EP1290026A2 - Menschliche sez6 nukleinsäure und polypeptide - Google Patents

Menschliche sez6 nukleinsäure und polypeptide

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Publication number
EP1290026A2
EP1290026A2 EP01930440A EP01930440A EP1290026A2 EP 1290026 A2 EP1290026 A2 EP 1290026A2 EP 01930440 A EP01930440 A EP 01930440A EP 01930440 A EP01930440 A EP 01930440A EP 1290026 A2 EP1290026 A2 EP 1290026A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
hsez6
nucleic acid
protein
cells
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.)
Withdrawn
Application number
EP01930440A
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English (en)
French (fr)
Inventor
Eric Wen Su
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Eli Lilly and Co
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Eli Lilly and Co
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Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1290026A2 publication Critical patent/EP1290026A2/de
Withdrawn legal-status Critical Current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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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 compounds and compositions comprising novel human SEZ6 (hSEZ ⁇ ) polypeptides, nucleic acids, host cells, transgenics, chimerics, antibodies, compositions, and methods of making and using thereof.
  • hSEZ ⁇ human SEZ6
  • axons The navigation of axons to their targets is a critical step in the patterning of neuronal projections.
  • the growth cones at the tips of developing axons are thought to select appropriate pathways by recognizing distinct guidance markers present in their environment.
  • axonal guidance depends on expression of both attractive and repulsive molecules on cells and in the extracellular matrix along the pathway of advancing axons (Dodd et al., Neuron, 1:105-116 (1988); Harrelson and
  • the extracellular domains of neuropilins are complex, usually consisting of two so-called CUB (extracellular complement-binding) domains, two domains having homology to coagulation factors V and VIII (b domains) , and a so-called MAM (meprin, A5, ⁇ ) domain. All of these domains have been implicated in mediating protein- protein interactions. More specifically, MAM domains are known to mediate homophilic interactions in receptor tyrosine phosphatases (Bork, P., and Beckmann, G., J. Mol . Biol . , 231:539-545 (1993); Zondag, G.C., et al . , J " . Biol . Chem.
  • SEZ6 polypeptides Most proteins that have SCRs are also involved in protein-protein interaction.
  • the presence of both SCRs, in addition to the CUB motifs, in SEZ6 polypeptides supports its putative role as a neuronal adhesion molecule structurally similar to the neuropilins.
  • SEZ6 splice variants which encode various secreted and membrane bound isoforms of SEZ6 polypeptides have also been identified. (Shimizu-Nishikawa, K. , et al. , (1995) ) .
  • the present invention further provides recombinant vectors, comprising 1-40 of said isolated hSEZ6 nucleic acid molecules of the present invention, host cells containing said nucleic acids and/or said recombinant vectors.
  • the present invention also provides methods of making or using hSEZ6 nucleic acids, and/or vectors, host cells, and transgenic animals comprising said nucleic acids.
  • hSEZ6 polypeptide can therefore be screened for such activities according to known methods.
  • An hSEZ6 polypeptide can thus be screened for a corresponding activity according to these and other methods known in the art.
  • the present invention also provides an hSEZ6 antibody, or fragment thereof, comprising a polyclonal and/or monoclonal antibody, or fragment thereof, that specifically binds at least one epitope specific to at least one hSEZ6 polypeptide as described herein.
  • the present invention also provides a method for producing an hSEZ6 antibody or antibody fragment, comprising generating the antibody or fragment thereof that binds at least one epitope that is specific to an isolated hSEZ6 polypeptide as described herein, the generating done by known recombinant, synthetic and/or hybridoma methods.
  • the present invention also provides methods for identifying polypeptides that bind an hSEZ6 polypeptide which comprises use of at least one isolated hSEZ6 polypeptide as described herein in at least one protein- protein interaction assays or reporter systems known in the art.
  • modifications in the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent, or possibly improved, second generation peptides, etc., that display equivalent or superior functional characteristics when compared to the original amino acid sequences.
  • Alterations in the hSEZ6 polypeptides of the present invention can include one or more amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, either from natural mutations or human manipulation, provided that the sequences produced by such modifications have substantially the same (or improved or reduced, as may be desirable) activity (ies) as the hSEZ6 analog sequences disclosed herein.
  • amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant peptide, etc., having similar biological activity, i.e., still retaining correct biological function.
  • amino acids having hydropathic indices within ⁇ 2 are preferably substituted for one another, those within ⁇ 1 are more preferred, and those within +0.5 are most preferred.
  • amino acid substitutions in the hSEZ ⁇ polypeptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc.
  • amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids.
  • amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral non- polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes that are not expected to be advantageous can also be useful if these result in the production of functional sequences.
  • biologically functional equivalents of the present hSEZ6 polypeptides can have any number of conservative or non-conservative amino acid changes that do not significantly affect their activity (ies) , or that increase or decrease activity as desired, 40, 30, 20, 10, 5, or 3 changes, or any range or value therein, may be preferred. In particular, 10 or fewer amino acid changes may be preferred; more preferably, seven or fewer amino acid changes may be preferred; most preferably, five or fewer amino acid changes may be preferred.
  • the encoding nucleotide sequences (gene, plasmid DNA, cDNA, synthetic DNA, or mRNA, for example) will, thus, have corresponding base substitutions, permitting them to code on expression for the biologically functional equivalent forms of the hSEZ6 polypeptides.
  • preferred hSEZ6 peptides, polypeptides, or proteins exhibit the same or similar biological or immunological activity (ies) as that (those) of the hSEZ6 polypeptides specifically disclosed herein, or increased or reduced activity, if desired.
  • the activity(ies) of ant variant hSEZ6 polypeptides can be determined by the methods described herein.
  • human antibody includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Rabat et al . (1991) .
  • Human antibodies are generated by various methods now routine to one skilled in the art.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vi tro or by somatic mutation in vivo) , for example in the CDRs and in particular CDR3.
  • Any human antibody can also be substituted at one or more positions with an amino acid, e.g., a biological property enhancing amino acid residue, which is not encoded by the human germline immunoglobulin sequence. In preferred embodiments, these replacements are within the CDR regions as described in detail below.
  • Human antibodies have at least three potential advantages over non-human and chimeric antibodies for use in human therapy:
  • the human immune system should not recognize the human antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign non-human antibody or a partially foreign chimeric antibody;
  • injected non-human antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of human antibodies.
  • Injected human antibodies will have a half-life essentially identical to naturally occurring human antibodies, allowing smaller and less frequent doses to be given.
  • hybridization refers to a process in which a partially or completely single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. Hybridization can occur under conditions of low, moderate or high stringency, with high stringency preferred. The degree of hybridization depends upon, for example, the degree of homology, the stringency conditions, and the length of hybridizing strands as known in the art.
  • 1 x 10 5 M" 1 preferably at least 1 x 10 6 M _1 to 1 x 10 7 M _1 or more, under suitable physiological conditions.
  • isolated used in reference to at least one polypeptide of the invention describes a state of isolation such that the peptide or polypeptide is not in a naturally occurring form and/or has been purified to remove at least some portion of cellular or non-cellular molecules with which the protein is naturally associated.
  • isolated may include the addition of other functional or structural polypeptides for a specific purpose, where the other peptide may occur naturally associated with at least one polypeptide of the present invention, but for which the resulting compound or composition does not exist naturally.
  • a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
  • crosslinking agents include, e.g., 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3 , 3 ' -dithiobis-
  • Antigenic epitope-bearing peptides and polypeptides of the invention are useful to raise antibodies, including monoclonal antibodies, or screen antibodies, including fragments or single chain antibodies, that bind specifically to a polypeptide of the invention. See, e.g., Wilson, et al., Cell 37:767-778 (1984) at 777.
  • Antigenic epitope- bearing peptides and polypeptides of the invention preferably contain a sequence of at least five, more preferably at least nine, and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • the hybridoma producing the MAbs of this invention may be cultivated in vi tro or in vivo . Production of high titers of MAbs in vivo makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/C mice to produce ascites fluid containing high concentrations of the desired MAbs.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable region derived from a murine MAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc . Natl . Acad. Sci . (USA) 71:3273-3277 (1984); Morrison et al., Proc. Natl . Acad. Sci . (USA) 81:6851-6855 (1984); Boulianne et al . , Nature 312:643646 (1984); Cabilly et al . , European Patent Application 125023
  • the anti-anti-Id may be epitopically identical to the original MAb which induced the anti-Id.
  • MAbs generated against a hSEZ6 protein or glycoprotein of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/C mice and/or any transgenically altered mouse capable of producing fully humanized MAbs. Spleen cells from such immunized mice are used produce anti-Id hybridomas secreting anti-Id MAbs.
  • the anti-Id MAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional similar mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original MAb specific for a hSEZ6 epitope.
  • KLH keyhole limpet hemocyanin
  • the anti-Id MAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as a hSEZ6 protein or glycoprotein.
  • a preferred method of isolating associating proteins is by contacting a hSEZ6 polypeptide to an antibody that binds the hSEZ6 polypeptide, and isolating resultant immune complexes .
  • These immune complexes may contain associating proteins bound to the hSEZ6 polypeptide.
  • the associating proteins may be identified and isolated by denaturing the immune complexes with a denaturing agent and, preferably, a reducing agent. The denatured, and preferably reduced, proteins can be separated on a polyacrylamide gel.
  • cDNA expression libraries usually comprise mammalian cDNA populations, typically human, mouse, or rat, and may represent cDNA produced from RNA of one cell type, tissue, or organ and one or more developmental stage.
  • Specific binding for screening cDNA expression libraries is usually provided by including one or more blocking agent (e.g., albumin, nonfat dry milk solids, etc.) prior to and/or concomitant with contacting the labeled hSEZ ⁇ polypeptide (and/or labeled anti-hSEZ6 antibody) .
  • a blocking agent e.g., albumin, nonfat dry milk solids, etc.
  • Another approach to identifying polypeptide sequences which bind to a predetermined polypeptide sequence i.e., hSEZ ⁇
  • Two-hybrid methods generally rely upon a positive association between two fusion proteins thereby reconstituting a functional transcriptional activator which then induces transcription of a reporter gene operably linked to an appropriate transcriptional activator binding site.
  • protein X protein X
  • protein Y protein Y
  • the host strain is engineered so that the reconstituted transcriptional activator drives the expression of a specific reporter gene, which provides the read-out for the protein-protein interaction (Field and Song, (1989); Chein et al . , (1991)).
  • a selectable or otherwise identifiable phenotypic change that characterizes a positive readout condition confers upon the organism (e.g., yeast, bacteria, mammalian cell) either: a selective growth advantage on a defined medium, a mating phenotype, a characteristic morphology or developmental stage, drug resistance, or a detectable enzymatic activity (e.g., ⁇ -galactosidase, luciferase, alkaline phosphatase).
  • organism e.g., yeast, bacteria, mammalian cell
  • a detectable enzymatic activity e.g., ⁇ -galactosidase, luciferase, alkaline phosphatase.
  • One advantage of a two-hybrid system for monitoring protein-protein interactions is their sensitivity in detection of physically weak, but physiologically important, protein-protein interactions. As such it offers a significant advantage over other methods for detecting protein-protein interactions (e.g., ELISA assay).
  • Various means for determining a compound's ability to modulate hSEZ ⁇ in the body of the transgenic animal are consistent with the invention. Observing the reversal of a pathological condition in the transgenic animal after administering a compound is one such means. Another more preferred means is to assay for markers of hSEZ ⁇ activity in the blood of a transgenic animal before and after administering an experimental compound to the animal.
  • the level of skill of an artisan in the relevant arts readily provides the practitioner with numerous methods for assaying physiological changes related to therapeutic modulation of hSEZ6 activity.
  • compounds of the present invention can be administered in a single daily dose or in multiple doses per day.
  • the treatment regime may require administration over extended periods of time.
  • the amount per administered dose or the total amount administered will be determined by the physician and depend on such factors as the nature and severity of the disease, the age and general health of the patient and the tolerance of the patient to the compound.
  • the instant invention further provides pharmaceutical formulations comprising a hSEZ ⁇ nucleic acid, polypeptide, and/or anti-hSEZ6 antibody of the present invention.
  • the proteins preferably in the form of a pharmaceutically acceptable salt, can be formulated for parenteral administration for the therapeutic or prophylactic treatment disorders commonly associated with aberrant hSEZ ⁇ activity.
  • compositions of the present invention maybe administered orally, parenterally, by inhalation spray, nasally, buccally, or via an implanted reservoir.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
  • the pH of the formulation maybe adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersion wetting agents and suspending agents .
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1, 3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • the invention further provides for the use of a hSEZ6 agonist, hSEZ ⁇ antagonist, hSEZ ⁇ polypeptide, hSEZ ⁇ nucleic acid, and/or hSEZ6 antibody in the manufacture of a medicament for the treatment or prevention of a disorder in which hSEZ6 activity is detrimental wherein said medicament further comprises another cytokine agonist, antagonist, polypeptide, nucleic acid, and/or antibody.
  • the invention further provides for the use of a hSEZ ⁇ agonist, hSEZ6 antagonist, hSEZ ⁇ polypeptide, hSEZ6 nucleic acid, and/or hSEZ ⁇ antibody in the manufacture of a medicament for the treatment or prevention of a neurological disorder selected from the group consisting of epilepsy, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, muscle injury, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed invertebrae disk syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies caused by lead, dapsone, ticks, or porphyria, peripheral myelin disorders, Alzheimer's disease, Gullain- Barre syndrome, Parkinson's disease, Parkinsonian disorders, ALS, multiple sclerosis, central myelin disorders, seizures, stroke, isch
  • Nucleic acids encoding hSEZ6 polypeptides may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example, for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.
  • the oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups .
  • a DNA fragment encoding a polypeptide can be inserted in such a way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
  • a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.
  • the amplified hSEZ6 nucleic acid fragments and the vector pQE60 are digested with appropriate restriction enzymes and the digested DNAs are then ligated together. Insertion of the hSEZ ⁇ DNA into the restricted pQE ⁇ O vector places an hSEZ ⁇ polypeptide coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG codon. The associated stop codon prevents translation of the six histidine codons downstream of the insertion point.
  • IPTG Isopropyl-b-D-thiogalactopyranoside
  • the protein is made soluble according to known method steps . After renaturation the polypeptide is purified by ion exchange, hydrophobic interaction and size exclusion chromatography.
  • baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
  • Such vectors are described, for instance, in Luckow, et al., Virology 170:31-39.
  • the cDNA sequence encoding the mature hSEZ ⁇ polypeptide in the deposited or other clone, lacking the AUG initiation codon and the naturally associated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non-limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of an hSEZ6 polypeptide, e.g., as presented in at least one of SEQ ID NOS:l OR 2, according to known method steps .
  • the plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean” BIO 101 Inc., La Jolla, CA) . This vector DNA is designated herein "VI” .
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, according to known methods. An agarose gel with "Blue Gal” (Life Technologies, Inc., Rockville, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue- stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies, Inc., Rockville, MD, page 9-10). After appropriate incubation, blue stained plaques are picked with a micropipettor tip (e.g., Eppendorf).
  • a micropipettor tip e.g., Eppendorf
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS .
  • the cells are infected with the recombinant baculovirus V-hSEZ6 at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available, e.g., from Life Technologies, Inc., Rockville, MD) . If radiolabeled polypeptides are desired, 42 hours later, 5 Ci of 35S- methionine and 5 mCi 35S-cysteine (available from Amersham) are added.
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation.
  • the polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled) . Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide.
  • a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV) . However, cellular elements can also be used (e.g., the human actin promoter) .
  • Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the transfected gene can also be amplified to express large amounts of the encoded polypeptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al . , Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
  • the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene .
  • Oligonucleotide primers containing Ascl or Nhel endonuclease restriction sites for the forward strands and EcoRV, EcoRI, or Pmel restriction sites for the reverse strands, are determined using ordinary skill of the art.
  • the resultant PCR-generated fragment is cleaved with the respective restriction enzymes then gel-purified.
  • the fragment is ligated into a mammalian expression vector that is digested with the appropriate restriction enzymes.
  • Expression vectors which are used include pPRl (a derivative of pJB02, Berry, J.; Gonzalez-DeWhitt, P.; Ryan, P.; Kovacevic, S.; and Amegadzie, B.Y.; unpublished), pEW1938 (a modified version of pJB02 with a FLIS epitope tag fused to the C-terminus), or pXenoFLIS.
  • the plasmid construct is designed to express a molecule (including the NH2-terminal amino acids which constitute the signal peptide) with the FLIS tag at the COOH-terminus of the protein. Protein expression is controlled by the CMV promoter.
  • hSEZ6 polypeptide For expression of the recombinant hSEZ6 polypeptide, cells are transiently transfected with the expression vector described above. All transfections are performed in spinner culture flasks utilizing cell lines that have been adapted to suspension growth in an animal protein-free medium (APFM) . Stock cells are maintained in 6-liter shake flask cultures (130 rpm, ' 37 °C incubator) at a working volume of approximately 2 liters and a cell density between 0.5 and 3.0 X 10 6 cells/mL.
  • APFM animal protein-free medium
  • transfections For 500 mL transfections, cells from the stock culture are centrifuged, washed, and seeded at 6.0 X 10 6 cells/mL into a 1 liter spinner flask containing a total of 450 mL APFM. In a separate container, the DNA are prepared for transfection by adding 250 ⁇ g of the appropriate plasmid DNA to 50 mL of APFM, followed by the addition of 500 ⁇ L of transfection reagent. A proprietary transfection reagent, X-tremeGENE Ro-1539 (Roche Diagnostics Corp.) , is used to introduce DNA into the cells.
  • the 50 mL DNA per transfection reagent mixture is added to the spinner flask containing the cells.
  • the flask is gassed with a 10% C0 2 /air mixture and incubated for 5 days in a non-C0 2 incubator at 37°C and 150 rpm.
  • the cells are removed by centrifugation at 2000 X g for 30 minutes, and the conditioned medium is submitted for purification by SDS-PAGE.
  • High throughput protein isolation is accomplished using affinity chromatography. Under a sterile hood, 1 mL of FLAG affinity resin is added to each flask containing the transfected cells in 500 mL media. The flasks are capped, and the media / resin slurry is shaken on an orbital shaker overnight at 4°C. After shaking, the media is poured into sterile, disposable columns attached to a vacuum manifold located inside a sterile hood. The resin is collected in the columns and washed with sterile, phosphate buffered saline (PBS) . The proteins are then eluted with 5 mL of sterile FLAG peptide solution at a concentration of 0.5 mM in PBS.
  • PBS phosphate buffered saline
  • the expression plasmid, phSEZ6HA is made by cloning a cDNA encoding hSEZ6 into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eucaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) or HIS tag (see, e.g, Ausubel, supra) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells
  • an ampicillin resistance gene for selection
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin polypeptide described by Wilson, et al., Cell 37:767-778 (1984) .
  • the fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizes the HA epitope.
  • pcDNAIII contains, in addition, the selectable neomycin marker.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme (s) and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the hSEZ6 -encoding fragment .
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook, et al . , Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expression of hSEZ6 by the vector.
  • hSEZ6-HA fusion polypeptide is detected by radio-labeling and immuno-precipitation, using methods described in, for example Harlow, et al . , Antibodies: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) .
  • the cells are labeled by incubation in media containing 35S-cysteine for 8 hours.
  • the cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson, et al . cited above.
  • Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody.
  • the precipitated polypeptides then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls .
  • Plasmid pC4 is used for the expression of hSEZ6 polypeptide.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146) .
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • MTX methotrexate
  • high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the hSEZ6 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • 5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 ⁇ g/ml G418.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • EXAMPLE 5 Directed Mutagenesis of hSEZ ⁇ polypeptides to provide DNA encoding specified substitutions, insertions or deletions of SEQ ID NO:l Using the Polymerase Chain Reaction
  • Unit 8.5 of Ausubel, supra contains two basic protocols for introducing base changes into specific DNA sequences.
  • Basic Protocol 1 as presented in the first section 8.5 of Ausubel, supra (entirely incorporated herein by reference) , describes the incorporation of a restriction site and Basic Protocol 2 , as presented below and in the second section of Unit 8.5 of Ausubel, supra, details the generation of specific point mutations (all of the following references in this example are to sections of Ausubel et al . , eds., Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-1999)).
  • An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
  • the entire amplified fragment can usually be sequenced from a single primer. If the fragment is somewhat longer, it is best to subclone the fragment into an M13-derived vector, so that both forward and reverse primers can be used to sequence the amplified fragment.
  • coli DNA polymerase I (UNIT 3.5 of Ausubel, supra), appropriate restriction endonucleases (Table 8.5.1), as well as, the reagents and equipment for synthesis, purification, and phosphorylation of oligonucleotides (UNITS 2.11, 2.12, & 3.10), electrophoresis on nondenaturing agarose and low gelling/melting agarose gels (UNITS 2.5A & 2.6), ligation of DNA fragments (UNIT 3.16), transformation of E. coli (UNIT 1.8), and preparation of plasmid DNA (UNIT 1.6) .
  • hSEZ6 polypeptide sequence of interest including at least one substitution, insertion or deletion selected from the group consisting of 261, 27T, 29E, 31H, 33T, 36R, 51S, 52D, 83R, 85E, 87A, 88P, 89Q, 98A, HIT, 115N, 126V, 129A, 134H, 136R, 138K, 141N, 142L, 145K, 146P, 148E, 150S, 153S, 154S, 167L, 169E, 171R, 172P, 179Q, 192D, 197P, 200M 202K, 203T, 204T, 206L,208V, 209E, 2131, 214T, 217G,
  • each amplified fragment 1 ⁇ l (500 ng) each flanking sequence primer (each 1 ⁇ M final)
  • Protein-induced cell responses are determined by monitoring tyrosine phosphorylation upon stimulation of cells by addition of hSEZ6 proteins. This is accomplished in two steps: cell manipulation and immunodetection.
  • ECV304 endothelial cell line
  • GLUTag SV40 Tag transformed enteroendocrine cell line
  • HDF skin fibroblasts
  • M07E leukemia cell line
  • the cells are plated into poly-D-lysine- coated, 96 well plates containing cell propagation medium [DMEM:F12 (3:1), 20 mM Hepes at pH 7.5, 5% FBS, and 50 ⁇ g/mL Gentamicin] .
  • the cells are seeded at a concentration of 20,000 cells per well in 100 ⁇ L medium.
  • the propagation medium in each well is replaced with 100 ⁇ L starvation medium containing DMEM-.F12 (3:1), 20mM Hepes at pH 7.5, 0.5% FBS, and 50 ⁇ g/mL Gentamicin.
  • the cells are incubated overnight .
  • pervanadate solution is made 10 minutes before cell lysis; pervanadate is prepared by mixing 100 ⁇ L of sodium orthovanadate (100 mM) and 3.4 ⁇ L of H 2 0 2 (producing 100X stock pervanadate solution) .
  • the lysis buffer is then prepared: 50mM Hepes at pH 7.5, 150 mM NaCI, 10% glycerol, 1% TRITON X-100, 1 mM EDTA, 1 mM pervanadate, and BM protease inhibitors.
  • the cells are stimulated by adding 10 ⁇ L of an hSEZ6 protein solution to the cells, and incubating for 10 minutes. Next, the medium is aspirated, and 75 ⁇ L lysis buffer are added to each well.
  • the cells are lysed at 4°C for 15 minutes, then 25 ⁇ L of 4X loading buffer are added to the cell lysates.
  • the resultant solution is mixed then heated to 95°C.
  • Detection of tyrosine phosphorylation is accomplished by Western immunoblotting. Twenty microliters of each cell sample are loaded onto SDS-PAGE 8-16% AA ready gels from Bio-Rad, and -the gels are run. The proteins are electrotransferred in transfer buffer (25 mM Tris base at pH 8.3, 0.2 M glycine, 20% methanol) from the gel to a nitrocellulose membrane using 250 mA per gel over a one hour period. The membrane is incubated for one hour at ambient conditions in blocking buffer consisting of TBST (20 mM TrisHCl at pH 7.5, 150 mM NaCI, 0.1% TWEEN-20) with 1% BSA. Next, the antibodies are added to the membrane.
  • transfer buffer 25 mM Tris base at pH 8.3, 0.2 M glycine, 20% methanol
  • the membrane is incubated overnight at 4°C with gentle rocking in primary antibody solution consisting of the antibody, TBST, and 1% BSA. The next day, the membrane is washed three times, five minutes per wash, with TBST. The membrane is then incubated in the secondary antibody solution consisting of the antibody, TBST, and 1% BSA for 1 hour at ambient conditions with gentle rocking. After the incubation, the membrane is washed four times with TBST, ten minutes per wash. Detection is accomplished by incubating the membrane with 10 to 30 mL of SuperSignal Solution for 1 minute at ambient conditions. After 1 minute, excess developing solution is removed, and the membrane is wrapped in plastic wrap.
  • the membrane is exposed to X-ray film for 20 second, 1 minute, and 2 minute exposures (or longer if needed) .
  • the number and intensity of immunostained protein bands are compared to bands for the negative control-stimulated cells (basal level of phosphorylation) by visual comparison.
  • GLUTag cells stimulated with hSEZ6 polypeptide induced tyrosine phosphorylation in those cells.
  • Example 7 Cell Stimulation with Detection Utilizing Reporters
  • Protein-induced cell responses are measured using reporters. The following cell line/reporter combinations are used:
  • positive controls are designed in the form of agonist cocktails. These cocktails included approximate maximal stimulatory doses of several ligands known to stimulate the regulated signal pathway. The following agonist cocktails are used as positive controls:
  • Cell lines and reporters with no exogenous stimulus added are used as negative controls .
  • the cells are transiently transfected with the reporter plasmids in tissue culture flasks using a standard optimized protocol for all cell lines (see Example 1) .
  • the cells are trypsinized and seeded into 96-well poly-D-lysine coated assay plates at a rate of 20,000 cells per well in growth medium.
  • the medium is replaced with serum-free growth medium.
  • stimulants for those reporters which required a 24-hour stimulation period are added.
  • stimulants for the reporters which required a 5-hour stimulation period are added. Five hours later, all conditions are lysed using a lysis/luciferm cocktail, and the fluorescence of the samples is determined using a Micro Beta reader.
  • Each assay plate is plated to contain 4 positive control wells, 16 negative control wells, and 64 test sample wells (2 replicates of 32 test samples) .
  • the threshold value for a positive "hit” is a fluorescence signal equal to the mean plus two standard deviations of the negative control wells. Any test sample that, in both replicates, generates a signal above that threshold is defined as a "hit . "
  • Example 8 Cell Proliferation and Cytotoxicity Determination Utilizing Fluorescence Detection
  • SK-N-MC (ATCC HTB-10) U373MG, MCF-7 (ATCC HTB-22)
  • GLUTag SV40 Tag transformed enteroendocrine cell line
  • TF.l ATCC CRL-2003
  • HDF skin fibroblasts
  • T1165 B cell line
  • cells Prior to assay, cells are incubated in an appropriate assay medium to produce a sub-optimal growth rate, e.g., a 1:10 or 1:20 dilution of normal culture medium.
  • a sub-optimal growth rate e.g., a 1:10 or 1:20 dilution of normal culture medium.
  • Cells are grown in T-150 flasks, then harvested by trypsin digestion and replated at 40 to 50% confluence into poly-D-lysine- treated 96-well plates. Cells are only plated into the inner 32 wells to prevent edge artifacts due to medium evaporation; the outer wells are filled with buffer alone.
  • hSEZ6 proteins are added to the appropriate wells. Each protein is assayed in triplicate at two different concentrations, IX and 0.1X dilution in assay medium.
  • the intensity of each well containing cells stimulated with an hSEZ6 protein is subtracted from the intensity of the wells containing assay medium only (controls) .
  • a positive number indicated stimulation of cell growth; a negative number indicated a reduction in growth.
  • confidence limits at 95 and 90% are calculated from the mean results.
  • Results lying outside the 95% .confidence limit are scored as "definite hits.” Results lying between the 95 and 90% confidence limits are scored as “ aybes.” The distinction between definite hits and maybes varied due to intraplate variability; thus, subjective scoring is used as a final determination for "hits.”
  • Cell assays performed using hSEZ6 polypeptide showed increased proliferation of SK-N-MC neuroblastoma cells and MCF7 breast cancer cells.
  • Tissues are prepared for analysis by removing the paraffin with xylene then gradually rehydrating the tissue with graded solutions of ethanol and water.
  • Antigen retrieval is performed to unmask antigenic. sites so that antibodies can recognize the antigen. This is accomplished by soaking the tissue in citrate buffer (Dako, Carpinteria, CA) for twenty minutes at 80 to 90°C followed 10 minutes at ambient temperature. The tissue is then washed in tris-buffered saline (TBS) containing 0.05% TWEENTM 20 and 0.01% thimerosol. To minimize non-specific background staining, the tissue is soaked in non-serum protein block (Dako) for 45 minutes, after which the protein block is removed by blowing air over the tissue.
  • Dako non-serum protein block
  • the tissue is exposed for 2 hours to the FLAG-HIS tagged hSEZ6 protein at 10 ⁇ g/mL. Following exposure, the tissue is washed twice with tris-buffered saline (TBS) containing 0.05% TWEENTM 20 and 0.01% thimerosol. The tissue sample is then incubated for one hour with mouse anti-FLAG antibody at 10 ⁇ g/mL. Subsequently, the tissue is washed twice with tris-buffered saline (TBS) containing
  • Example 10 Treatment or Prevention of Neurological Diseases or Neuropathologies with hS ⁇ Z ⁇ Polypeptides
  • hSEZ6 polypeptide or fragment or variant thereof subcutaneously at a dose of 0.5 mg/day, to ensure a slower release into the bloodstream.
  • the treatment is continued until the patient is relieved of the symptoms of the disorder.
  • Yet another protocol is a controlled trial in patients with Alzhemier's Disease and is treated with hSEZ6 polypeptide or fragment or variant thereof as described herein.
  • hSEZ6 polypeptide or fragment or variant thereof subcutaneously at a dose of 0.5 mg/day, to ensure a slower release into the bloodstream. The treatment is continued until the patient is relieved of the symptoms of the disorder.
  • the particular dose of hSEZ6 polypeptide or fragment or variant thereof administered and the route of administration is adjusted by the attending physician evaluating the particular circumstances surrounding the case, including the compound administered, the particular condition being treated, the patient characteristics and similar considerations .

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