Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to an isolated human smooth muscle cell-derived migration factor (SDMF) gene, to essentially pure human SDMF protein, and to compositions and methods of producing and using human SDMF sequences and proteins .
• SDMF smooth muscle cell-derived migration factor
• SMC smooth muscle cells
• rat SDMF is a potent SMC migration factor which does not enhance proliferation of SMC
• the 58 kDa SMC-secreted protein differed biochemically from other known SMC migration factors and was reported to induce migration by an autocrme mechanism
• Sequencing and/or cloning of the rat SDMF was not reported SMC migration is involved in a number of blood vessel pathologies
• SMC autocrme migratory factor SDMF is thought to play an important role in blood vessel pathology e g , the formation of in imal thickening of atherosclerotic lesions.
• the involvement of SDMF in the regulation of SMC migration necessitates the full identification of SDMF and its cDNA.
• a need also exists for compounds which modulate the activity of SDMF for methods to identify such modulators and for reagents useful in such methods.
• one aspect of the present invention is an isolated polynucleotide selected from the group consisting of :
• Another aspect of the invention is a functional polypeptide encoded by the polynucleotides of the invention.
• Another aspect of the invention is a method for preparing essentially pure human SDMF protein comprising culturing a recombmant host cell comprising a vector comprising a polynucleotide of the invention under conditions promoting expression of the protein and recovery thereof .
• Another aspect of the invention is an antisense oligonucleotide comprising a sequence which is capable of binding to the polynucleotide of the invention.
• Another aspect of the invention is a modulator of the polypeptides of the invention.
• Another aspect of the invention is a method for assaying a medium for the presence of a substance that modulat2 es SDMF activity comprising the steps of- (a) providing a SDMF protein having the ammo acid sequence of SDMF (SEQ ID NO: 2 ) or a functional derivative thereof and SMC a chamber;
• Another aspect of the invention is a method for assaying a medium for the presence of a substance that modulates SDMF activity comprising the steps of:
• Another aspect of the invention is SDMF protein modulating compounds identified by the methods of the invention
• Another aspect of the invention is a method for the treatment of a patient having need to modulate SDMF activity comprising administering to the patient a therapeutically effective amount of the modulating compounds of the invention
• Another aspect of the invention is a method for the treatment of a patient having need of SDMF comprising administering to the patient a therapeutically effective amount of the polypeptide of the invention
• Another aspect of the invention is a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier
• Another aspect of the invention is a method of diagnosing conditions associated with SDMF protein deficiency which comprises.
• Another aspect of the invention is a method of treating conditions which are related to insufficient
• SDMF protein function which comprises administering the polynucleotide of claim 1 to a patient deficient in SDMF protein function wherein a SDMF protein is expressed and alleviates the condition
• Yet another aspect of the invention is a transgenic non-human animal capable of expressing in any cell thereof the polynucleotide of the invention
• Figure 1 is an ammo acid sequence alignment of human SDMF protein with murme pl4 protein
• Figure 2 is an ammo acid sequence alignment of human SDMF protein with human PCO CE protein
• Figure 3 is a graph of experimental results demonstrating the biological activity of purified human SDMF
• the term 'SDMF gene' refers to DNA molecules comprising a nucleotide sequence that encodes smooth muscle cell-derived migration factor
• the human SDMF gene sequence is listed in SEQ ID NO .1
• the coding region of the SDMF gene consists of nucleotides 94-1440 of SEQ ID NO.1
• the deduced 449 ammo acid sequence of the SDMF gene product is listed in SEQ ID NO 2
• the term "functional fragments" when used to modify a specific gene or gene product means a less than full length portion of the gene or gene product which retains substantially all of the biological function associated with the full length gene or gene product to which it relates
• fragments are generated by well-known nucleolytic or proteolytic techniques and the fragments tested for the described biological function
• an "antigen' refers to a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen-speciflc response
• immunogen immunogen
• epitope' refers to the site on an antigen or hapten to which a specific antibody molecule binds
• the term is also used herein interchangeably with “antigenic determinant ' or "antigenic determinant site"
• 'monoclonal antibody' is understood to include antibodies derived from one species (e g , murme, rabbit, goat, rat, human, etc ) as well as antibodies derived from two, or perhaps more, species (e.g , chimeric and humanized antibodies)
• a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having ammo acids derived from both coding sequences
• the coding sequences need not be contiguous to one another so long as the expressed sequence is ultimately processed to produce the desired protein f
• 'recombmant' polypeptides refer to polypeptides produced by recombmant DNA techniques, l e , produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide 'Synthetic' polypeptides are those prepared by chemical synthesis.
• a "replicon” is any genetic element (e g , plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vi vo , i.e , capable of replication under its own control
• a "vector” is a replicon, such as a plasmid, phage, or cos id, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
• a "reference” gene refers to the human SDMF gene sequence of the invention and is understood to include the various sequence polymorphisms that exist, wherein nucleotide substitutions in the gene sequence exist, but do not affect the essential function of the gene product .
• a "mutant” gene refers to human
• nucleotide sequence encoding' a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences
• a "promoter sequence' is a DNA regulatory region capable of binding RNA polymerase m a cell and initiating transcription of a downstream (3' direction) coding sequence
• the promoter sequence is bound at the 3 terminus by a translation start codon (e g ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
• a transcription initiation site (conveniently defined by mapping with nuclease ⁇ l) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
• Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
• Prokaryotic promoters contain Shme- Dalgarno sequences addition to the -10 and -35 consensus sequences .
• DNA "control sequences” refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers and the like, which collectively provide for the expression (i.e , the transcription and translation) of a coding sequence a host cell
• a control sequence directs the expression of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
• a "host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.
• a cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane
• Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
• the exogenous DNA may be maintained on an episomal element, such as a plasmid
• a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
• transfection refers to a process by which cells take up foreign DNA and integrate that foreign DNA into their chromosome. Transfection can be accomplished, for example, by various techniques in which cells take up DNA (e.g. , calcium phosphate precipitation, electroporation, assimilation of liposomes, etc.) or by infection, in which viruses are used to transfer DNA into cells.
• a "target cell” is a cell(s) that is selectively transfected over other cell types (or cell lines) .
• a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
• a "cell line” is a clone of a primary cell that is capable of stable growth in vi tro for many generations .
• a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heterologous region encodes a gene, the gene will usually be flanked by DNA that does not flank the gene in the genome of the source animal.
• heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene) . Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
• a "modulator" of a polypeptide is a substance which can affect the polypeptide function.
• An aspect of the present invention is isolated polynucleotides encoding a human SDMF protein and substantially similar sequences. Isolated polynucleotide sequences are substantially similar if
• Moderately stringent conditions is a term understood by the skilled artisan and has been described in, for example, Sambrook et al . Molecular Cl oning : A Labora tory Manual , 2nd edition, Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press (1989) .
• An exemplary hybridization protocol using moderately stringent conditions is as follows.
• Nitrocellulose filters are prehybridized at 65°C in a solution containing 6X SSPE, 5X Denhardt ' s solution (lOg Ficoll, lOg BSA and lOg polyvinylpyrrolidone per liter solution) , 0.05% SDS and 100 ug/ l tRNA.
• Hybridization probes are labeled, preferably radiolabelled (e.g., using the Bios TAG-IT® kit) . Hybridization is then carried out for approximately 18 hours at 65°C. The filters are then washed twice in a solution of 2X SSC and 0.5% SDS at room temperature for 15 minutes.
• the filters are washed at 58°C, air-dried and exposed to X-ray film overnight at 70°C with an intensifying screen.
• Degenerate DNA sequences encode the same ammo acid sequence as SEQ ID NO: 2 or the proteins encoded by that sequence capable of hybridizing under moderately stringent conditions to SEQ ID NO: 1, but have variation (s) the nucleotide coding sequences because of the degeneracy of the genetic code.
• the degenerate codons UUU and UUC both code for the ammo acid phenylalanine, whereas the four codons GGX all code for glycme.
• substantially similar sequences are defined as those sequences m which about 66%, preferably about 75% and most preferably about 90%, of the nucleotides or ammo acids match over a defined length of the molecule.
• substantially similar refers to the sequences having similar identity to the sequences of the instant invention.
• nucleotide sequences that are substantially the same can be identified by hybridization or by sequence comparison
• Protein sequences that are substantially the same can be identified by techniques such as proteolytic digestion, gel electrophoresis and/or microsequencmg.
• Embodiments of the isolated polynucleotides of the invention include DNA, genomic DNA and RNA, preferably of human origin.
• a method for isolating a nucleic acid molecule encoding a SDMF protein is to probe a genomic or cDNA library with a natural or artificially designed probe using art recognized procedures See, e g ,
• SEQ ID NO 1 or fragments thereof comprising at least 15 contiguous nucleotides are particularly useful probes It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe
• Useful reagents include, but are not limited to, radioisotopes fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product
• the probes would enable the ordinarily skilled artisan are to isolate complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding SDMF proteins from human mammalian or other animal sources or to screen such sources for related sequences, e g , additional members of the family type and/or subtype, including transcriptional regulatory and control elements as well as other stability, processing, translation and tissue specificity-determining regions from
• Another aspect of the invention is functional polypeptides encoded by the polynucleotides of the invention.
• An embodiment of a functional polypeptide of the invention is the human SDMF protein having the ammo acid sequence set forth m SEQ ID NO: 2.
• Another aspect of the invention is a method for preparing essentially pure human SDMF protein
• Yet another aspect is the human SDMF protein produced by the preparation method of the invention
• This protein has the ammo acid sequences listed in SEQ ID NO.2 and include variants with a substantially similar ammo acid sequence that have the same function.
• the proteins of this invention are preferably made by recombmant genetic engineering techniques by cultur g a recombmant host cell containing a vector encoding the polynucleotides of the invention under conditions promoting the expression of the protein and recovery thereof
• the isolated polynucleotides can be introduced into expression vectors by operatively linking the DNA to the necessary expression control regions, e g , regulatory regions, required for gene expression
• the vectors can be introduced into an appropriate host cell such as a prokaryotic, e g , bacterial, or eukaryotic, e g , yeast or mammalian cell by methods well known in the art See Ausubel et al , supra
• the coding sequences for the desired proteins, having been prepared or isolated, can be cloned into any suitable vector or replicon Numerous cloning vectors are known to those of skill in the art and the selection of an appropriate cloning vector is a matter of choice
• recombmant DNA vectors for cloning and host cells which they can transform include but are not limited to, the bacteriophage ⁇ ( E col l ) , pBR322 ( E
• the gene can be placed under the control of control elements such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired protein is transcribed into RNA m the host cell transformed by a vector containing the expression construct
• control elements such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired protein is transcribed into RNA m the host cell transformed by a vector containing the expression construct
• the coding sequence may or may not contain a signal peptide or leader sequence
• the proteins of the present invention can be expressed using, for example, the E col i tac promoter or the protein A gene ⁇ spa ) promoter and signal sequence Leader sequences can be removed by the bacterial host m post-translational processing See, e g , U S Patent Nos 4,431,739, 4,425,437 and 4,338,397
• regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell Regulatory sequences are known to those of skill the art Exemplary are those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus including the presence of a regulatory compound
• Other types of regulatory elements may also be present m the vector, for example enhancer sequences
• An expression vector is constructed so that the particular coding sequence is located m the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
• Modification of the sequences encoding the particular antigen of interest may be desirable to achieve this end. For example, m some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
• control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
• a vector such as the cloning vectors described above.
• the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
• Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence.
• Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., T. Maniatis et al . , supra ; "DNA Cloning, "
• the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed.
• Preferred mammalian cells include human embryonic kidney cells (293) , monkey kidney cells, fibroblast (COS) cells, Chinese hamster ovary (CHO) cells, Drosophi la or munne L-cells.
• the protein can be purified directly from the media If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction
• Another aspect of this invention is the operative linking of the polynucleotides of the invention to regulatory elements which are differentially responsive to various temperature or metabolic conditions thereby effectively turning on or off the phenotypic expression m response to those conditions
• proteins of the present invention are by constructing gene libraries, using the resulting clones to transform E col i and pooling and screening individual colonies using polyclonal serum or monoclonal antibodies to human SDMF
• the proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis on an automated peptide synthesizer, using known ammo acid sequences or ammo acid sequences derived from the DNA sequence of the genes of interest Such methods are known to those skilled m the art
• modulators of the polypeptides of the invention Functional modulation of SDMF by a substance includes partial to complete inhibition of function, identical function, as well as enhancement of function
• modulators of the invention include antibodies, peptides, oligonucleotides and small organic molecules including peptidomimetics
• the proteins of the present invention or their fragments comprising at least one epitope can be used to produce antibodies, both polyclonal and monoclonal, directed to epitopes corresponding to ai ⁇ no acid
• polyclonal antibodies are desired, a selected mammal such as a mouse, rabbit, goat or horse is immunized with a protein of the present invention, or its fragment, or a mutant protein. Serum from the immunized animal is collected and treated according to known procedures. Serum polyclonal antibodies can be purified by immunoaffmity chromatography or other known procedures .
• Monoclonal antibodies to the proteins of the present invention, and to the fragments thereof, can also be readily produced by one skilled m the art.
• the general methodology for making monoclonal antibodies by using hybridoma technology is well known.
• Immortal antibody-producing cell lines can be created by cell fusion and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al . , “Hybridoma Techniques” (1980) ; Hammerling et al . , “Monoclonal Antibodies and T-cell Hybridomas” (1981) ; Kennett et al . , "Monoclonal
• Antibodies (1980) ; and U.S. Patent Nos . 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500; 4,491,632; and 4, 493 , 890.
• Panels of monoclonal antibodies produced against the antigen of interest, or fragment thereof, can be screened for various properties, i.e., for isotype, epitope, affinity, etc.
• Monoclonal antibodies are useful purification, using immunoaffmity techniques, of the individual antigens which they are directed against.
• genes encoding the monoclonals of interest may be isolated from the hybridomas by PCR techniques known in the art and cloned and expressed in the appropriate vectors.
• the antibodies of this invention whether polyclonal or monoclonal have additional utility in that they may be employed as reagents in im unoassays, RIA, ELISA, and the like.
• the antibodies of the invention can be labeled with an immunoaffmity techniques
• X analytically detectable reagent such as a radioisotope, fluorescent molecule or enzyme.
• An additional use of monoclonal antibodies is to treat various pathologies arising from overproduction or inappropriate production of SDMF. Such pathologies may include restenosis and atherosclerosis.
• a therapeutically effective amount of an SDMF-modulating monoclonal antibody is administered to a patient having a need to modulate SDMF activity.
• Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions may also be used in assays or therapeutically.
• a therapeutic monoclonal antibody would be "humanized" as described in Jones et al.
• antisense oligonucleotides comprising a sequence which is capable of binding to the polynucleotides of the invention.
• Synthetic oligonucleotides or related antisense chemical structural analogs can be designed to recognize and specifically bind to and prevent transcription of a target nucleic acid encoding SDMF protein by those of ordinary skill in the art. See generally, Cohen, J.S. , Trends in Pharm. Sci., 10, 435(1989) and Weintraub,
• Another aspect of the invention is a method for assaying a medium for the presence of a substance that inhibits or otherwise modulates SDMF protein function by interfering with the binding of SDMF protein to binding partners.
• a substance that inhibits or otherwise modulates SDMF protein function by interfering with the binding of SDMF protein to binding partners. Examples include, but are not limited to, a cell surface receptor, soluble receptor, binding protein, antibodv and any fragments thereof as well as
• SDMF protein having the ammo acid sequence of human SDMF (SEQ ID NO: 2) or a functional derivative thereof and SMC are provided in a chemotaxis assay chamber as described in Example 3, infra , or m any other migration assay known to those skilled the art such as that described by Koyama et al . in J. Bi ol .
• test substance which is suspected of modulating SDMF activity is added to either the SDMF or SMC, the chamber is incubated under conditions which permit the migration of SMC and the number of migrated and nonmigrated SMC counted The result is compared to a control to determine the effect of the test substance.
• a SDMF protein having the ammo acid sequence of human SDMF (SEQ ID NO: 2 ) or a functional derivative thereof together with a binding partner
• the mixture is incubated with a test substance which is suspected of modulating SDMF activity, under conditions which permit the formation of a SDMF gene product/binding partner complex.
• An assay is performed for the presence of the complex, free SDMF protein or free binding partner and the result compared to a control to determine the effect of the test substance.
• Modulation of SDMF function would be expected to have effects on SMC migration. Any modulators so identified would be expected to be useful as a therapeutic for the treatment and prevention of atherosclerosis, restenosis or other blood vessel pathologies where SMC migration is involved
• SMC migration factors interact with specific receptor proteins SDMF could be used to isolate proteins which interact with it and this interaction could be a target for interference Inhibitors of protein-protein interactions between SDMF and other factors could lead to the development of pharmaceutical agents for the modulation of SDMF activity.
• yeast two-hybrid system provides methods for detecting the interaction between a first test protein and a second test protein, m vi vo , using reconstitution of the activity of a transcriptional activator. The method is disclosed in, e.g. , U.S. Patent No. 5,283,173; reagents are available from Clontech and Stratagene. Briefly, SDMF cDNA is fused to a Gal 4 transcription factor DNA binding domain and expressed in yeast cells.
• cDNA library members obtained from cells of interest are fused to a transactivation domain of Gal 4 cDNA clones which express proteins which can interact with SDMF will lead to reconstitution of Gal 4 activity and transactivation of expression of a reporter gene such as Gal l -l acZ .
• An alternative method is screening of ⁇ gtll, ⁇ ZAP (Stratagene) or equivalent cDNA expression libraries with recombmant SDMF.
• Recombmant SDMF protein or fragments thereof are fused to small peptide tags such as FLAG, HSV or GST.
• the peptide tags can possess convenient phosphorylation sites for a k ase such as heart muscle creatine k ase or they can be biot ylated.
• Recombmant SDMF can be phosphorylated with 2 [p] or use ⁇ 5 unlabeled and detected with streptavidin or antibodies against the tags ⁇ gtllcDNA expression libraries are made from cells of interest and are incubated with the recombmant SDMF, washed and cDNA clones isolated which interact with SDMF. See, e.g , T Maniatis et a 1 , supra
• Another method is the screening of a mammalian expression library in which the cDNAs are cloned into a vector between a mammalian promoter and polyadenylation site and transiently transfected in COS or 293 cells followed by detection of the binding protein 48 hours later by incubation of fixed and washed cells with a labelled SDMF, prefereably iodinated, and detection of bound SDMF by autoradiography (See Sims et al . , Sci ence 241 , 585-589 (1988) and McMahan et al . , EMBO J. 10, 2821-2832 (1991)) .
• pools of cDNAs containing the cDNA encoding the binding protein of interest can be selected and the cDNA of interest can be isolated by further subdivision of each pool followed by cycles of transient transfection, binding and autoradiography.
• the cDNA of interest can be isolated by transfecting the entire cDNA library into mammalian cells and panning the cells on a dish containing SDMF bound to the plate. Cells which attach after washing are lysed and the plasmid DNA isolated, amplified in bacteria, and the cycle of transfection and panning repeated until a single cDNA clone is obtained (See Seed et al , Proc . Na tl . Acad . Sci .
• binding protein If the binding protein is secreted, its cDNA can be obtained by a similar pooling strategy once a binding or neutralizing assay has been established for assaying supernatants from transiently transfected cells. General methods for screening supernatants are disclosed in Wong et al . , Science 228, 810-815 (1985) .
• Another alternative method is isolation of proteins interacting with SDMF directly from cells. Fusion proteins of SDMF with GST or small peptide tags are made and immobilized on beads.
• Biosynthetically labeled or unlabeled protein extracts from the cells of interest are prepared, incubated with the beads and washed with buffer. Proteins interacting with SDMF are eluted specifically from the beads and analyzed by ⁇ DS-PAGE.
• Binding partner primary amino acid sequence data are obtained by microsequencing.
• Another alternative method is immunoaffinity purification.
• Recombinant SDMF is incubated with labeled or unlabeled cell extracts and immunoprecipitated with anti-SDMF antibodies.
• the immunoprecipitate is recovered with protein A-Sepharose and analyzed by SDS-PAGE. Unlabelled proteins are labeled by biotinylation and detected on SDS gels with streptavidin. Binding partner proteins are analyzed by microsequencing. Further, standard biochemical purification steps known to those skilled m the art may be used prior to microsequencing.
• Yet another alternative method is screening of peptide libraries for binding partners.
• Recombinant tagged or labeled SDMF is used to select peptides from a peptide library which interact with SDMF. Sequencing of the peptides leads to identification of consensus peptide sequences which might be found in interacting proteins .
• SDMF binding partners identified by any of these methods or other methods which would be known to those of ordinary skill in the art as well as those putative binding partners discussed above can be used in the assay method of the invention.
• Assaying for the presence of SDMF/binding partner complex are accomplished by, for example, the yeast two-hybrid system, ELISA or immunoassays using antibodies specific for the complex. In the presence of test substances which interrupt or inhibit formation of SDMF/binding partner interaction, a decreased amount of complex will be determined relative to a control lacking the test substance .
• Assays for free SDMF or binding partner are accomplished by, for example, ELISA or immunoassay us q specific antibodies or by incubation of radiolabeled SDMF with cells or cell membranes followed by centrifugation or filter separation steps. In the presence of test substances which interrupt or inhibit formation of SDMF/binding partner interaction, an increased amount of free SDMF or free binding partner will be determined relative to a control lacking the test substance.
• compositions comprising an effective amount of SDMF protein of the invention and a pharmaceutically acceptable carrier.
• Pharmaceutical compositions of proteinaceous drugs of this invention are particularly useful for parenteral administration, i.e. , subcutaneously, intramuscularly or intravenously.
• the SDMF protein is surrounded by a membrane bound vesicle, such as a liposome.
• the compositions for parenteral administration will commonly comprise a solution of the proteins of the invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
• aqueous carriers may be employed, e.g. , water, buffered water, 0.4% saline, 0.3% glycine and the like.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
• concentration of the protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight, and will be selected primarily based on fluid volumes, viscosities, etc. according to the particular mode of administration selected.
• a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water and 50 mg of a protein of the invention.
• a pharmaceutical composition of the invention for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and 150 g of a protein of the invention.
• Actual methods for preparing parenterally admmistrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, e.g., Remington ' s Pharmaceu ti cal Science , 15th ed. , Mack Publishing Company, Easton, Pennsylvania.
• the proteins described herein can be lyophilized for storage and reconstituted a suitable carrier prior to use. This technique has been shown to be effective with conventional proteins and art-known lyophilization and reconstitution techniques can be employed.
• the physician will determine the dosage of the present therapeutic agents which will be most suitable and it will vary with the form of administration and the particular compound chosen, and furthermore, it will vary with the particular patient under treatment. Generally, the physician will wish to initiate treatment with small dosages substantially less than the optimum dose of the compound and increase the dosage by small increments until the optimum effect under the circumstances is reached. It will generally be found that when the composition is administered orally, larger quantities of the active agent will be required to produce the same effect as a smaller quantity given parenterally.
• the therapeutic dosage will generally be from 1 to 10 milligrams per day and higher although it may be administered m several different dosage units.
• the pharmaceutical composition of the invention can be administered for prophylactic and/or therapeutic treatments.
• compositions are administered to a patient already suffering from a disease m an amount sufficient to cure or at least partially arrest the disease and its complications.
• compositions containing the present compounds or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance to the disease.
• the pharmaceutical composition of the invention should provide a quantity of the compounds of the invention sufficient to effectively treat the patient. Additionally, some diseases result from inherited defective genes. These genes can be detected by comparing the sequence of the defective gene with that of a normal one. Individuals carrying mutations the SDMF gene may be detected at the DNA level by a variety of techniques Nucleic acids used for diagnosis (genomic DNA, mRNA, etc.) may be obtained from a patient's cells, such as from blood, urine, saliva or tissue biopsy, e.g., chorionic villi sampling or removal of amniotic fluid cells and autopsy material.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR) , strand displacement amplification (SDA) , etc. prior to analysis.
• LCR ligase chain reaction
• SDA strand displacement amplification
• RNA or cDNA may also be used for the same purpose.
• PCR primers complementary to the nucleic acid of the instant invention can be used to identify and analyze SDMF mutations For example, deletions and insertions can be detected by a change m size of the amplified product m comparison to the normal SDMF genotype.
• Point mutations can be identified by hybridizing amplified DNA to rabiolabeled SDMF RNA of the invention or al ernatively, radiolabelled SDMF antisense DNA sequences of the invention Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A
• Tm melting temperatures
• point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by yet other well-known techniques, e.g., direct DNA sequencing, single-strand conformational polymorphism. See Orita et al . , Genomics , 5 , 874-879 (1989) .
• a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
• the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent- tags .
• Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
• the presence of nucleotide repeats may correlate to a causative change in SDMF activity or serve as marker for various polymorphisms.
• DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis . DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures. See, e.g. , Myers et al . , Science, 230 , 1242 (1985) .
• sequence alterations may be detected as changes in the migration pattern of DNA heteroduplexes in non- denaturing gel electrophoresis such as heteroduplex electrophoresis. See, e.g. , Nagamine et al . , Am . J. Hum . Gene t . , 45 , 337-339 (1989) . Sequence changes at specific locations may also be revealed by nuclease
• X protection assays such as RNase and SI protection or the chemical cleavage method as disclosed by Cotton et al in Proc Na tl Acad Sci . USA, 85 , 4397-4401 (1985)
• the detection of a specific DNA sequence may be achieved by methods such as hybridization (e g , heteroduplex electroporation, see, White et al , Genomics, 12, 301-306 (1992) , RNAse protection (e g , Myers et ai . , Sci ence, 230, 1242 (1985) ) chemical cleavage (e g., Cotton et al .
• restriction enzymes e.g., restriction fragment length polymorphisms (RFLP) m which variations in the number and size of restriction fragments can indicate insertions, deletions, presence of nucleotide repeats and any other mutation which creates or destroys an endonuclease restriction sequence Southen blotting of genomic DNA may also be used to identify large, I e , greater than 100 base pair deletions and insertions
• mutations such as microdeletions , aneuploidies, translocations and inversions, can also be detected by in si tu analysis. See, e g., Keller et al , DNA Probes, 2nd Ed., Stockton Press, New York, N Y , USA (1993) That is, DNA or RNA sequences in cells can be analyzed for mutations without isolation and/or immobilization onto a membrane.
• Fluorescence in si tu hybridization is presently the most commonly applied method and numerous reviews of FISH have appeared See, e g , Trachuck et al , Sci ence, 250 , 559-562 (1990), and Trask et al , Trends , Gene t , 7,
• some diseases are a result of, or are characterized by, changes m gene expression which can be detected by changes the mRNA
• the SDMF gene can be used as a reference to identify individuals expressing an increased or decreased level of SDMF protein, e.g. , by Northern blotting or m si t u hybridization.
• probes can vary widely but it is preferred that the probe be at least 15 nucleotides in length. It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
• useful reagents include but are not limited to radioisotopes , fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. As a general rule, the more stringent the hybridization conditions, the more closely related genes will be that are recovered.
• Gene therapy means gene supplementation where an additional reference copy of a gene of interest is inserted into a patient's cells.
• the reference copy would be a wild-type form of the SDMF gene or a gene encoding a protein or peptide which modulates the activity of the endogenous SDMF.
• Gene therapy of the present invention can occur m vi vo or ex vivo .
• MMLV mouse Moloney leukemia virus
• the therapeutic gene is typically "packaged" for administration to a patient such as in liposomes or in a replication-deficient virus such as adenovirus as described by Berkner, K.L. , in Curr . Top . Microbiol . Immunol . , 158 , 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N. , in Curr . Top . Mi crobi ol . Immunol . , 158 , 97-129 (1992) and U.S. Patent No. 5,252,479.
• Another approach is administration of "naked DNA” in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
• Another approach is administration of "naked DNA” in which the therapeutic gene is introduced into the target tissue by microparticle bombardment using gold particles coated with the DNA.
• Cell types useful for gene therapy of the present invention include lymphocytes, hepatocytes, myoblasts, fibroblasts, any cell of the eye such as retinal cells, epithelial and endothelial cells.
• Transfection of pulmonary epithelial cells can occur via inhalation of a neubulized preparation of DNA vectors in liposomes, DNA- protein complexes or replication-deficient adenoviruses . See, e.g., U.S. Patent No. 5,240,846.
• Transgenic, non-human mammals capable of expressing the polynucleotides of the invention in any cell.
• Transgenic, non-human animals may be obtained by transfecting appropriate fertilized eggs or embryos of a host with the polynucleotides of the invention or with mutant forms found in human diseases. See, e.g., U.S. Patent Nos . 4,736,866; 5,175,385; 5,175,384 and 5,175,386.
• the resultant transgenic animal may be used as a model for the study of SDMF gene function.
• Particularly useful transgenic animals are those which display a detectable phenotype associated with the expression of the SDMF
• /7 protein Drug development candidates may then be screened for their ability to reverse or exacerbate the relevant phenotype.
• Rat SDMF protein was purified by the method of Koyama et al . in J. Biol Chem , supra The purified 60 kDa protein was subjected to automated Edman degradation sequencing and found to contain a blocked N-termmus
• a search of a random cDNA sequence database consisting of short partial sequences known as expressed sequence tags (ESTs) with the murine pl4 sequence disclosed many ESTs which encoded parts of the putative human homologue of pl4 or SDMF
• ESTs expressed sequence tags
• a cDNA containing an EST which matched the 5 end of the murine pl4 cDNA sequence and which contained a start codon was selected and further sequenced (SEQ ID NO: 1) . This cDNA was
• Sequence analysis of the SDMF cDNA revealed a 1,347 nucleotide open reading frame (SEQ ID NO: 1) encoding a 449 amino acid protein with a predicted molecular mass of 49.4 kDa (SEQ ID NO: 2) , starting with an ATG at position 94 and terminating with a TGA at position 1441.
• the murine pi4 DNA sequence (SEQ ID NO: 4) reported by Lecain et al . was determined by Takahara et al . , supra , to omit two guanosine residues. One additional residue was located between nucleotides 1213 and 1214 and the other between nucleotides 1375 and 1376 of the sequence published by Lecain et al .
• the corrected murine pl4 DNA and amino acid sequences are shown in SEQ ID NOs : 6 and 7, respectively.
• a search of the GenEMBL database with the murine pl4 amino acid sequence disclosed a 449 amino acid human procollagen C-proteinase enhancer protein (PCOLCE), accession number L33799 (SEQ ID NOs : 8 and 9) , having 90.1% identity.
• PCOLCE was reported in Takahara et al . , supra .
• Alignment of the deduced amino acid sequence of human SDMF (SEQ ID NO: 2) with the human PCOLCE sequence (SEQ ID NO: 9) was accomplished using the GCG program Bestfit. The overall amino acid identity was 99% with zero gaps and is shown in Fig. 2 (top, human SDMF; bottom, human PCOLCE) .
• the putative human SDMF cDNA was isolated from its cloning vector (Bluescript) by digestion with EcoRl (5 1 end) and Kpnl (3' end) and inserted into the mammalian expression vector pCDN (Aiyar et al . , Mol ecul ar and Cel l ular Biochemis try 131 , 75-86 (1994)) . 60ug of this vector DNA was transfected into 8x10 * COS cells plated the previous day into a 150mm flask using the DEAE dextran/chloroquine method of Maniatis et al , supra, followed by a 10% DMSO shock (eg Maniatis et al) .
• RASMC Rat aortic smooth muscle cells
• DMEM Dulbecco ' s modified Eagle's medium
• BSA bovine serum albumin
• the lower compartment of the chamber contained 0.6 L of DMEM supplemented with 0.2% BSA.
• Human SDMF which was purified to near homogeneity from COS cell culture supernatant was either coated on the lower surface of the filter or added to the lower compartment.
• RASMC were subjected to either platelet-derived growth factor (PDGF) at a concentration (1 nM) which induces maximum smooth muscle cell migration (control) or various concentrations of purified SDMF.
• PDGF platelet-derived growth factor
• Incubation was at 37" C in an atmosphere of 95% air and 5% C0 2 for 20 hours. After incubation, nonmigrated cells on the upper surface were scraped gently and washed with PBS three times. The filters were fixed and stained with Giemsa stain.
• the number of SMC that had migrated to the lower surface of the filters was determined microscopically and one randomly chosen high-power field (HPF) was counted per filter. Experiments were performed in triplicate. The results in Figure 1 show that purified SDMF enhanced the migration of rat smooth muscle cells dose dependently and its maximum activity was at least 4-5 times that of PDGF which is consistent with published observations by Koyama et al . , J . Bi ol . Chem . , supra .
• ATCATCGCGC CCCCGGACCA GGTCATCGCG CTGACCTTCG AGAAGTTTGA CCTGGAGCCG 720
• Trp Thr lie Thr Val Pro Glu Gly Gin Thr Val Ser Leu Ser Phe Arg 65 70 75 80
• Pro Pro Asp Gin Val lie Ala Leu Thr Phe Glu Lys Phe Asp Leu Glu 195 200 205
• CAAA 1 504 (2) INFORMATION FOR SEQ ID NO : 5 :
• CAACCAGGAC CAGATCCTCA ATAACCTAAG CAAGAGGAAG TGTCCCTCCC AACCTAGGAC 1440
• CTCTGCAAAA TTCAGCTGCT GCCTCTGTCT TGAGGACCCC AGCGCCTTTC CCCCGGGGCC 60
• CCTTTTGCCC AGGGCCAGAC CCCCAACTAC ACCAGACCCG TGTTCCTGTG CGGAGGGGAT 180

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