invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy.
  • the therapeutic method includes administering a pyrogen-free, physiologically acceptable form of the composition topically, systematically, locally or in association with an implant or device. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site tissue damage. Additional useful agents may also optionally be included in the composition, as described above, or may be administered simultaneously or sequentially with the composition in the methods of the invention.
  • compositions can include a matrix capable of delivering the polypeptide-containing composition to the site tissue damage, providing a structure for the developing tissue and optimally capable of being resorbed into the body.
  • the choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.
  • Potential matrices for the compositions include calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomo ⁇ hometric determinations and tetracycline labeling.
  • compositions comprising an MPD polypeptide, fragment, antibody, or MPD binding partner is useful in the treatment of disease conditions in non-human animals, such as pets (dogs, cats, birds, primates, and the like), domestic farm animals (horses cattle, sheep, pigs, birds, and the like).
  • an appropriate dose may be determined according to the animal's body weight. For example, a dose of 0.2-1 mg/kg may be used.
  • the dose is determined according to the animal's surface area, an exemplary dose ranging from 0.1-20 mg/m 2 , or more preferably, from 5-12 mg/m 2 .
  • a suitable dose is 0.4 mg/kg.
  • an MPD polypeptide, fragment, antibody, or MPD binding partner (preferably constructed from genes derived from the same species as the subject), is administered by injection or other suitable route one or more times per week until the animal's condition is improved, or it may be administered indefinitely.
  • the invention also relates to the use an MPD polypeptide, fragment, and variant; polynucleotide encoding an MPD polypeptide, fragment, and variant; agonists or antagonists of an MPD polypeptide such as antibodies; an MPD polypeptide binding partner; complexes formed from an MPD polypeptide, fragment, variant, and binding partner, and the like, in the manufacture of a medicament for the prevention or therapeutic treatment of a disease or disorder.
  • the invention provides a computer readable medium having stored thereon a member selected from the group consisting of a polypeptide comprising a sequence as set forth in SEQ ED Nos: 1-26, or 27; and a set of polypeptide sequences wherein at least one of said sequences comprises a sequence as set forth in SEQ ID Nos: 1-27.
  • One embodiment of the invention comprises a computing environment and a plurality of algorithms selectively executed to analyze a polypeptide or polynucleotide of the invention.
  • analyses of an ADAM polypeptide include, without limitation, displaying the amino acid sequence of a polypeptide in the set, comparing the amino acid sequence of one polypeptide in the set to the amino acid sequence of another polypeptide in the set, predicting the structure of a polypeptide in the set, determining the nucleotide sequences of nucleic acids encoding a polypeptide in the set, and identifying a gene corresponding to a polypeptide in the set.
  • EXAMPLE 1 Identification of MPD Polypeptides A data set was received from Celera Genomics (Rockville, Maryland) containing amino acid sequences predicted to be encoded by the human genome. This data set was searched using a BLAST algorithm to identify ADAM family polypeptides. The polypeptides set forth in Figure 1 were identified as having substantially homology to metalloproteinase-disintegrin polypeptides, including the ADAM family of polypeptides.
  • EXAMPLE 2 RACE Analysis
  • Polynucleotides encoding an MPD polypeptide are identified by rapid amplification of cDNA ends (RACE) analysis. All RACE products are cloned into vectors and sequenced. Sequence analysis of the RACE products provides a number of clones having substantially identical sequences. RACE Analysis kits are available from a number of companies including Roche Molecular Systems. Primers were designed based upon consensus sequences found by RACE product comparison. A primer pair comprising nucleotides reverse transcribed from the polypeptide sequences of Figure 1 are used to PCR amplify a cDNA library from lymph node cells, bone marrow cells, as well as other cell types known in the art. The resulting PCR products are cloned and sequenced using standard protocols.
  • RACE rapid amplification of cDNA ends
  • SEQ ED Nos:4-5, 10, 14, 21-22, and 25-26 comprise the sequence HexGHxxGxxHD (SEQ ID NO:28) at residues 208 to 219, 15 to 26, 229 to 240, 555 to 566, 3 to 14, 269 to 280, and 154 to 165, respectively.
  • SEQ ID NO:24 comprises a sequence that has substantial identity to the conserved HexGHxxGxxHD motif.
  • a polypeptide comprising SEQ ED NO: 4, 5, 10, 14, 21, 22, 24, 25, or 26 is predicted to have metalloproteinase activity.
  • SEQ ED Nos:4, 8, 10, 14, 18-19, 21, and 25, as well as some known metalloproteinases, share homology to a sequence comprising LNIx(I/V)(A/V)LVGLE(V/I)WT and thus a polypeptide comprising SEQ ED NO: 4, 8, 10, 14, 18-19, 21, or 25 is predicted to have metalloproteinase activity.
  • the invention also provides SEQ ED Nos:2, 3, 7, 9, 17, 20, and 27 which have a high degree of homology to the Testicular Metalloproteinase-like, Disintegrin-like, Cysteine rich (TMDC) protein family, including TMDC m, TMDC EVA, and TMDC EVC.
  • TMDC Testicular Metalloproteinase-like, Disintegrin-like, Cysteine rich
  • Table 1 shows the relative identity of representative polypeptides of the invention with TMDC protein family members.
  • polypeptides of Figure 1 have homology to the ADAM (A Disintegrin And Metalloproteinase) family of proteins.
  • ADAM A Disintegrin And Metalloproteinase
  • Such polypeptides include metalloproteinase domains characterized as having the highly conserved HExGHxxGxxHD motif (SEQ ED NO: 28) and/or a LNlx( ⁇ A/V)LVGLE(V/ ⁇ )WT motif (SEQ ED NO:29).
  • SEQ ID Nos:4, 10, 14, 21, 25, and 26 comprise a sequence from about amino acids residues 65 to 274 of SEQ ED NO:4; 24 to 235 of SEQ ED NO: 10; 85 to 290 of SEQ ID NO: 14; 202 to 411 of SEQ ED NO:21; 123 to 332 of SEQ ED NO:25; and 118 to 215 of SEQ ID NO:26.
  • a number of polypeptides of Figure 1 have homology to disintegrin domain sequences typically characterized as containing a conserved motif having a sequence CGN(G/K)x(L/V)(E/D)x(G/N)EECDCG (SEQ ED NO:30) (herein after the "CGN-GEEC" motif).
  • SEQ D Nos:4, 10, 14, 21, and 24-26 contain the CGN-GEEC motif and thus a polypeptide comprising SEQ ID NO: 4, 10, 14, 21, 24, 25, or 26 is predicted to have disintegrin activity.
  • SEQ ED NO: 11 has a putative CGN-GEEC sequence at residues 43 to 57 and thus a polypeptide comprising SEQ ED NO: 11 is predicted to have disintegrin activity.
  • ADAM family of proteins are characterized as having a number of conserved cysteine residues in their disintegrin and cysteine-rich domains.
  • Table 2, above provides a summary of the relative domains and residues characterizing the domains of some of the polypeptides of the invention.
  • Variants of the MPD polypeptide sequences can be identified based upon the sequences provided herein. A number of variants are provided herein. Amino acid substitutions and other alterations (deletions, insertions, and the like) to MPD amino acid sequences are predicted to be more likely to alter or disrupt MPD polypeptide activities if they result in changes to the conserved residues of the amino acid sequences as shown in Figure 1, and particularly if those changes do not substitute an amino acid of similar structure (such as substitution of any one of the aliphatic residues - Ala, Gly, Leu, lie, or Val - for another aliphatic residue).
  • a substantially purified MPD polypeptide can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U.S. Patent 4,411,993.
  • Mice are immunized with an MPD polypeptide immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 ⁇ g subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional MPD polypeptide emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule.
  • Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for an MPD polypeptide antibody by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of binding of an MPD polypeptide to an MPD polypeptide binding partner.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • positive animals are provided one last intravenous injection of an MPD polypeptide in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580).
  • Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin and thymidine
  • hybridoma cells are screened by ELISA for reactivity against a substantially pure MPD polypeptide by adaptations of the techniques disclosed in Engvall et al, (Immunochem. 8:871, 1971) and in U.S. Patent 4,703,004.
  • a preferred screening technique is the antibody capture technique described in Beckmann et al, (J. Immunol. 144:4212, 1990).
  • Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-MPD monoclonal antibody.
  • hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques.
  • Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography.
  • affinity chromatography based upon binding of antibody to Polypeptide A or Polypeptide G can also be used, as can chromatography based upon binding to MPD polypeptide.
  • the gene corresponding to an MPD polypeptide is mapped using PCR-based mapping strategies.
  • Initial human chromosomal assignments are made using an MPD-specific PCR primers such as those described Example 8 and a BIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (New Haven, CT), following the manufacturer's instructions. More detailed mapping is performed using a Genebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville, AL (see, e.g., Walter, MA et al, Nature Genetics 7:22-28, 1994).
  • MPDdis-Fc polypeptide a nucleic acid encoding a disintegrin domain such as amino acid residues of, for example, SEQ EDNO:6; SEQ ED NO:6 from about residue 43 to 148; SEQ ID NO:8 from about residue 1 to 366; SEQ ED NO:8 from about residue 38 to 366; SEQ ED NO: 11; SEQ ED NO: 13; SEQ ED NO: 14 from about residue 1 to 622; SEQ ED NO: 14 from about residue 84 to 622; SEQ ED NO: 14 from about residue 299 to 622; SEQ ID NO: 16; SEQ ED NO:21 from about residue 1 to 701; SEQ ED NO:23; SEQ ED NO:24; SEQ ED NO:24 from about residue 278 to 435; SEQ ID NO:25 from about residue 1 to 627; SEQ ID NO:26; or SEQ ID NO:26 from about residue 224 to 383, beginning with the CGN-GEEC sequence and ending prior to
  • the construct can use the igKappa leader, which is cleaved by the signal peptidase after the C-terminal G (Glycine) amino acid.
  • the soluble form of the molecule is then predicted to start after this residue.
  • a few residues correspoding to codon(s) of the restiction site can be present at either end of the disintegrin domain used to link a disintegrin domain of to the Fc domain.
  • a mouse corneal pocket assay is used to quantitate the inhibition of angiogenesis by MPDdis-Fc polypeptides in vivo.
  • agents to be tested for angiogenic or anti- angiogenic activity are immobilized in a slow release form in a hydron pellet, which is implanted into micropockets created in the corneal epithelium of anesthetized mice.
  • Vascularization is measured as the appearance, density, and extent of vessel in growth from the vascularized corneal limbus into the normally avascular cornea.
  • Hydron pellets as described in Kenyon et al, Invest Opthamol. & Visual Science 37:1625, 1996, inco ⁇ orate sucralfate with bFGF (90 ng/pellet), bFGF and IgG (11 ⁇ g/pellet, control), or bFGF and a range of concentrations of the agent to be tested (e.g., MPDdis-Fc polypeptide).
  • the pellets are surgically implanted into corneal stromal micropockets created by micro-dissection 1 mm medial to the lateral corneal limbus of 6-8 week old male C57BL mice.
  • the corneas are photographed using a Zeiss slit lamp at an incipient angle of 35-50° from the polar axis in the meridian containing the pellet. Images are digitized and processed by subtractive color filters (Adobe Photoshop 4.0) to delineate established microvessels by hemoglobin content. Image analysis software (Bioquant, Arlington, TN) is used to calculate the fraction of the corneal image that is vascularized, the vessel density within the vascularized area, and the vessel density within the total cornea. The inhibition of bFGF-induced corneal angiogenesis, as a function of the dose of MPD disintegrin-Fc polypeptide, is determined.
  • the donor heart tissue is grafted into the left ear pinnae of the recipient on day 0 and the mice are divided into two groups.
  • the control group receives human IgG (Hu IgG) while the other group receives MPDdis-Fc, both intraperitoneally.
  • the treatments are continued for five consecutive days.
  • the functionality of the grafts is determined by monitoring visible pulsatile activity on days 7 and 14 post-engraftment.
  • the inhibition of functional engraftment, as a function of the dose of MPDdis-Fc, is determined.
  • the histology of the transplanted hearts is examined is order to visualize the effects of MPDdis-Fc on edema at the ' site of transplant and host and donor tissue vasculature (using, e.g., Factor VIII staining).
  • MPDdis-Fc are tested in animal models of solid tumors. The effect of the MPDdis-Fc is determined by measuring tumor frequency and tumor growth. The biological activity of MPDdis- Fc is also demonstrated in other in vitro, ex vivo, and in vivo assays known in the art, such as calcium mobilization assays and assays to measure platelet activation, recruitment, or aggregation.
  • Oligonucleotides corresponding to MPD polypeptide coding region can be used to assess MPD mRNA expression using a panel of human tissue and cell line cDNAs ("MTC cDNA," Clontech).
  • the forward primer and reverse primer are designed to amplify a predicted coding region fragment of a desired length. Tissues and cell lines that expressed MPD mRNA, as evidenced by the presence of an amplified DNA fragment of the desired length are identified. Because an MPD polypeptide of the invention is not expressed in every tissue, the invention provides a method of tissue-typing by utilizing antibodies to the polypeptides of the invention or by utilizing oligonucleotide primers or probes specific for polynucleotides of the invention.
  • a forward primer: 5'-CTGCTGCTGTGGCTGGGAGTG-3' (SEQ ED NO:43) and a reverse primer: 5'-GTCATACCCAAATTATGACCAAGCTCAGG-3' (SEQ ED NO:44) the following tissues were found to express an mRNA having a sequence encoding SEQ ED NO: 8: placenta, liver, kidney, pancreas, testis stomach, lyph node, heart, lung, colon adenocarcinoma, fetal liver, fetal lung, fetal spleen, fetal skeletal muscle, fetal thymus, fetal kidney, prostate, thymus, ovary, leukocyte, and esophagus.

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Abstract

L'invention concerne un nouveau polypeptide disintégrine, des procédés de fabrication de ces polypeptides et des procédés d'utilisation de ceux-ci, en vue de traiter des conditions et des troubles associés à la disintégrine et également d'identifier des agents capables de moduler les activités du polypeptide métalloprotéinase-disintégrine.
EP01957300A 2000-07-28 2001-07-27 Polypeptides metalloproteinase-disintegrine et procedes de fabrication et procede d utilisation de ceux-ci Withdrawn EP1307568A2 (fr)

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