EP0996628A2 - Proteine de mammifere de type facteur de croissance nerveuse - Google Patents

Proteine de mammifere de type facteur de croissance nerveuse

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Publication number
EP0996628A2
EP0996628A2 EP98930397A EP98930397A EP0996628A2 EP 0996628 A2 EP0996628 A2 EP 0996628A2 EP 98930397 A EP98930397 A EP 98930397A EP 98930397 A EP98930397 A EP 98930397A EP 0996628 A2 EP0996628 A2 EP 0996628A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
leu
arg
gly
ala
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.)
Ceased
Application number
EP98930397A
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German (de)
English (en)
Inventor
Paul O. Sheppard
Laura J. Jelinek
Theodore E. Whitmore
Hal Blumberg
Joyce M. Lehner
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Zymogenetics Inc
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Zymogenetics Inc
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Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP0996628A2 publication Critical patent/EP0996628A2/fr
Ceased legal-status Critical Current

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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
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e . g. estrogen, testosterone) , parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF) , epidermal growth factor (EGF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , erythropoietin (EPO) and calcitonin.
  • Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.
  • the present invention addresses this need by providing a novel neuro-growth factor like polypeptide called Zneul and related compositions and methods.
  • the present invention provides an isolated polynucleotide encoding a mammalian polypeptide termed
  • the mature human Zneul polypeptide is comprised of a sequence of amino acids approximately 254 amino acids long.
  • Amino acid residue 20 of SEQ ID NO: 2, a threonine is the initial amino acid of the mature polypeptide.
  • amino residues 1-19 comprise a signal sequence
  • the mature Zneul polypeptide is represented by the amino acid sequence comprised of residues 20-254.
  • the mature Zneul polypeptide is further represented by SEQ ID NO: 3.
  • Mouse Zneul is defined by SEQ ID NOs:18 and 19. Having a signal sequence of amino acid residues 1-23, and the mature mouse Zneul is from 24-278 represented by SEQ ID NO: 24.
  • the polypeptide further comprises an affinity tag.
  • the polynucleotide is DNA.
  • an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zneul polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.
  • a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a protein polypeptide encoded by the DNA segment.
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond.
  • the first portion of the chimeric polypeptide consists essentially of (a) a Zneul polypeptide as shown in SEQ ID NO: 2 (b) allelic variants of SEQ ID NO: 2; and (c) protein polypeptides that are at least 90% identical to (a) or (b) .
  • the second portion of the chimeric polypeptide consists essentially of another polypeptide such as an affinity tag.
  • the affinity tag is an immunoglobulin F c polypeptide.
  • the invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
  • an antibody that specifically binds to a Zneul polypeptide as disclosed above, and also an anti- idiotypic antibody which neutralizes the antibody to a Zneul polypeptide.
  • the present invention is also directed domains of the polypeptide including SEQ ID N0s:8, 9, 10, 11, 12, 13, 14, 15, and 16.
  • An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention.
  • Specific examples of said polypeptides are defined by the amino acid sequences of SEQ ID NOs : 20-23. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
  • Another embodiment of the present invention relates to a method for producing an antibody which binds to a peptide or polypeptide defined by SEQ ID NOs: 2-3,8, 9, 11-16, and 19-24 or to a peptide or polypeptide which is at least 90% identical to said peptide or polypeptide comprising inoculating an animal with said peptide or polypeptide or with a nucleic acid which encodes said peptide or polypeptide, wherein said animal produces antibodies to said peptide or polypeptide; and isolating said antibody.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • “Operably linked” when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e . g. transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • polynucleotide is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
  • promoter is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "soluble protein” is a protein polypeptide that is not bound to a cell membrane.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID N0:1, or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is about 0.02 M or less at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for isolating DNA and RNA are well known in the art.
  • Total RNA can be prepared using guanidine HCI extraction followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al . , Biochemistry 28:52-94 (1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc . Na tl . Acad . Sci . USA 63:1408-1412 (1972).
  • Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods.
  • Polynucleotides encoding Zneul polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • the polynucleotides of the present invention can be synthesized using DNA synthesizer.
  • DNA synthesizer Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • sequences disclosed in SEQ ID NOS : 1 , 2 and 3 represent a single allele of the human. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures .
  • the present invention further provides counterpart proteins and polynucleotides from other species ("species orthologs").
  • species orthologs are Zneul polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primates .
  • Species orthologs of the human Zneul protein can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a protein-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human or mouse cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the protein. Similar techniques can also be applied to the isolation of genomic clones.
  • an isolated polynucleotide which encodes a polypeptide includes all allelic variants and species orthologs of the polypeptide of SEQ ID NO : 2.
  • the present invention also provides isolated protein polypeptides that are substantially homologous to the polypeptide of SEQ ID NO: 3 and its species orthologs.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • substantially homologous is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO: 2, or its species orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 3 , or its species orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull . Math . Bio . 48 : 603-616 (1986) and
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, protein A, Nilsson et al . , EMBO J.
  • an affinity tag such as a poly-histidine tract, protein A, Nilsson et al . , EMBO J.
  • Acidic glutamic acid aspartic acid
  • Polar glutamine asparagine
  • Hydrophobic leucine isoleucine
  • continued valine Aromatic: phenylalanine tryptophan tyrosine
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244 , 1081-1085, (1989); Bass et al . , Proc . Natl . Acad . Sci . USA 88:4498-4502, (1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244 , 1081-1085, (1989); Bass et al . , Proc . Natl . Acad . Sci . USA 88:4498-4502, (1991).
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-protein interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al . , Science 255:306-312, (1992); Smith et al . , J. Mol . Biol . 224:899-904, (1992); Wlodaver et al . , FEBS Lett . 303:59-64, (1992). The identities of essential amino acids can also be inferred from analysis of homologies with related proteins .
  • Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized proteins in host cells.
  • Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
  • Mutagenized DNA molecules that encode active proteins or portions thereof e.g., ligand-binding fragments
  • polypeptides that are substantially homologous to SEQ ID NO: 3 or allelic variants thereof and retain the properties of the wild-type protein.
  • a polypeptide as defined by SEQ ID NO: 2 includes all allelic variants and species orthologs of the polypeptide .
  • the protein polypeptides of the present invention including full-length proteins, protein fragments (e.g. ligand-binding fragments), and fusion polypeptides can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • a DNA sequence encoding a Zneul polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of the this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • a region of a protein to which an antibody can bind is defined as an "antigenic epitope” . See for instance, Geysen, H.M. et al., Proc . Natl . Acad Sci . USA 81:3998-4002 (1984) .
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein.
  • the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided) ; and sequences containing proline residues are particularly preferred.
  • All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides defined by SEQ ID NOs : 20-24.
  • Polynucleotides generally a cDNA sequence, of the present invention encode the above-described polypeptides.
  • a cDNA sequence which encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows.
  • Alanine (Ala) is encoded by GCA, GCC, GCG or
  • Cysteine (Cys) is encoded by TGC or TGT;
  • Aspartic acid is encoded by GAC or GAT
  • Glutamic acid (Glu) is encoded by GAA or GAG Phenylalanine (Phe) is encoded by TTC or TTT
  • Glycine is encoded by GGA, GGC, GGG or GGT;
  • Histidine is encoded by CAC or CAT;
  • Isoleucine (lie) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;
  • Methionine (Met) is encoded by ATG;
  • Asparagine is encoded by AAC or AAT; Proline (Pro) is encoded by CCA, CCC, CCG or
  • Glutamine (Gin) is encoded by CAA or CAG;
  • Arginine is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is encoded by AGC, AGT, TCA, TCC,
  • Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
  • Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and
  • Tyrosine (Tyr) is encoded by TAC or TAT.
  • mRNA messenger RNA
  • T thymine
  • U uracil nucleotide
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the protein, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
  • the secretory signal sequence is joined to the Zneul DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830).
  • Cultured mammalian cells are preferred hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al . , Cell 14:725, (1978); Corsaro and Pearson, Soma tic Cell Genetics 7:603, (1981): Graham and Van der Eb, Virology 52:456, (1973), electroporation, Neumann et al . , EMBO J. 1:841- 845, (1982), DEAE-dextran mediated transfection, Ausubel et al . , eds .
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293, ATCC No. CRL 1573; Graham et al . , J. Gen . Virol . 36 : 59 - 12 , (1977) and Chinese hamster ovary (e.g. CH0-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • promoters from SV-40 or cytomegalovirus are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288.
  • suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4 , 601, 978 , and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like.
  • Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • hygromycin resistance e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222; Bang et al . , U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . (Bangalore) 11:47-58, (1987).
  • Fungal cells including yeast cells, and particularly cells of the genus Sa ccharomyces , can also be used within the present invention, such as for producing protein fragments or polypeptide fusions.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in yeast is the P0T1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • a novel protein is produced by a cultured cell, and the cell is used to screen for a receptor or receptors for the protein, including the natural receptor, as well as agonists and antagonists of the natural ligand.
  • Expressed recombinant polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affini ty Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, (1988) .
  • the polypeptides of the present invention can be isolated by exploitation of their properties .
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins. Briefly, a gel is first charged with divalent metal ions to form a chelate, E. Sulkowski, Trends in Biochem . 3 : 1 - 1 , (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • the Zneul polypeptide shown in SEQ ID NO: 2 has a signal peptide including amino acid residues 1-19.
  • Amino acid residues 20-104 define a hydrophilic domain homologous to an HSMHC3W5A domain, SEQ ID NO: 17, (GenBank No. gl401159) .
  • Amino acid residues 105-135 define a domain homologous to an Epidermal Growth Factor (EGF) domain.
  • EGF Epidermal Growth Factor
  • Amino acid residues 136-177 define another domain homologous to an EGF domain; and amino acid residues 178- 273 define a domain also homologous to an HSMHC3W5A domain .
  • EGF1 of Zneul SEQ ID NO : 9 which corresponds to amino acid residues 105 to 135 of SEQ ID NO: 2
  • EGF1 in Zneul is about 56% similar to the HSMHC3W5A_6 domain, its closest human relative.
  • EGF2 of Zneul SEQ ID NO: 10 which corresponds to amino acid residues 136 to 177 of SEQ ID NO: 2
  • EGF2 of Zneul is about 48% similar to PIR_S31101 fibrillin, its closest human relative.
  • HSM1 HSMHC3W5A-like (HSM1) domain of Zneul, SEQ ID NO: 8 which corresponds to amino acid residues 20- 104 of SEQ ID NO: 2.
  • SEQ ID NO: 8 is approximately 38% similar to HSMHC3W5A, its closest human relative.
  • HSM2 HSMHC3W5A-like domain
  • SEQ ID NO: 11 which corresponds to amino acid residues 178-273 of SEQ ID NO: 2
  • HSM2A-like domain SEQ ID NO: 11 which corresponds to amino acid residues 178-273 of SEQ ID NO: 2
  • tissue specificity of Zneul expression indicates that Zneul can be used as a growth, maintenance, or differentiation factor in the spinal cord, heart, spleen, testis, thyroid and lymph nodes.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the Zneul gene has been mapped on chromosome 9q34.3.
  • a Zneul nucleic acid probe could be used to check for abnormalities in chromosome 9. In a normal chromosome 9, one would predict that a Zneul nucleic acid probe would hybridize to chromosome 9. If the probe does not hybridize to chromosome 9, this would indicate an abnormality in chromosome 9.
  • Zneul ' s closest human homolog is HSMHC3W5A a gene in the HLA class III region, which is contained in a cosmid which contains Notch 4.
  • Zneul is also homologous to Notch 4 in its EGF-like domains. Zneul may be involved in EGF-receptor pathways.
  • Notch The original member of this gene family was the Drosophila gene Notch which controls cell fate decisions in the development of the peripheral nervous system.
  • Notch is a cell surface receptor with a single transmembrane domain.
  • All members of the Notch family have large numbers of EGF-like motifs (29-39 in mouse, 10-13 in C. elegans) and three or more copies of LNR (linl2/ Notch repeats) in the extracellular domain.
  • Notch family members also contain six copies of the cdclO/SWI6 motif (also called ankyrin repeats) and a PEST protein degradation sequence in the intracellular domain.
  • EGF repeats are involved in ligand binding.
  • LNR may be regulatory domains which bind ligand when high ligand concentrations exist and cause decreased activity of Notch.
  • Cdcl ⁇ /SWI6 domains are involved in protein- protein interactions with components of the Notch- activated signal transduction pathway.
  • TAN-1 oncogene is a fusion of part of the ⁇ T cell receptor with a small region of the human Notch 1 extracellular domain and the entire intracellular domain.
  • TAN-1 is an activated form of Notch which causes T- lymphoblastic leukemias .
  • the int-3 oncogene is caused by ⁇ integration of the mouse mammary tumor virus into the Notch 4 gene resulting in expression of the intact intracellular domain. Int-3 also is an activated form of Notch which leads to mammary carcinoma.
  • Notch family members The function of Notch family members has been extensively studied in Drosophila and C. elegans . These proteins control binary decisions that depend on cell-cell interactions. Notch proteins act consistent with their proposed role as a receptor. Gain-of-function and loss- of-function Notch alleles result in opposite cell fate decisions. Notch receptors and their ligands play important roles in lateral inhibition, the process whereby signaling between neighboring cells is amplified by a feedback loop between Notch and its ligand. This process results in increased receptor activity in some cells and increased ligand activity in others leading to the distinction between signaling cells and receiving cells.
  • CADASIL cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
  • chromosome 19 Mutations in a gene region called CADASIL (for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) on chromosome 19 are associated with a type of stroke and dementia whose key features include recurrent subcortical ischaemic events and vascular dementia.
  • Notch3 has been mapped to this region, and mutations in CADASIL patients indicate that Notch3 could be the defective protein in CADASIL patients, Joutel, A. et al . Na ture 383 : 101 - 110 (1996) .
  • ligands for the Notch receptor family There is also a conserved family of ligands for the Notch receptor family. Multiple ligands are able to activate the same receptor. For example, delta and serrate each act as ligands for Drosophila Notch. These ligands all contain EGF repeats (from 1-14), a DSL domain (delta, serrate, lag-2) and a transmembrane domain. Therefore, receptor and ligand are homologous to one another. In addition, receptor and ligand are often coexpressed and are associated with each other in vesicles .
  • Zneul is similar to Notch and its ligands in having two EGF repeats. However, it has a small number of EGF repeats and lacks a membrane spanning domain, linl2/Notch domains and ankyrin repeats. Based on structure/function experiments of Notch, one would predict that Zneul would antagonize Notch function. If the EGF repeats in zneul could bind receptor, it could inhibit ligand binding on neighboring cells. Furthermore, Zneul may have its own target receptor for which it would be an agonist .
  • Zneul is widely expressed in adult human tissues. Zneul is most highly expressed in heart, placenta, spleen, testis, thyroid, spinal cord and lymph node. Dot blots indicate that Zneul is also expressed in a variety of fetal tissues. There are at least three mRNA sizes :
  • Zneul Since the sequence of Zneul is from the 1.3 kb mRNA in brain, it is difficult to predict what types of molecules the larger transcripts encode. It is possible that larger forms could encode soluble Zneul proteins with more EGF repeats and other domains observed in Notch or Notch ligands. Alternatively, the extra sequences could encode transmembrane and intracellular domains.
  • Zneul may alter the binary decisions in differentiation of stem cells into specific lineages or may alter the cell fate decisions of adjacent cells.
  • Zneul may have nothing to do with Notch. Many proteins have EGF repeats. Zneul may act as a growth factor for a different class of receptor.
  • Notch4 and HSMHC3W5A are also linked at the MHC III locus, i.e., duplication of an authentic Notch receptor and a 2 EGF-repeat novel protein.
  • Zneul and its antagonists can be used as therapeutic reagents for the following.
  • the Sell2 gene was identified as a suppresser of a linl2 gain-of-function mutant.
  • Sell2 is a homolog of a positional cloned human early-onset familial Alzheimer's disease gene. Therefore, Zneul could affect a pathway affecting this disease and it is expressed in brain, albeit at lower levels than most other tissues.
  • Zneul Hodgkin's lymphoma and acute myeloid leukemia.
  • a probe for Zneul which does not properly hybridize to chromosome 9q34 would indicate an abnormality of chromosome 9 and would indicate a possible predilection of the individual for developing cancer.
  • an endothelial-specific gene Zneul could be involved in promoting or inhibiting endothelial cell tumors such as hemangiopericytomas? Another possibility is in angiogenesis since blocking a tumor's blood supply would be an effective cancer treatment.
  • Zneul may play a role in Nerve regeneration since Notch plays a role in neurogenesis in both flies and mammalian cells.
  • the present invention also provides reagents with significant therapeutic value.
  • the Zneul polypeptide naturally occurring or recombinant
  • fragments thereof, antibodies and anti-idiotypic antibodies thereto, along with compounds identified as having binding affinity to the Zneul polypeptide, should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a Zneul polypeptide should be a likely target for an agonist or antagonist of the Zneul polypeptide.
  • Antibodies to the Zneul polypeptide can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in pharmaceutically acceptable carriers or diluents along with physiologically innocuous stabilizers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies, binding fragments thereof or single-chain antibodies of the antibodies including forms which are not complement binding.
  • the quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medications administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vi tro may provide useful guidance in the amounts useful for in vivo administration of these reagents . Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Methods for administration include oral, intravenous, peritoneal, intramuscular, or transdermal administration. Pharmaceutically acceptable carriers will include water, saline, buffers to name just a few. Dosage ranges would ordinarily be expected from l ⁇ g to lOOO ⁇ g per kilogram of body weight per day.
  • the doses by be higher or lower as can be determined by a medical doctor with ordinary skill in the art .
  • Remington ' s Pharmaceutical Sciences 11 Ed., (Mack Publishing Co., Easton, Penn., 1990), and Goodman and Gilman ' s : The Pharmacological Bases of Therapeutics , 9 Ed. (Pergamon Press 1996) .
  • a gene encoding a Zneul polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell.
  • defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al . , Mol ec . Cell . Neurosci . , 2 :320-330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin . Invest . , 90 :626-630 (1992), and a defective adeno- associated virus vector [Samulski et al . , J. Virol . , 61:3096-3101 (1987); Samulski et al . J. Virol . , 63:3822- 3828 (1989) ] .
  • HSV1 herpes virus 1
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . , Cell , 33:153 (1983); Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J " . Virol . , 62:1120 (1988); Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al . ; and Blood, 82:845 (1993).
  • a retroviral vector e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . , Cell
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner et al . , Proc . Na tl . Acad . Sci . USA, 84:7413-7417 (1987); see Mackey et al . , Proc . Natl . Acad . Sci . USA, 85:8027-8031 (1988) ] .
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
  • directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter [see, e.g., Wu et al . , J. Biol . Chem . ,
  • ZNEU1 polypeptides can also be used to prepare antibodies that specifically bind to Zneul epitopes, peptides or polypeptides.
  • the Zneul polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the Zneul polypeptide encoded by SEQ ID NO: 2 or 3 or at least a contiguous 9 amino acid fragment thereof.
  • Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al . (eds.), National Institutes of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al . , Molecular Cloning: A Labora tory Manual , Second Edi tion (Cold Spring Harbor, NY, 1989); and Hurrell, J. G. R. , Ed., Monoclonal Hybridoma Antibodies : Techniques and Applica tions (CRC Press, Inc., Boca Raton, FL, 1982) .
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zneul polypeptide or a fragment thereof.
  • the immunogenicity of a Zneul polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zneul or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full- length molecule or a portion thereof. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 anc Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen- binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non- human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) . In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to Zneul protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zneul protein or peptide) .
  • Genes encoding polypeptides having potential Zneul polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Random peptide display libraries can be screened using the Zneul sequences disclosed herein to identify proteins which bind to Zneul.
  • binding proteins which interact with Zneul polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zneul "antagonists" to block Zneul binding and signal transduction in vi tro and in vivo . These anti-Zneul binding proteins would be useful for down regulating the effect of Zneul.
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • antibodies herein specifically bind if they bind to a Zneul polypeptide, peptide or epitope with a binding affinity
  • binding affinity of an antibody - can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis.
  • antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides.
  • Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zneul but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) .
  • Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-16) , Zneul polypeptides, and non-human Zneul.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides.
  • antibodies raised to Zneul are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zneul will flow through the matrix under the proper buffer conditions.
  • Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al .
  • assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay.
  • ELISA enzyme-linked immunosorbent assay
  • antibodies can be screened for binding to wild-type versus mutant Zneul protein or polypeptide .
  • Antibodies to Zneul may be used for tagging cells that express Zneul; for isolating Zneul by affinity purification; for diagnostic assays for determining circulating levels of Zneul polypeptides; for detecting or quantitating soluble Zneul as marker of underlying pathology or disease; in analytical methods employing
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to Zneul or fragments thereof may be used in vi tro to detect denatured Zneul or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • An additional embodiment of the present invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide having an amino acid sequence described above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zneul polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention.
  • Examples of said polypeptides are defined by the amino acid sequences of SEQ ID NOs: 20-23. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.
  • Zneul was identified from expressed sequence tag (EST) SEQ ID NO: 4.
  • the cDNA clone containing the EST was discovered in a brain cDNA library which contained the EST.
  • the cDNA was isolated from E . coli transfected with the plasmid and then streaked out on an LB 100 ⁇ g/ml ampicillin and 100 ⁇ g/ml methicillin plate.
  • the cDNA insert was sequenced. The insert was determined to be 1514 base pairs long with a 274 amino acid open reading frame and a putative 19 amino acid signal peptide.
  • the GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
  • a publicly available WWW server http://www-genome.wi.mit.edu/cgi- bin/contig/rhmapper .pi) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.
  • Each of the 95 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR ' reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA) , 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA) , 1 ⁇ l sense primer, SEQ ID NO: 6, 1 ⁇ l antisense primer, SEQ ID NO: 7, 2 ⁇ l "RediLoad” (Research Genetics, Inc., Huntsville, AL) , 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and x ⁇ l ddH20 for a total volume of 20 ⁇ l .
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 70°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .
  • GCCCCCATGC CCCTGCCCAA CATGCTGGGG GTCCAGAAGC CACCTCGGGG TGACTGAGCG 996
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • Trp Glu Gly His Ser Leu Ser Ala Asp Gly Thr Leu Cys Val Pro Lys 145 150 155 160
  • Trp Glu Gly His Ser Leu Ser Ala Asp Gly Thr Leu Cys Val 145 150 155
  • GTC TAC AGA CCC AGC CGT AGA GTG TGT ACT GTG GGG ATT TCC GGA GGT 386 Val Tyr Arg Pro Ser Arg Arg Val Cys Thr Val Gly He Ser Gly Gly 30 35 40
  • GAG GAG GTG TAC AGG CTG CAG GCT CGG GTT GAT GTG CTA GAA CAG AAA 914 Glu Glu Val Tyr Arg Leu Gin Ala Arg Val Asp Val Leu Glu Gin Lys 205 210 215
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • Trp Glu Gly Gin Ser Pro Ser Ala Asp Gly Thr Arg Cys Leu Ser Lys 145 150 155 160

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Abstract

L'invention concerne de nouveaux polypeptides de mammifère de type facteur de croissance nerveuse, des polynucléotides codant les polypeptides et des compositions et des procédés associés comprenant des anticorps et des anticorps anti-idiotypes.
EP98930397A 1997-06-18 1998-06-18 Proteine de mammifere de type facteur de croissance nerveuse Ceased EP0996628A2 (fr)

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NZ525914A (en) * 1998-03-10 2004-03-26 Genentech Inc Novel polypeptides and nucleic acids encoding the same
US7723488B2 (en) 1998-03-27 2010-05-25 Genentech, Inc. Monoclonal antibodies to secreted and transmembrane polypeptides
PT1241186E (pt) * 1998-04-22 2009-03-05 Genentech Inc Proteína gene 6 específico de paragem do crescimento (gas-6) humano e ácidos nucleicos que a codificam
AU3965799A (en) * 1998-04-23 1999-11-08 Millennium Pharmaceuticals, Inc. Novel molecules of the t125-related protein family and uses thereof
AU2883600A (en) * 1999-03-08 2000-09-28 Genentech Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
AU2224800A (en) * 1999-03-08 2000-09-28 Genentech Inc. Compositions and methods for the treatment of tumor
EP1214426A2 (fr) * 1999-08-31 2002-06-19 Novozymes A/S Nouvelles proteases et leurs variants
US7217554B2 (en) 1999-08-31 2007-05-15 Novozymes A/S Proteases and variants thereof
CA2384749A1 (fr) * 1999-09-13 2001-03-22 Curagen Corporation Nouvelles proteines humaines, polynucleotides codant celles-ci et procedes d'utilisation des memes
EP2275549A1 (fr) * 2000-06-23 2011-01-19 Genentech, Inc. Compositions et procédés pour le traitement et le diagnostic des troubles impliquant une angiogénèse
FR2851249A1 (fr) * 2003-02-17 2004-08-20 Commissariat Energie Atomique Nouveau facteur soluble secrete par les cellules endotheliales des vaisseaux sanguins, capable d'inhiber le recrutement des cellules perivasculaires du type musculaire lisse et ses applications
WO2005117968A2 (fr) * 2004-04-14 2005-12-15 Genentech, Inc. Compositions et methodes destinees a la modulation du developpement vasculaire
DE102007019162A1 (de) * 2007-04-20 2008-10-23 Johann Wolfgang Goethe-Universität Frankfurt am Main Verwendung von EGFL7 zur Modulation von Zellen
TWI428142B (zh) 2009-05-08 2014-03-01 Genentech Inc 人類化之抗－ｅｇｆｌ７抗體及其使用方法
US9789213B2 (en) 2012-03-27 2017-10-17 London Health Sciences Centre Research Inc. EGFL7 targeting and/or binding polypeptides and methods for inhibiting angiogenesis

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US6337387B1 (en) * 1995-11-17 2002-01-08 Asahi Kasei Kabushiki Kaisha Differentiation-suppressive polypeptide

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