EP1140120A2 - Brainiac-5 humains - Google Patents

Brainiac-5 humains

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Publication number
EP1140120A2
EP1140120A2 EP99966502A EP99966502A EP1140120A2 EP 1140120 A2 EP1140120 A2 EP 1140120A2 EP 99966502 A EP99966502 A EP 99966502A EP 99966502 A EP99966502 A EP 99966502A EP 1140120 A2 EP1140120 A2 EP 1140120A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
brainiac
amino acid
seq
sequence
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Withdrawn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
EP99966502A
Other languages
German (de)
English (en)
Other versions
EP1140120A4 (fr
Inventor
Paul E. Young
Steven M. Ruben
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
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Filing date
Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP1140120A2 publication Critical patent/EP1140120A2/fr
Publication of EP1140120A4 publication Critical patent/EP1140120A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P25/24—Antidepressants
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present invention relates to a novel human gene encoding a polypeptide related to the Notch family. More specifically, isolated nucleic acid molecules are provided encoding a human polypeptide named Brainiac-5. Brainiac-5 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to the immune and nervous systems, and therapeutic methods for treating and/or preventing such disorders.
  • the invention further relates to screening methods for identifying agonists and antagonists of Brainiac-5 activity.
  • Control of cell division is a basic aspect of multicellular existence that depends upon a programmed series of events.
  • One factor in cellular proliferation and its control is the presence of various polypeptide growth factors.
  • Growth factors are essential components of growth media for in vitro cell culture and are involved in cell survival in vivo.
  • a partial list of growth factors identified to date include platelet-derived growth factor (PDGF; implicated in the repair of the vascular system in vivo); epidermal growth factor (EGF; which acts as a mitogen for cells of ectodermal and mesodermal origin); transforming growth factor (TGF)-alpha (which acts as a mitogen similarly to EGF, with the exception that it enables normal cells to grow in soft-agar); transforming growth factor (TGF)-beta (a mitogen for some cells and a growth inhibitor for others); and nerve growth factor (NGF; which is involved in the development and maintenance of sympathetic and embryonic neurons).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • TGF-alpha which acts as a mitogen similarly to EGF, with the exception that it enables normal cells to grow in soft-agar
  • TGF transforming growth factor
  • TGF-beta a mitogen for some cells and a growth inhibitor for others
  • NGF nerve growth factor
  • Peptide growth factors are produced and secreted from a variety of tissues.
  • the target cells are typically located near the site of release of the growth factor (paracrine response).
  • growth factors elicit a wide variety of effects on their target cells and are involved in processes such as inflammation, immune reactions, and wound repair.
  • the Notch family of transmembrane receptor proteins have been demonstrated to mediate cell fate decisions, and mutations in mammalian Notch genes have been implicated in leukemia, cervical cancer, colon cancer, breast cancer, stroke, and dementia.
  • Normal hypertrophy is a compensatory action which functions to maintain the pumping action of the heart.
  • Abnormal hypertrophy occurs in a number of situations including hypertension, myocardial infarction, valve disease, and cardiomyopathy.
  • the effects of peptide growth factors on cardiac myocytes are reflected in differentiated patterns of gene expression. For example, stimulation of the alpha-adrenergic receptor induces hypertrophy of cultured cardiac myocytes and produces specific changes in gene expression at the level of transcription.
  • Organ and clonal cell systems have been developed to analyze cardiomyogenic differentiation. (See, for example, Bader, D. et al., Molecular Biology ofthe Cardiovascular System, Roberts, R. et al., ed.: 41-49 (1990).) Differentiation in these systems can be monitored by in vitro analysis of cardiac myogenesis and monoclonal antibodies that have been raised against muscle-specific protein.
  • polypeptide growth factors are very important cell culture reagents for stimulating cellular growth and aiding survival of the cells in vitro. Homology with other members of the Fringe family and indication that mammalian Fringe family members play an evolutionarily conserved role in the Notch signaling pathway suggests that these polypeptides, as well as Brainiac-5 polypeptides, have uses which include the treatment or prevention of disorders of cell fate or differentiation (e.g., cancerous conditions, such as, leukemia, cervical cancer, colon cancer, breast cancer), treatment or prevention of disorders of the nervous system, and stimulation of tissue repair and regeneration.
  • disorders of cell fate or differentiation e.g., cancerous conditions, such as, leukemia, cervical cancer, colon cancer, breast cancer
  • the search continues to exist for polypeptides that stimulate and/or inhibit growth of particular cells for both in vitro and in vivo uses.
  • novel tissue specific markers that can be employed qualitatively to help identify a particular cell or tissue type and employed qualitatively to assess whether cells, tissues or organs are abnormal in their expression of a particular polypeptide.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the Brainiac-5 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 203572 on January 11, 1999.
  • Nucleic acid molecules of the invention include those encoding the complete amino acid sequence shown in SEQ ID NO:2 excepting an N-terminal methionine residue, or the complete amino acid sequence encoded by the cDNA clone in ATCC Deposit Number 203572 excepting an N-terminal methionine, which molecules also can encode additional amino acids fused to the N-terminus and/or C-terminus of the Brainiac-5 amino acid sequence.
  • one embodiment of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the Brainiac-5 polypeptide having the complete amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to 278 of SEQ ID NO:2); (b) a nucleotide sequence encoding the Brainiac-5 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • nucleic acid molecules that comprise a polynucleotide having (i.e., comprising, or alternatively consisting of) a nucleotide sequence at least 90% identical, and more preferably at least 92%, 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c) or (d), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c) or (d), above.
  • This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
  • An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Brainiac-5 polypeptide having an amino acid sequence in (a), (b) or (c), above.
  • a further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions.
  • a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide to have an amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Brainiac-5 polypeptides or peptides by recombinant techniques.
  • a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human Brainiac-5 nucleic acid sequence, under conditions promoting expression of said polypeptide and subsequent recovery of said polypeptide.
  • the invention also provides an isolated Brainiac-5 polypeptide comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the Brainiac-5 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 (i.e., positions 1-278 of SEQ ID NO:2); (b) the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 203572; (c) the complete amino acid sequence of the predicted mature Brainiac-5 polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 203572.
  • polypeptides of the present invention also include polypeptides having (i.e., comprising, or alternatively consisting of) an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 92%, 95%, 96%, 97%, 98% or 99% identical to those described in (a), (b) or (c), above.
  • An additional embodiment of the invention relates to a peptide or polypeptide which comprises, or alternatively consists of, the amino acid sequence of an epitope-bearing portion of a Brainiac-5 polypeptide having (i.e., comprising, or alternatively consisting of) an amino acid sequence described in (a), (b) or (c), above.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Brainiac-5 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
  • a further embodiment of the invention relates to a peptide or polypeptide which comprises, or alternatively consists of, the amino acid sequence of a Brainiac-5 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions.
  • a peptide or polypeptide in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of a Brainiac-5 polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • the invention provides an isolated antibody that binds specifically to a Brainiac-5 polypeptide having an amino acid sequence described in (a), (b) or (c), above.
  • the invention further provides methods for isolating antibodies that bind specifically to a Brainiac-5 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
  • the invention also provides for pharmaceutical compositions comprising
  • Brainiac-5 polypeptides particularly human Brainiac-5 polypeptides, which may be employed, for instance, to treat, prevent, and/or diagnose immune and/or nervous system diseases and disorders. Methods of treating individuals in need of Brainiac-5 polypeptides are also provided.
  • compositions comprising a Brainiac-5 polynucleotide or polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism.
  • the compositions comprise a Brainiac-5 polynucleotide for expression of a Brainiac-5 polypeptide in a host organism for treatment, prevention, and/or diagnosis of disease.
  • Particularly preferred in this regard is expression in a human patient for treatment, prevention, and/or diagnosis of a dysfunction associated with aberrant endogenous activity of a Brainiac-5 polynucleotide and/or polypeptide.
  • the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of the Brainiac-5 polypeptide, which involves contacting a receptor whose activity is inhibited or enhanced by the Brainiac-5 polypeptide with the candidate compound in the presence of a Brainiac-5 polypeptide, assaying cell division activity of the receptor in the presence of the candidate compound and of Brainiac-5 polypeptide, and comparing the receptor activity to a standard level of activity, the standard being assayed when contact is made between the receptor and in the presence of the Brainiac-5 polypeptide and the absence of the candidate compound In this assay, an increase in receptor activity over the standard indicates that the candidate compound is an agonist of Brainiac-5 activity and a decrease in receptor activity compared to the standard indicates that the compound is an antagonist of Brainiac-5 activity.
  • a screening assay for agonists and antagonists involves determining the effect a candidate compound has on Brainiac-5 binding to another member of the Notch family (e.g., fringe, Serrate, Delta, Jagged, Radical Fringe, Lunatic Fringe, and Maniac Fringe).
  • the method involves contacting the Notch family member with a Brainiac-5 polypeptide and a candidate compound and determining whether Brainiac-5 polypeptide binding to the Notch family member is increased or decreased due to the presence of the candidate compound.
  • an increase in binding of Brainiac-5 over the standard binding indicates that the candidate compound is an agonist of Brainiac-5 binding activity and a decrease in Brainiac-5 binding compared to the standard indicates that the compound is an antagonist of Brainiac-5 binding activity.
  • the antagonists may be employed to treat and/or prevent, diseases, disorders or conditions including, but not limited to, septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis, cachexia (wasting or malnutrition), immune system function, lymphoma, and autoimmune disorders.
  • the Brainiac-5 polypeptide(s) may bind to a cell surface polypeptide which also function as a viral receptor or coreceptor.
  • Brainiac-5, or agonists or antagonists thereof may be used to regulate viral infectivity at the level of viral binding or interaction with the Brainiac-5 receptor or coreceptor or during the process of viral internalization or entry into the cell. It has been discovered that Brainiac-5 is expressed not only in ovarian tumor, but also (using BLAST analysis of the HGS EST database) in bone marrow stromal cells and synovial sarcoma.
  • nucleic acids of the invention are useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample.
  • polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s).
  • Brainiac-5 gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" Brainiac-5 gene expression level, i.e., the Brainiac-5 expression level in healthy tissue from an individual not having the immune system disorder.
  • tissues e.g., cancerous and wounded tissues
  • bodily fluids e.g., serum, plasma, urine, synovial fluid or spinal fluid
  • the invention provides a diagnostic method useful during diagnosis of such a disorder, which involves: (a) assaying Brainiac-5 gene expression level in cells or body fluid of an individual; (b) comparing the Brainiac-5 gene expression level with a standard Brainiac-5 gene expression level, whereby an increase or decrease in the assayed Brainiac-5 gene expression level compared to the standard expression level is indicative of disorder in the immune system.
  • Another embodiment of the invention is related to a method for treating or diagnosing an individual in need of an increased level of Brainiac-5 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Brainiac-5 polypeptide of the invention or an agonist thereof.
  • a further embodiment of the invention is related to a method for treating or diagnosing an individual in need of a decreased level of Brainiac-5 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a Brainiac-5 antagonist.
  • Preferred antagonists for use in the present invention are Brainiac-5-specific antibodies.
  • Figures 1A and IB show the nucleotide sequence (SEQ ID NO: l) and deduced amino acid sequence (SEQ ID NO:2) of Brainiac-5. A single potential asparagine-linked glycosylation site is marked in the amino acid sequence of Brainiac-5. The potential site of glycosylation begins at asparagine-79 in Figures 1A and IB (SEQ ID NO:2).
  • the potential glycosylation site is marked with a bold pound symbol (#) above the nucleotide sequence coupled with a bolded one letter abbreviation for the asparagine (N) in the amino acid sequence in Figures 1 A and IB.
  • Regions of high identity between Brainiac-5 and the closely related Drosophila Brainiac and human UDP-galactose-2-acetamido-2-deoxy-D-glucose-3-beta- galactosyltransferase an alignment of these sequences is presented in Figures 2A, 2B, and 2C) are underlined in Figures 1A and IB.
  • FIGS. 1A and IB show the regions of identity between the Brainiac-5 amino acid sequence and the translation product of the Drosophila melanogaster mRNA for Brainiac (SEQ ID NO:3; GenBank Accession No.
  • U41449 human UDP-galactose-2-acetamido-2-deoxy-D-glucose-3-beta-galactosyltransferase (SEQ ID NO:4; GenBank Accession No. Y15014), as determined by the computer program MegAlign (DNA*STAR nucleotide and amino acid sequence analysis package) using the default parameters.
  • Figure 3 shows an analysis of the Brainiac-5 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • the positive peaks indicate locations of the highly antigenic regions of the Brainiac-5 polypeptide, i.e., regions from which epitope- bearing peptides of the invention can be obtained.
  • IX Hydrophobicity Plot - Hopp-Woods
  • X Alpha, Amphipathic Regions - Eisenberg
  • XI Beta, Amphipathic Regions - Eisenberg
  • XII Flexible Regions - Karplus-Schulz
  • XIII Antigenic Index - Jameson-Wolf
  • XIV Surface Probability Plot - Emini.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding a Brainiac-5 polypeptide having the amino acid sequence shown in SEQ ID NO:2, which was determined by sequencing a cloned cDNA.
  • the nucleotide sequence shown in Figures 1 A and IB (SEQ ID NO: l) was obtained by sequencing the HOGCC45 cDNA clone, which was deposited on January 11, 1999 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and given ATCC accession number 203572.
  • the deposited clone is contained in the pCMVSPORT 2.0 plasmid (Life Technologies, Inc., Gaithersburg, MD).
  • the Brainiac-5 polypeptides of the present invention share sequence homology with the translation products of the Drosophila melanogaster mRNA which encodes Brainiac ( Figure 2; SEQ ID NO:3) human mRNA which encodes UDP-galactose-2- acetamido-2-deoxy-D-glucose-3-beta-galactosyltransferase ( Figure 2; SEQ ID NO:4).
  • Drosophila Brainiac is thought to be an important neurogenic secreted molecule that is believed to play a role in the differentiation of embryonic cells into neurons.
  • Brainiac-5 polynucleotides and polypeptides of the invention exert an effect on the differentiation of cells in the early stages of cell and tissue development, and may serve to aid in the differentiation of embryonic cells into dendritic or other immune system cells or neurons or other cells of the nervous system.
  • nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • nucleic acid molecule of the present invention encoding a Brainiac-5 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNA using mRNA as starting material.
  • nucleic acid molecule described in Figures 1 A and IB was discovered in a cDNA library derived from ovarian tumor cells. Additional clones of the same gene were also identified in cDNA libraries from the following tissues: bone marrow stromal cells and synovial sarcoma cells.
  • the determined nucleotide sequence of the Brainiac-5 cDNA of Figures 1A and IB contains a partial open reading frame encoding a polypeptide of 278 amino acid residues, initiating with a valine codon at nucleotide positions 1-3 of the nucleotide sequence in Figures 1A and IB (SEQ ID NO:l), and a deduced molecular weight of about 30,475 Daltons.
  • the amino acid sequence of the Brainiac-5 polypeptide shown in SEQ ID NO:2 is about 37.7% identical to Drosophila melanogaster mRNA for Brainiac ( Figure 2), which can be accessed as GenBank Accession No. U41449.
  • Brainiac-5 polypeptides encoded by the respective deposited cDNA clones may be somewhat longer or shorter. More generally, the actual open reading frames comprising Brainiac-5 may be anywhere in the range of ⁇ 20 amino acids, more likely in the range of ⁇ 10 amino acids, of that predicted from the valine codon at the N-terminus shown in Figures 1A and IB (SEQ ID NO:l). It will further be appreciated that, depending on the analytical criteria used for identifying various functional domains, the exact "address" of a signal sequence or other particular domains of the Brainiac-5 polypeptides may differ slightly from the predicted positions herein.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • the polynucleotides of the invention are less than
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of Brainiac-5 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1A and IB (SEQ ID NO: l).
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100. or at least 250, at least 500, or at least 1000 contiguous nucleotides of Brainiac-5 coding sequence, but do not comprise all or a portion of any Brainiac-5 intron.
  • the nucleic acid comprising Brainiac-5 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the Brainiac-5 gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • an "isolated" nucleic acid molecule does not encompass a chromosome isolated or removed from a cell or a cell lysate (e.g., a "chromosome spread", as in a karyotype).
  • an "isolated" nucleic acid molecule does not encompass a cDNA or genomic library which contains a sequence which encodes Brainiac-5.
  • an "isolated" nucleic acid molecule does not encompass any collection of vectors which contain exceptionally large DNA, RNA, or cDNA inserts with respect to the Brainiac-5 sequence disclosed herein (for example, cDNA or genomic libraries, or YAC or BAC artificial chromosomes, and the like) which contain a sequence encoding Brainiac-5.
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF), or comprising a partial ORF, initiating with a valine codon at positions 1-3 of the nucleotide sequence shown in Figures 1A and IB (SEQ ID NO: l).
  • isolated nucleic acid molecules of the invention include DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode Brainiac-5 polypeptides of the invention.
  • Brainiac-5 variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are preferred.
  • the genetic code and species-specific codon preferences are well known in the art.
  • the invention provides isolated nucleic acid molecules encoding the Brainiac-5 polypeptide having (i.e., comprising, or alternatively consisting of) an amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 203572 on January 11, 1999.
  • this nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA clone.
  • the invention further provides an isolated nucleic acid molecule having (i.e., comprising, or alternatively consisting of) the nucleotide sequence shown in Figures 1A and IB (SEQ ID NO:l) or the nucleotide sequence of the Brainiac-5 cDNA contained in the above-described deposited clone, or a nucleic acid molecule having (i.e., comprising, or alternatively consisting of) a sequence complementary to one of the above sequences.
  • isolated molecules, particularly DNA molecules are useful, for example, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the Brainiac-5 gene in human tissue, for instance, by Northern blot analysis.
  • the present invention is further directed to nucleic acid molecules encoding portions of the nucleotide sequences described herein as well as to fragments of the isolated nucleic acid molecules described herein.
  • the invention provides a polynucleotide having (i.e., comprising, or alternatively consisting of) a nucleotide sequence representing the portion of SEQ ID NO: 1 which consists of positions 1-977 of SEQ ID NO: 1.
  • the invention includes a polynucleotide comprising, or alternatively consisting of, any portion of at least about 30 nucleotides, preferably at least about 50 nucleotides, of SEQ ID NO: 1 from positions 1 -977 of SEQ ID NO: 1 , excluding the sequences of the following related cDNA clones, and any subfragments therein: HOGCC45RA (SEQ ID NO:5); HTEDM56R (SEQ ID NO:6); HSSET36R (SEQ ID NO:7); HSOBD70R (SEQ ID NO:8); HI 8701 (SEQ ID NO:9); and (SEQ ID NO: 10). Further, the invention includes a polynucleotide comprising, or alternatively consisting of, any portion of at least about 25 nucleotides, preferably at least about 30
  • the invention includes a polynucleotide comprising, or alternatively consisting of, nucleotides 1-600; 25-600; 50-600; 75-600; 100-600; 125-600; 150-600; 175-600;
  • 375-400 1-375; 25-375; 50-375; 75-375; 100-375; 125-375; 150-375; 175-375; 200-375; 225-375; 250-375; 275-375; 300-375; 325-375; 350-375; 1-350; 25-350;
  • these fragments encode a polypeptide which has biological activity, and/or a Brainiac-5 functional activity (e.g., activation of the Notch signaling pathway, mediation of protein-protein interactions (e.g., between Brainiac-5 and Su(H) or a human homolog of Su(H)), and/or binding of an antibody specific to Brainiac-5).
  • a Brainiac-5 functional activity e.g., activation of the Notch signaling pathway, mediation of protein-protein interactions (e.g., between Brainiac-5 and Su(H) or a human homolog of Su(H)), and/or binding of an antibody specific to Brainiac-5).
  • fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nt in length which are useful, for example, as diagnostic probes and primers as discussed herein.
  • fragments 50-300 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figures 1A and IB (SEQ ID NO: 1).
  • a fragment at least 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figures 1A and IB (SEQ ID NO:l).
  • “about” in the phrase “at least about” is meant the recited value and values that are larger or smaller by several, a few, a small number, 5, 4, 3, 2 or 1.
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the Brainiac-5 polypeptides as identified in Figure 3, and described in more detail below.
  • Brainiac-5 functional activity is meant, for example, activation of the Notch signaling pathway, mediation of protein-protein interactions (e.g., between Brainiac-5 and Su(H) or a human homolog of Su(H)), and/or binding of an antibody specific to Brainiac-5.
  • Brainiac-5 polypeptides and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123.
  • physiological correlates of Brainiac-5 binding to its substrates can be assayed.
  • polynucleotides of the invention encode a polypeptide comprising, or alternatively consisting of, amino acids 7 to 20, 7 to 33, 61 to 83, 105 to 119, 139 to 148, 160 to 171, 187 to 196 of SEQ ID NO:2.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the polynucleotides of the invention encode one, two, three, four or more functional attributes of Brainiac-5.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of Brainiac-5.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to determine regions of Brainiac-5 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or IV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Figure 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures 1 A and IB.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson-Wolf regions of high antigenic index.
  • Tyr 65 A A -0.73 0.51 . -0.60 0.35
  • Trp 71 A A -0.59 1.26 * -0.60 0.24
  • Trp 90 A A -1.10 1.27 . -0.60 0.27
  • Trp 249 A A B -0.44 1.31 * -0.60 0.24
  • Trp 252 A A B -0.92 1.50 * * -0.60 0.26
  • fragments in this regard are those that comprise, or alternatively consist of, regions of Brainiac-5 that combine several structural features, such as several of the features set out above.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the sequence complementary to the coding sequence and/or noncoding sequence depicted in SEQ ID NO:l, the Brainiac-5 cDNA clone contained in ATCC Deposit No. 203572, or fragments (such as, for example, the open reading frame or a fragment thereof) of these sequences, as described herein.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • a portion of a polynucleotide of "at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in Figures 1A and IB (SEQ ID NO:l).
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly (A) tract of the Brainiac-5 cDNA shown in Figures 1 A and IB (SEQ ID NO: 1), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double- stranded cDNA clone).
  • nucleic acid molecules of the present invention which encode a Brainiac-5 polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; and the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences.
  • nucleic acids of the invention are the above polypeptide sequences together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • additional, non-coding sequences including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide or which may function in secretion of the fused polypeptide from a cell.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described by Gentz and colleagues (Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson and coworkers (Cell 37:767 (1984)).
  • other such fusion proteins include the Brainiac-5 polypeptides fused to Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the Brainiac-5 polypeptides. Variants may occur naturally, such as a natural allelic variant.
  • an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to ohgonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res.
  • cassette mutagenesis see e.g., Wells et al, Gene 34:315 (1985)
  • restriction selection mutagenesis see e.g., Wells er al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).
  • Such variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Brainiac-5 polypeptides or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • Additional embodiments of the invention are directed to isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide (e.g., a Brainiac-5 fragment described herein) having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions, 10-20 conservative amino acid substitutions, 5-10 conservative amino acid substitutions, 1-5 conservative amino acid substitutions, 3-5 conservative amino acid substitutions, or 1-3 conservative amino acid substitutions.
  • a Brainiac-5 polypeptide e.g., a Brainiac-5 fragment described herein
  • a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide to have an amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • nucleic acid molecules encoding a mature polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the mature Brainiac-5 amino acid sequence encoded by the deposited cDNA clone.
  • one embodiment of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the Brainiac-5 polypeptide having the complete amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to 278 of SEQ ID NO:2); (b) a nucleotide sequence encoding the Brainiac-5 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
  • nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 92%, 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c) or (d), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c) or (d), above.
  • This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
  • An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Brainiac-5 polypeptide having an amino acid sequence in (a), (b) or (c), above.
  • a further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions.
  • a polynucleotide which encodes the amino acid sequence of a Brainiac-5 polypeptide to have an amino acid sequence which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Brainiac-5 polypeptides or peptides by recombinant techniques.
  • a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a Brainiac-5 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequences encoding the Brainiac-5 polypeptides.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequences shown in Figures 1A and IB or to the nucleotides sequence of the deposited cDNA clone
  • Bestfit program uses the local homology algorithm of Smith and Waterman to find the best segment of homology between two sequences (Advances in Applied Mathematics 2:482-489 (1981)).
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)).
  • RNA sequence In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
  • nucleic acid molecules comprising or alternatively consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A and IB
  • nucleic acid sequence of the deposited cDNA or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having Brainiac-5 activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having Brainiac-5 activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having Brainiac-5 activity include, inter alia, (1) isolating the Brainiac-5 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the Brainiac-5 gene, as described by Verma and colleagues (Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988)); and Northern Blot analysis for detecting Brainiac-5 mRNA expression in specific tissues.
  • FISH in situ hybridization
  • nucleic acid molecules having (i.e., comprising, or alternatively consisting of, sequences at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1A and IB (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having Brainiac-5 polypeptide functional activity.
  • a polypeptide having Brainiac-5 polypeptide functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the mature Brainiac-5 polypeptide of the invention, as measured in a particular biological assay.
  • the Brainiac-5 polypeptides of the present invention modulate cellular growth and differentiation.
  • biological activity of Brainiac-5 polypeptides can be examined in organ culture assays or in colony assay systems in agarose culture. Stimulation or inhibition of cellular proliferation may be measured by a variety of assays.
  • For observing cell growth inhibition one can use a solid or liquid medium. In a solid medium, cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In a liquid medium, growth inhibition can be screened by measuring culture broth turbidity or incorporation of labeled thymidine in DNA.
  • the incorporation of a nucleoside analog into newly synthesized DNA is employed to measure proliferation (i.e., active cell growth) in a population of cells.
  • proliferation i.e., active cell growth
  • bromodeoxyuridine BrdU
  • anti-BrdU mouse monoclonal antibody clone BMC 9318 IgG,
  • This antibody binds only to cells containing DNA which has incorporated bromodeoxyuridine.
  • detection methods may be used in conjunction with this assay including immunofluorescence, immunohistochemical, ELISA, and colorimetric methods.
  • Kits that include bromodeoxyuridine (BrdU) and anti-BrdU mouse monoclonal antibody are commercially available from Boehringer Mannheim (Indianapolis, IN).
  • the effect upon cellular differentiation can be measured by contacting embryonic cells with various amounts of a Brainiac-5 polypeptide and observing the effect upon differentiation of the embryonic cells. Tissue-specific antibodies and microscopy may be used to identify the resulting cells.
  • Brainiac-5 polypeptides modulate immune and/or nervous system cell proliferation and differentiation in a dose-dependent manner in the above-described assays.
  • a polypeptide having Brainiac-5 polypeptide activity includes polypeptides that also exhibit any of the same growth and differentiation regulating activities in the above-described assays in a dose-dependent manner.
  • a polypeptide having Brainiac-5 polypeptide activity will exhibit substantially similar dose-dependence in a given activity as compared to the Brainiac-5 polypeptide (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity relative to the reference Brainiac-5 polypeptide).
  • nucleic acid molecules having a sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in Figures 1A and IB (SEQ ID NO: l) will encode a polypeptide "having Brainiac-5 polypeptide functional activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of Brainiac-5 polypeptides or fragments thereof by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli,
  • Streptomyces and Salmonella typhimu ⁇ um cells
  • fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells.
  • yeast cells e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, 293 and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Vectors preferred for use in bacteria include pHE4-5 (ATCC Accession No.
  • pQE70, pQE60 and pQE-9 QIAGEN, Inc., supra
  • pBS vectors Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, CA).
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl, and pSG (Stratagene); and pSVK3, pBPV, pMSG and pSVL (Pharmacia).
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • yeast Pichia pastoris is used to express Brainiac-5 protein in a eukaryotic system.
  • Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
  • a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 . Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXI) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S.B., et al, Mol. Cell. Biol.
  • a heterologous coding sequence such as, for example, a Brainiac-5 polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOXI regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
  • the plasmid vector pPIC9K is used to express DNA encoding a Brainiac-5 polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998.
  • This expression vector allows expression and secretion of a Brainiac-5 protein of the invention by virtue of the strong AOXI promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
  • PHO alkaline phosphatase
  • yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
  • high-level expression of a heterologous coding sequence such as, for example, a Brainiac-5 polynucleotide of the present invention
  • a heterologous coding sequence such as, for example, a Brainiac-5 polynucleotide of the present invention
  • an expression vector such as, for example, pGAPZ or pGAPZalpha
  • Enhancers are -acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman (Cell 23:175 (1981)), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., Brainiac-5 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with Brainiac-5 polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous Brainiac-5 polynucleotides.
  • endogenous genetic material e.g., Brainiac-5 coding sequence
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous control regions e.g., promoter and/or enhancer
  • endogenous Brainiac-5 polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci.
  • the host cells described infra can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • cell-free translation systems can also be employed to produce the polypeptides of the invention using RNAs derived from the DNA constructs of the present invention.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • a preferred fusion protein comprises a heterologous region from immunoglobuhn that is useful to stabilize and purify polypeptides.
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobuhn molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (Bennett, D., et al, J. Molecular Recognition 8:52-58 (1995); Johanson, K., et al, J. Biol Chem. 270:9459-9471 (1995)).
  • polynucleotides encoding Brainiac-5 polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein inco ⁇ orated by reference in their entireties.
  • a preferred fusion protein of the invention comprises a heterologous region from immunoglobuhn that is useful to stabilize and purify proteins.
  • EP-A-O 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobuhn molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins, such as hIL-5 has been fused with Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 5:52-58 (1995) and K. Johanson et al, J. Biol Chem. 270:9459-9471 (1995).
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • Polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310: 105-111).
  • a peptide corresponding to a fragment of the complete Brainiac-5 polypeptides of the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the Brainiac-5 polynucleotide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b- alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca- methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the
  • the invention encompasses Brainiac-5 polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of
  • polypeptides of the invention may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • polypeptides of the invention may be modified by iodination.
  • Brainiac-5 polypeptides of the invention may also be labeled with biotin.
  • biotinylated Brainiac-5 polypeptides of the invention may be used, for example, as an imaging agent or as a means of identifying one or more Brainiac-5 receptor(s) or other coreceptor or coligand molecules.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art, e.g., EP 0401 384, herein inco ⁇ orated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • Preferred for therapeutic pu ⁇ oses is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol As an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • the Brainiac-5 polypeptides of the invention can be recovered and purified by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the invention further provides an isolated Brainiac-5 polypeptide having (i.e., comprising, or alternatively consisting of) the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide or polypeptide comprising a portion of the above polypeptides.
  • the Brainiac-5 polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the Brainiac-5 polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
  • the polypeptides of the invention are monomers, dimers, trimers or tetramers.
  • the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
  • Multimers encompassed by the invention may be homomers or heteromers.
  • the term homomer refers to a multimer containing only Brainiac-5 polypeptides of the invention (including Brainiac-5 fragments, variants, and fusion proteins, as described herein). These homomers may contain Brainiac-5 polypeptides having identical or different amino acid sequences.
  • a homomer of the invention is a multimer containing only Brainiac-5 polypeptides having an identical amino acid sequence.
  • a homomer of the invention is a multimer containing Brainiac-5 polypeptides having different amino acid sequences.
  • the multimer of the invention is a homodimer (e.g., containing Brainiac-5 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing Brainiac-5 polypeptides having identical or different amino acid sequences).
  • the multimer of the invention is a homotrimer.
  • the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • heteromer refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the Brainiac-5 polypeptides of the invention.
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
  • the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
  • Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers of the invention such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
  • multimers of the invention are formed by covalent associations with and/or between the Brainiac-5 polypeptides of the invention.
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:2 or contained in the polypeptide encoded by the clone deposited in connection with this application).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a Brainiac-5 fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a Brainiac-5-Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No.
  • WO 98/49305 the contents of which are herein inco ⁇ orated by reference in its entirety).
  • two or more Brainiac-5 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby inco ⁇ orated by reference). Proteins comprising multiple Brainiac-5 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • Brainiac-5 polypeptides of the invention involves use of Brainiac-5 polypeptides fused to a leucine zipper polypeptide sequence.
  • Leucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins.
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric Brainiac-5 proteins are those described in PCT application WO 94/10308, hereby inco ⁇ orated by reference.
  • Recombinant fusion proteins comprising a soluble Brainiac-5 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric Brainiac-5 is recovered from the culture supernatant using techniques known in the art.
  • Trimeric Brainiac-5 may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby inco ⁇ orated by reference.
  • Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric Brainiac-5.
  • proteins of the invention are associated by interactions between the Flag® polypeptide sequence contained in Flag®-Brainiac-5 fusion proteins of the invention. In a further embodiment, proteins of the invention are associated by interactions between the heterologous polypeptide sequence contained in Flag®-Brainiac-5 fusion proteins of the invention and anti-Flag® antibody.
  • the multimers of the invention may be generated using chemical techniques known in the art.
  • polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety). Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art.
  • polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be inco ⁇ orated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • the invention provides an isolated Brainiac-5 polypeptide having (i.e., comprising, or alternatively consisting of) the amino acid sequence encoded by the cDNA clone contained in ATCC No. 203572, or the amino acid sequence in Figures 1A and IB (SEQ ID NO:2), or a peptide or polypeptide comprising a portion (i.e., a fragment) of the above polypeptides.
  • Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2, encoded by the cDNA contained in the plasmid having ATCC accession number 203572, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or the complementary strand of the nucleotide sequence shown in Figures 1A-B (SEQ ID NO:l).
  • Polypeptide fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments that comprise or alternatively, consist of, from about amino acid residues: 1 to 15, 16-30, 31-46, 47-55, 56-72, 73-104, 105-163, 163-188, 186-210 and 210-278 of the amino acid sequence disclosed in SEQ ID NO:2.
  • polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context, "about” means several, a few, 5, 4, 3, 2 or 1. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
  • Additional polypeptide fragments of the invention comprise, or alternatively consist of, amino acids 7 to 20, 7 to 33, 61 to 83, 105 to 119, 139 to 148, 160 to 171, 187 to 196 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • Brainiac-5 polypeptide fragments comprising, or alternatively consisting of, one, two, three, four, five or more functional regions of polypeptides of the invention, such as the Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Ka ⁇ lus-Schulz flexible regions, Emini surface-forming regions and Jameson-Wolf regions of high antigenic index set out in Figures 3 and in Table I and as described herein.
  • functional regions of polypeptides of the invention such as the Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic
  • polypeptide fragments of the invention are antigenic.
  • the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to routinely determine regions of Brainiac-5 which exhibit a high degree of potential for antigenicity.
  • Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or IV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response (e.g., a polypeptide comprising amino acid residues from about Val-1 to about Val-11 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Thr- 14 to about Gln-22 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Val-34 to about His-53 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Phe-94 to about Val- 108 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Ala- 120 to about Gin- 126 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Arg- 138 to about He- 149 in S
  • fragments of the invention are those that comprise regions of Brainiac-5 that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a polypeptide that elicits an antibody response when the complete or whole polypeptide is the immunogen.
  • an antigenic epitope a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes (see, for instance, Geysen, et al, Proc. Natl Acad. Sci. USA 81:3998-4002 (1983)).
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a polypeptide molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a polypeptide sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked polypeptide (see, for instance, Sutcliffe, J. G., et al, Science 219:660-666 (1983)).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a polypeptide, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact polypeptides (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • 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 (see, for instance, Wilson, et al, Cell 37:767-778 (1984)).
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Brainiac-5-specific antibodies include: a polypeptide comprising amino acid residues from about Val-1 to about Val-11 in SEQ ID NO:2; from about Thr- 14 to about Gln-22 in SEQ ID NO:2; from about Val-34 to about His-53 in SEQ ID NO:2; from about Phe-94 to about Val- 108 in SEQ ID NO:2; from about Ala- 120 to about Gln-126 in SEQ ID NO:2; from about Arg- 138 to about He- 149 in SEQ ID NO:2; ; from about Leu-202 to about Ala-211 in SEQ ID NO:2; and from about Phe-274 to about Ser-278 in SEQ ID NO:2.
  • polypeptide fragments have been determined to bear antigenic epitopes of the Brainiac-5 polypeptide by the analysis of the Jameson-Wolf antigenic index, as shown in Figure 3 and/or Table I, above.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means (see, for example, Houghten, R. A., et al, Proc. Natl Acad. Sci. USA 52:5131-5135 (1985); and U.S. Patent No. 4,631,211 to Houghten, et al. (1986)).
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art (see, for instance, Sutchffe, et al, supra; Wilson, et al, supra; Chow, M., et al, Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J., et al, J. Gen. Virol. 66:2347-2354 (1985)).
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art (see, for instance, Geysen, et al, supra). Further still, U.S. Patent No. 5,194,392, issued to Geysen, describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No.
  • Brainiac-5 polypeptide-specific antibodies for use in the present invention can be raised against the intact Brainiac-5 polypeptide or an antigenic polypeptide fragment thereof, which may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to Brainiac-5 polypeptides.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl, et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the Brainiac-5 polypeptides or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of Brainiac-5 polypeptide is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or Brainiac-5 polypeptide-binding fragments thereof).
  • monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al, Nature 256:495 (1975); Kohler, et al, Eur. J. Immunol. 6:511 (1976); Kohler, et al, Eur. J. Immunol. 6:292 (1976); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681)).
  • such procedures involve immunizing an animal (preferably a mouse) with a Brainiac-5 polypeptide antigen or, more preferably, with a Brainiac-5 polypeptide-expressing cell.
  • Suitable cells can be recognized by their capacity to bind anti-Brainiac-5 polypeptide antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C), and supplemented with about 10 ⁇ g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • a suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Manassas, Virginia.
  • SP2O parent myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands and colleagues (Gastroenterology 80:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the Brainiac-5 polypeptide antigen.
  • Brainiac-5 polypeptide-specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the Brainiac-5 polypeptide-specific antibody can be blocked by the Brainiac-5 polypeptide antigen.
  • Such antibodies comprise anti-idiotypic antibodies to the Brainiac-5 polypeptide-specific antibody and can be used to immunize an animal to induce formation of further Brainiac-5 polypeptide-specific antibodies.
  • Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • Brainiac-5 polypeptide-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • chimeric monoclonal antibodies For in vivo use of anti-Brainiac-5 in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in deposited clone 203572 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO: 1 or contained in deposited clone HOGCC45 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:l), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
  • Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutchffe et al., Science 219:660-666 (1983)).
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutchffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
  • a preferred immunogenic epitope includes the secreted protein.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutchffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • CH2, CH3, or any combination thereof and portions thereof resulting in chimeric polypeptides.
  • Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins.
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
  • Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • HA hemagglutinin
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the invention provides a peptide or polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide of the invention.
  • polypeptides or polypeptides are also encompassed by the invention.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 57:3998- 4002 (1983).
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutchffe, J. G., Shinnick, T. M., Green,
  • 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.
  • 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. See, for instance, Wilson et al, Cell 37:1 1 -lie) (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least six, at least seven, at least eight, more preferably at least nine, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Brainiac-5-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Val-1 to about Val-11 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-14 to about Gln-22 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Val-34 to about His-53 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-94 to about Val-108 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ala-120 to about Gln-126 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Arg- 138 to about He- 149 in S
  • polypeptide fragments have been determined to bear antigenic epitopes of the Brainiac-5 polypeptide by the analysis of the Jameson-Wolf antigenic index, as shown in Figure 3 and Table I, above.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • “about” is meant, the particularly recited ranges and ranges larger or smaller at the N- and/or C-terminus by several, a few, 5, 4, 3, 2 or 1 residue.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. See, e.g., Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 52:5131-5135; this "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U. S. Patent No. 4,631,211 to Houghten et al. (1986). Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art.
  • SMPS Simultaneous Multiple Peptide Synthesis
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. See, for instance, Geysen et al., supra. Further still, U.S. Patent No.
  • polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI CH2, CH3, and any combination thereof, including both entire domains and portions thereof
  • chimeric polypeptides facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker et al, Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric Brainiac-5 polypeptides or polypeptide fragments alone (Fountoulakis et al, J. Biochem. 270:3958-3964 (1995)).
  • Brainiac-5 polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunecker, et al, Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric Brainiac-5 polypeptide or polypeptide fragment alone (Fountoulakis, et al, J. Biochem. 270:3958-3964 (1995)).
  • the Brainiac-5 polypeptides of the present invention and the epitope-bearing fragments thereof are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid).
  • a heterologous antigen e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid.
  • the heterologous antigen is an immunogen.
  • the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling may be employed to modulate the activities of Brainiac-5 thereby effectively generating agonists and antagonists of Brainiac-5. See generally, U.S. Patent Nos.
  • alteration of Brainiac-5 polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments into a desired Brainiac-5 molecule by homologous, or site-specific, recombination.
  • Brainiac-5 polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of Brainiac- 5 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No.
  • WO 96/14328 AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (International Publication No. WO 98/07880), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No.
  • the heterologous molecules are any member of the TNF family.
  • Brainiac-5 polypeptides protein engineering may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant polypeptides or muteins including single or multiple amino acid substitutions, deletions, additions or fusion polypeptides.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • the Brainiac-5 polypeptide of the invention is a member of the Brainiac polypeptide family
  • deletions of N-terminal amino acids up to the arginine at position 8 of SEQ ID NO:2 may retain some biological activity such as the ability to modulate cell growth and differentiation.
  • Polypeptides having further N- terminal deletions including the arginine-8 residue in SEQ ID NO: 2 may not retain such biological activities, or may exhibit an alterred biological activity, because it is known that this residue in a Brainiac -related polypeptide is in the beginning of the conserved domain believed to be required for biological activities.
  • the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature form of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form of the polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the Brainiac-5 polypeptide shown in SEQ ID NO:2, up to the arginine residue at position number 8, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-278 of SEQ ID NO:2, where n 1 is an integer in the range of 1 to 8, and 9 is the position of the first residue from the N-terminus of the complete Brainiac-5 polypeptide (shown in SEQ ID NO:2) believed to be required for modulation of cell growth and differentiation activity of the Brainiac-5 polypeptide.
  • the invention provides nucleic acid molecules encoding polypeptides having (i.e., comprising, or alternatively consisting of) the amino acid sequence of residues of 1-278; 2-278; 3-278; 4-278; 5-278; 6-278; 7-278; or 8-278 of SEQ ID NO:2.
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above nucleic acid molecule sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules are also provided. Similarly, many examples of biologically functional C-terminal deletion muteins are known. For instance, Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli, et al, J. Biotechnology 7: 199-216 (1988)).
  • the Brainiac-5 polypeptide of the invention is a member of the Brainiac polypeptide family
  • deletions of C-terminal amino acids up to the cysteine at position 263 of SEQ ID NO:2 may retain some biological activity such as the ability to modulate cell growth and differentiation.
  • Polypeptides having further C-terminal deletions including the cysteine residue at position 263 of SEQ ID NO: 2 may not retain such biological activities because this residue is in the beginning of the conserved domain required for biological activities.
  • deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained.
  • the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature form of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the Brainiac-5 polypeptide shown in SEQ ID NO:2, up to the cysteine residue at position 263 of SEQ ID NO:2, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides having (i.e., comprising, or alternatively consisting of) the amino acid sequence of residues 1-m 1 of the amino acid sequence in SEQ ID NO:2, where m 1 is any integer in the range of 263 to 278, and residue 262 is the position of the first residue from the C- terminus of the complete Brainiac-5 polypeptide (shown in SEQ ID NO:2) believed to be required for the cell growth and differentiation modulatory activities of the Brainiac-5 polypeptide.
  • the invention provides nucleic acid molecules encoding polypeptides having (i.e., comprising, or alternatively consisting of) the amino acid sequence of residues 1-278; 1-277; 1-276; 1-275; 1-274; 1-273; 1-272; 1-271; 1-270; 1-269; 1-268; 1-267; 1-266; 1-265; 1-264; or 1-263 of SEQ ID NO:2.
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules are also are provided.
  • the invention also provides nucleic acid molecules encoding polypeptides having (i.e., comprising, or alternatively consisting of) one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n'-m 1 of SEQ ID NO:2, where n 1 and m 1 are integers as described above e.g., a polypeptide comprising, or alternatively consisting of, amino acids 8 to 263 of SEQ ID NO:2).
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.
  • nucleotide sequence encoding a polypeptide consisting of a portion of the complete Brainiac-5 amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 203572, where this portion excludes any integer from 1 to about 7 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 203572, or any integer from 1 to about 15 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 203572.
  • “about” means the recited value and values that are larger or smaller by several, a few, a small number, 5, 4, 3, 2 or 1.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a Brainiac-5 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six Brainiac-5 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the Brainiac-5 amino acid sequence shown in Figures 1A and IB (i.e., SEQ ID NO:2), up to the proline residue at position number 273 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues n 2 -278 of Figures 1A and IB (SEQ ID NO:2), where n 2 is an integer in the range of 2 to 273, and 273 is the position of the first residue from the N-terminus of the complete Brainiac-5 polypeptide believed to be required for at least immunogenic activity of the Brainiac-5 polypeptide.
  • the invention provides nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of A-2 to S-278; E-3 to S-278; D-4 to S-278; F-5 to S-278; E-6 to S-278; R-7 to S-278; R-8 to S-278; Q-9 to S-278; A-10 to S-278; V-l 1 to S-278; R-12 to S-278; Q-13 to S-278; T-14 to S-278; W-15 to S-278; G-16 to S-278; A-17 to S-278; E-18 to S-278; G-19 to S-278; R-20 to S-278; V-21 to S-278; Q-22 to S-278; G-23 to S-278; A-24 to S-278; L-25 to S-278; V-26 to S-278; R-27 to S-278; R-28 to S-278; V-29 to S-278; F-30 to S-278; L-31 to S-278;
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.
  • Brainiac-5 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six Brainiac-5 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the Brainiac-5 polypeptide shown in Figures 1A and IB (SEQ ID NO:2), up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising the amino acid sequence of residues 1-m 2 of Figures 1A and IB (SEQ ID NO:2), where ⁇ r is an integer in the range of 6 to 278, and 6 is the position of the first residue from the C-terminus of the complete Brainiac-5 polypeptide believed to be required for at least immunogenic activity of the Brainiac-5 polypeptide.
  • the invention provides nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues V-l to D-277; V-l to W-276; V-l to Q-275; V-l to F-274; V-l to P-273; V-l to G-272; V-l to A-271; V-l to A-270; V-l to V-269; V-l to P-268; V-l to Q-267; V-l to P-266; V-l to H-265; V-l to A-264; V-l to C-263; V-l to A-262; V-l to P-261; V-l to G-260; V-l to H-259; V-l to P-258; V-l to G-257; V-l to H-256; V-l to L-255; V-l to L-254; V-l to R-253; V-l to W-252; V-l to M-251; V-l
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molcules are also provided.
  • the invention also provides nucleic acid molcules encoding polypeptides having (i.e., comprising, or alternatively consisting of) one or more amino acids deleted from both the amino and the carboxyl termini of a Brainiac-5 polypeptide, which may be described generally as having residues n 2 -m 2 of Figures 1A and IB (SEQ ID NO:2), where n 2 and m 2 are integers as described above.
  • the present application is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules are also provided by the invention.
  • the invention further includes variations of the Brainiac-5 polypeptides which show substantial Brainiac-5 polypeptide activity or which include regions of Brainiac-5 polypeptides such as the polypeptide portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity.
  • guidance concerning how to make phenotypically silent amino acid substitutions is provided wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change (Bowie, J. U., et al, Science 247:1306-1310 (1990)),.
  • the first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.
  • the second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • the fragment, derivative or analog of the polypeptide of SEQ ID NO:2, or those encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which either the Brainiac-5 mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a proprotein sequence.
  • the Brainiac-5 polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table II).
  • Amino acids in the Brainiac-5 polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro proliferative activity.
  • Replacement of amino acids can also change the selectivity of the binding of a ligand to cell surface receptors (for example, Ostade, et al, Nature 361:266-268 (1993)) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors.
  • Sites that are critical for ligand- receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al, J. Mol. Biol. 224:899-904 (1992); de Vos, et al. Science 255:306-312 (1992)).
  • the invention also encompasses Brainiac-5 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate Brainiac-5 polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • amino acid residues of the polypeptides of the invention may be deleted or substituted with another residue to elminate undesired processing by proteases such as, for example, furins or kexins.
  • the polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. Recombinantly produced versions of the Brainiac-5 polypeptides can be substantially purified by the one-step method described by Smith and Johnson (Gene 67:31-40 (1988)).
  • Polypeptides of the invention also can be purified from natural or recombinant sources using anti- Brainiac-5 antibodies of the invention in methods which are well known in the art of protein purification.
  • Recombinant DNA technology known to those skilled in the art (see, for instance, DNA shuffling supra) can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the co ⁇ esponding natural polypeptide, at least under certain purification and storage conditions.
  • the invention also provides an isolated Brainiac-5 polypeptide comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the Brainiac-5 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 (i.e., positions 1-278 of SEQ ID NO:2); (b) the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit No. 203572; (c) the complete amino acid sequence of the predicted mature Brainiac-5 polypeptide encoded by the cDNA clone contained in the ATCC Deposit No. 203572.
  • polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 92%, 95%, 96%, 97%, 98% or 99% identical to those described in (a), (b) or (c), above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above.
  • polypeptides of the present invention include polypeptides which have (i.e., comprise, or alternatively consist of) at least 90% similarity, more preferably at least 92% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above.
  • polypeptides of the invention also comprise those which are at least 80% identical, more preferably at least 90%, 92% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA or to the polypeptide of SEQ ID NO:2, and also include portions of such polypeptides with at least 15 amino acids, more preferably at least 30 amino acids, even more preferably at least 40 amino acids, still even more preferably at least 50 amino acids, still more preferably at least 60 amino acids, and yet even more preferably at least 75 amino acids.
  • a further embodiment of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of a Brainiac-5 polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions.
  • a peptide or polypeptide it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of a Brainiac-5 polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-278 or 261 to the end of the coding region of SEQ ID NO:2 or to a polypeptide expressed from the deposited cDNA clone which expresses Brainiac-5.
  • polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino acids in length.
  • “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • the invention also provides an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10, 30 or 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:2.
  • % similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a Brainiac-5 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the Brainiac-5 polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • whether any particular polypeptide is at least 90%, 92%,
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the pu ⁇ oses of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
  • polypeptide of the present invention could be used, for example, as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting Brainiac-5 polypeptide expression as described below or as agonists and antagonists capable of enhancing or inhibiting Brainiac-5 polypeptide function.
  • polypeptides can be used in the yeast two-hybrid system to "capture" Brainiac-5 polypeptide-binding polypeptides which are also candidate agonists and antagonists according to the present invention.
  • the yeast two hybrid system is described by Fields and Song (Nature 340:245-246 (1989)).
  • polypeptides of the invention can also be expressed in transgenic animals.
  • mice Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
  • transgene i.e., polynucleotides of the invention
  • transgene i.e., polynucleotides of the invention
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson, et al, Appl Microbiol Biotechnol. 40:691-698 (1994);
  • transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810- 813 (1997)).
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic or chimeric animals.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the pu ⁇ ose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
  • Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of Brainiac-5 polypeptides, studying conditions and/or disorders associated with aberrant Brainiac-5 expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the
  • the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
  • the cells can be inco ⁇ orated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
  • genetically engineered fibroblasts can be implanted as part of a skin graft
  • genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
  • the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • Antibodies The present invention further relates to antibodies and T-cell antigen receptors
  • TCR which immunospecifically bind a polypeptide, preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobuhn molecules and immunologically active portions of immunoglobuhn molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobuhn molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobuhn molecule.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobuhn and include antibodies isolated from human immunoglobuhn libraries or from animals transgenic for one or more human immunoglobuhn and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681 ; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148: 1547-1553 (1992).
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention that they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
  • Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies that bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5X 10 "2 M, 10 "2 M, 5X 10 "3 M, 10 " M, 5X 10 "4 M, 10 " 4 M, 5X10 “5 M, 10 "5 M, 5X10 “6 M, 10 “6 M, 5X10 "7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 “9 M, 10 " 9 M, 5X10 10 M, 10 10 M, 5X10"M, 10"M, 5X10 12 M, 10 I2 M, 5X10 I3 M, 10 13 M, 5X10 " 14 M, 10 14 M, 5X10 ',5 M, and 10 15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • the invention features both receptor-specific antibodies and ligand-specific antibodies.
  • the invention also features receptor- specific antibodies which do not prevent ligand binding but prevent receptor activation.
  • Receptor activation i.e., signaling
  • receptor activation may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981- 1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161 (4): 1786- 1794 (1998); Zhu et al., Cancer Res.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (inco ⁇ orated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
  • the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references inco ⁇ orated by reference in their entireties).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobuhn molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulf ⁇ de stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobuhn constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are inco ⁇ orated herein by reference in their entireties.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and framework regions from a human immunoglobuhn molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805- 814 (1994); Roguska.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobuhn sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is inco ⁇ orated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobuhn genes.
  • the human heavy and light chain immunoglobuhn gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobuhn genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobuhn loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobuhn transgenes harbored by the transgenic mice rea ⁇ ange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti- idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized ohgonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping ohgonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those ohgonucleotides, and then amplification of the ligated ohgonucleotides by PCR.
  • chemically synthesized ohgonucleotides e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobuhn may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an ohgonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • a suitable source e.g., an antibody cDNA library, or
  • Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
  • the nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobuhn constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038- 1041). Methods of Producing Antibodies.
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transfe ⁇ ed to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobuhn molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, 1984, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the co ⁇ ect modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobuhn molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446- 2452(1991), which are inco ⁇ orated by reference in their entireties.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
  • polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
  • polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 1251, 1311, l l lln or 99Tc.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM- CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is inco ⁇ orated herein by reference in its entirety.
  • an antibody with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100,
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
  • One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
  • immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Cmrent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS- PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • labeled antigen e.g., 3H or 1251
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
  • a labeled compound e.g., 3H or 1251
  • the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the described disorders.
  • Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein).
  • the antibodies of the invention can be used to treat, inhibit or prevent diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, [insert diseases and disorders].
  • the treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
  • lymphokines or hematopoietic growth factors such as, e.g., IL-2, IL-3 and IL-7
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is prefe ⁇ ed. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 "2 M, 10 "2 M, 5X10 '3 M, 10 "3 M, 5X10 '4 M, 10 "4 M, 5X10 "5 M, 10 "5 M, 5X10 " 6 M, 10 "6 M, 5X10 "7 M, 10 “7 M, 5X10 '8 M, 10 “8 M, 5X10 "9 M, 10 “9 M, 5X10 '°M, 10 ,0 M, 5X10 ' ⁇ M, 10-"M, 5X10 12 M, 10 12 M, 5X10 I M, 10 13 M, 5X10 ,4 M, 10 14 M, 5X10 15 M, and 10 15 M.
  • nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
  • the nucleic acids produce their encoded protein that mediates a therapeutic effect.
  • the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
  • nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
  • nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl.
  • the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody. Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.
  • nucleic acid- ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.); WO93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)).
  • the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
  • a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Cu ⁇ . Opin. in Genetics and Devel. 3:110-114.
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus- based gene therapy.
  • adenovirus vectors are used.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No. 5,436,146).
  • Another approach to gene therapy involves transfe ⁇ ing a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transfe ⁇ ed gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • Such introduction can be ca ⁇ ied out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • Recombinant blood cells are preferably administered intravenously.
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
  • the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity.
  • the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
  • the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing
  • in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
  • the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
  • the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobuhn are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
  • Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable ca ⁇ ier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • ca ⁇ ier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical ca ⁇ iers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a prefe ⁇ ed ca ⁇ ier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid ca ⁇ iers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and ca ⁇ iers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical earners are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the compounds of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic pu ⁇ oses to detect, diagnose, or monitor diseases and/or disorders associated with the abe ⁇ ant expression and/or activity of a polypeptide of the invention.
  • the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of abe ⁇ ant expression.
  • the invention provides a diagnostic assay for diagnosising a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • a diagnostic assay for diagnosising a disorder comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as.iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as.iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
  • Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
  • monitoring of the disease or disorder is ca ⁇ ied out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050).
  • kits The present invention provides kits that can be used in the above methods.
  • a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
  • the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
  • kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
  • the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
  • a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
  • the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
  • a kit may include a control antibody that does not react with the polypeptide of interest.
  • a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
  • a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry).
  • the kit may include a recombinantly produced or chemically synthesized polypeptide antigen.
  • the polypeptide antigen of the kit may also be attached to a solid support.
  • the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
  • a kit may also include a non-attached reporter-labeled anti-human antibody.
  • binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
  • the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention.
  • the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
  • the antibody is attached to a solid support.
  • the antibody may be a monoclonal antibody.
  • the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
  • the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti- human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adso ⁇ tion of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
  • the invention provides an assay system or kit for ca ⁇ ying out this diagnostic method.
  • the kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
  • the invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Included are both receptor- specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.
  • the antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • These antibodies act as Brainiac-5 agonists as reflected in an increase in cellular proliferation in response to binding of Brainiac-5 to a Brainiac-5 receptor in the presence of these antibodies.
  • the above antibody agonists can be made using methods known in the art. See e.g., WO 96/40281; US Patent 5,811,097; Deng, B. et al., Blood 92(6): 1981-1988 (1998); Chen, Z. et al., Cancer Res. 58(16):3668- 3678 (1998); Ha ⁇ op, J.A. et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu, Z. et al., Cancer Res.
  • the antibodies of the invention fix complement.
  • the antibodies of the invention or fragments thereof are associated with heterologous polypeptides or nucleic acids (e.g. toxins, such as, compounds that bind and activate endogenous cytotoxic effecter systems, and radioisotopes; and cytotoxic prodrugs).
  • antibodies to the Brainiac-5 polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the Brainiac - 5, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1989), and Nissinoff, J. Immunol 147(8):2429-2438 (1991)).
  • antibodies which bind to Brainiac-5 and competitively inhibit the Brainiac-5 multimerization and/or binding to ligand can be used to generate anti- idiotypes that "mimic" the Brainiac-5 mutimerization and/or binding domain and, as a consequence, bind to and neutralize Brainiac-5 and/or its ligand.
  • Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize Brainiac-5 ligand.
  • anti-idiotypic antibodies can be used to bind Brainiac-5 on the surface of the cell, and thereby block Brainiac-5- mediated cellular activation, proliferation, and/or differentiation.
  • Brainiac-5 is expressed not only in ovarian tumor cells, but also (using BLAST analysis of the HGS EST database) in bone ma ⁇ ow stromal cells and synovial sarcoma cells.
  • substantially altered (increased or decreased) levels of Brainiac-5 gene expression can be detected in immune and/or nervous system tissue or other cells or bodily fluids (e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" Brainiac-5 gene expression level, that is, the Brainiac-5 expression levels in immune and/or nervous system tissues or bodily fluids from an individual not having the immune and/or nervous system disorder.
  • bodily fluids e.g., sera, plasma, urine, synovial fluid or spinal fluid
  • the invention provides a diagnostic method useful during diagnosis of a immune and/or nervous system disorder, which involves measuring the expression level of the gene encoding the Brainiac-5 polypeptides in immune and/or nervous system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard Brainiac-5 gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune and/or nervous system disorder.
  • Brainiac-5 polypeptides and mRNA encoding the Brainiac-5 polypeptides when compared to a co ⁇ esponding "standard" level.
  • enhanced levels of the Brainiac-5 polypeptides can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with such a cancer when compared to sera from mammals of the same species not having the cancer.
  • cancers that may be treated, prevented, and/or diagnosed using Brainiac-5 polynucleotides, polypeptides, and/or agonists and/or antagonists of the invention include, but are not limited to, leukemia; acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (specific examples thereof include myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)); chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, and chronic lymphocytic leukemia); polycythemia vera; lymphoma (e.g., Hodgkin's disease, and non-Hodgkin's disease); multiple myeloma; Waldenstrom's macroblogulinemia; heavy chain disease; and/or solid tumors (e.g., sarcomas and carcinomas (specific
  • the invention provides a diagnostic method useful during diagnosis of an immune and/or nervous system disorder, including cancers of these systems, which involves measuring the expression level of the gene encoding the Brainiac-5 polypeptides in immune and/or nervous system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard Brainiac-5 gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune and/or nervous system disorder.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed Brainiac-5 gene expression will experience a worse clinical outcome relative to patients expressing the genes at a level nearer the standard level.
  • testing the expression level of the genes encoding the Brainiac-5 polypeptides is intended qualitatively or quantitatively measuring or estimating the level of the Brainiac-5 polypeptides or the level of the mRNA encoding the Brainiac-5 polypeptides in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the Brainiac-5 polypeptide levels or mRNA levels in a second biological sample).
  • the Brainiac-5 polypeptides level or mRNA level in the first biological sample is measured or estimated and compared to a standard Brainiac-5 polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder of the immune and/or nervous systems.
  • a standard Brainiac-5 polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains Brainiac-5 polypeptides or mRNA.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain free Brainiac-5 polypeptides, immune and/or nervous system tissue, and other tissue sources found to express complete or mature Brainiac-5 polypeptides or a Brainiac-5 receptor.
  • body fluids such as sera, plasma, urine, synovial fluid and spinal fluid
  • tissue sources found to express complete or mature Brainiac-5 polypeptides or a Brainiac-5 receptor.
  • the present invention is useful for treating, preventing, and/or diagnosing various immune and/or nervous system-related disorders in mammals, preferably humans.
  • mammals preferably humans.
  • a nonexclusive list of preferred mammals includes monkeys, apes, cats, dogs, cows, pigs, horses, rabbits, and humans. Humans are particularly prefe ⁇ ed mammals.
  • Such disorders include any disregulation of immune and/or nervous system cell and/or tissue function including, but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses , autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception, neuronal survival; synapse formation; conductance; neural differentiation, autoimmunity, arthritis, leukemias, lymphomas, immunosuppression, immunity, humoral immunity, inflammatory bowel disease, myelosuppression, lymphoproliferative disorders, in the maintenance and differentiation of various hematopoietic lineages from early hematopoietic stem and committed progenitor cells, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, bone ma ⁇ ow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution
  • Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (Anal Biochem. 162: 156-159 (1987)). Levels of mRNA encoding the Brainiac-5 polypeptides are then assayed using any appropriate method. These include Northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription in combination with the polymerase chain reaction
  • RT-LCR reverse transcription in combination with the ligase chain reaction
  • Brainiac-5 polypeptide levels in a biological sample can occur using antibody-based techniques.
  • Brainiac-5 polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol. 101:976-985 (1985); Jalkanen, ML, et al, J. Cell. Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting Brainiac-5 gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 1 ,2 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 1 ,2 In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • Brainiac-5 polypeptides can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of Brainiac-5 polypeptides include those detect
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a Brainiac-5-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 1 1 I, u2 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder.
  • a radioisotope for example, 1 1 I, u2 In, 99m Tc
  • a radio-opaque substance for example, parenterally, subcutaneously or intraperitoneally
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain Brainiac-5 polypeptides.
  • In vivo tumor imaging is described by Burchiel and coworkers (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S. W. and Rhodes, B. A., eds., Masson Publishing Inc. (1982)).
  • Brainiac-5 polynucleotides and polypeptides are useful for diagnosis of conditions involving abnormally high or low expression of Brainiac-5 activities. Given the cells and tissues where Brainiac-5 polypeptides are expressed as well as the activities modulated by Brainiac-5 polypeptides, it is readily apparent that a substantially altered (increased or decreased) level of expression of Brainiac-5 polypeptides in an individual compared to the standard or "normal" level produces pathological conditions related to the bodily system(s) in which Brainiac-5 polypeptides are expressed and/or is active.
  • cellular receptor molecules may also often be exploited by a virus as a means of initiating entry into a potential host cell.
  • a virus for example, it was recently discovered by Wu and colleagues (J. Exp. Med. 185: 1681-1691 (1997)) that the cellular chemokine receptor CCR5 functions not only as a cellular chemokine receptor, but also as a receptor for macrophage-tropic human immunodeficiency virus (HIV)-l.
  • HIV human immunodeficiency virus
  • the invention also provides a method of treating, preventing, and/or diagnosing an individual exposed to, or infected with, a virus through the prophylactic or therapeutic administration of Brainiac-5 polypeptides, or an agonist or antagonist thereof, to block or disrupt the interaction of a viral particle with the Brainiac-5 receptors and, as a result, block the initiation or continuation of viral infectivity.
  • the Brainiac-5 polypeptides of the present invention binds to the Brainiac-5 receptor and, as such, is likely to block immune-tropic viral infections. Agonists and antagonists of the Brainiac-5/Brainiac-5 Receptor interaction are also likely to interfere with immune-tropic viral infection. As a result, such an interaction is likely to interfere with the infectious life cycle of one or more immune-tropic viruses such as HIV- 1 , HIV-2, HTLV-III, HS V- 1 , HS V-2, and the like.
  • immune-tropic viruses such as HIV- 1 , HIV-2, HTLV-III, HS V- 1 , HS V-2, and the like.
  • Brainiac-5 polypeptides of the present invention or agonists or antagonists thereof, to prophylactically or therapeutically block viral infection may be easily tested by the skilled artisan.
  • Simmons and coworkers Science 276:276-279 (1997)) and Arenzana-Seisdedos and colleagues (N ⁇ twre 383:400 (1996)) each outline a method of observing suppression of HIV-1 infection by an antagonist of the CCR5 chemokine receptor and of the CC chemokine RA ⁇ TES, respectively, in cultured peripheral blood mononuclear cells. Cells are cultured and infected with a virus, HIV-1 in both cases noted above.
  • CC chemokine or its receptor An agonist or antagonist of the CC chemokine or its receptor is then immediately added to the culture medium.
  • Evidence of the ability of the agonist or antagonist of the chemokine or cellular receptor is determined by evaluating the relative success of viral infection at 3, 6, and 9 days postinfection.
  • compositions comprising an amount of an isolated Brainiac-5 polypeptide, or an agonist or antagonist thereof, of the invention to an individual either infected with a virus or at risk for infection with a virus is performed as described below.
  • the Brainiac-5 polypeptides of the invention is a member of the Brainiac family
  • the mature secreted form of the polypeptide may be released in soluble form from the cells which express the Brainiac-5 polypeptides by proteolytic cleavage. Therefore, when the mature form of a Brainiac-5 polypeptide is added from an exogenous source to cells, tissues or the body of an individual, the polypeptide will exert its physiological activities on its target cells of that individual.
  • the invention also provides a method of treatment, prevention, and/or diagnosis of an individual in need of an increased level of Brainiac-5 activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated Brainiac-5 polypeptide of the invention, particularly a mature form of the Brainiac-5 polypeptides of the invention, effective to increase the Brainiac-5 polypeptide activity level in such an individual.
  • Brainiac-5 polypeptides are believed to elicit a potent cellular response including any genotypic, phenotypic, and/or mo ⁇ hologic change to the cell, cell line, tissue, tissue culture or patient. As indicated, such cellular responses include not only
  • Apoptosis-programmed cell death- is a physiological mechanism involved in the deletion of peripheral B and/or T lymphocytes of the immune system, and its disregulation can lead to a number of different pathogenic processes (J.C. Ameisen, AIDS 5: 1197-1213 (1994); P.H. Krammer et ⁇ /., Curr. Opin. Immunol. 6:279-289 (1994)).
  • cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus erythematosus and immune-related glomerular glomerular glomerular glomerular glomerular glomerular glomerular glomerular grafta, axoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclas
  • Brainiac-5 polynucleotides or polypeptides of the invention are used to treat, prevent, and/or diagnose autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas.
  • lymphocytic leukemias including, for example, MLL and chronic lymphocytic leukemia (CLL)
  • CLL chronic lymphocytic leukemia
  • Brainiac-5 polynucleotides or polypeptides of the invention are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
  • cancerous cells or tissue e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma
  • cancer therapy e.g., chemotherapy or radiation therapy.
  • Brainiac-5 polynucleotides or polypeptides of the invention are used to inhibit the growth, progression, and or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxo
  • AIDS Diseases associated with increased apoptosis include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
  • Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
  • myelodysplastic syndromes such as aplastic anemia
  • ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
  • toxin-induced liver disease such as that caused by alcohol
  • septic shock cachexia and anorexia.
  • Brainiac-5 polypeptides of the invention inhibit the growth of human histiocytic lymphoma U-937 cells in a dose-dependent manner. In additional preferred embodiments, Brainiac-5 polypeptides of the invention inhibit the growth of PC-3 cells, HT-29 cells, HeLa cells, MCF-7 cells, and A293 cells. In highly preferred embodiments, Brainiac-5 polynucleotides or polypeptides of the invention are used to inhibit growth, progression, and/or metastasis of prostate cancer, colon cancer, cervical carcinoma, and breast carcinoma. Because Brainiac-5 belongs to the Brainiac family, the polypeptides should also modulate angiogenesis.
  • Brainiac-5 inhibits immune cell functions, the polypeptides will have a wide range of anti-inflammatory activities.
  • Brainiac-5 may be employed as an anti-neovascularizing agent to treat, prevent, and/or diagnose solid tumors by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophages and by inhibiting the angiogenesis of tumors.
  • host defense cells e.g., cytotoxic T cells and macrophages
  • angiogenesis of tumors e.g., cytotoxic T cells and macrophages
  • Those of skill in the art will recognize other non-cancer indications where blood vessel proliferation is not wanted. They may also be employed to enhance host defenses against resistant chronic and acute infections, for example, myobacterial infections via the attraction and activation of microbicidal leukocytes.
  • Brainiac-5 may also be employed to inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-immune diseases and lymphocytic leukemias (including, for example, chronic lymphocytic leukemia (CLL)). Brainiac-5 may also be employed to stimulate wound healing, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells. In this same manner, Brainiac-5 may also be employed to treat, prevent, and or diagnose other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis.
  • CLL chronic lymphocytic leukemia
  • Brainiac-5 also increases the presence of eosinophils that have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis. It may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various hematopoietic progenitor cells, for example, to release mature leukocytes from the bone ma ⁇ ow following chemotherapy, i.e., in stem cell mobilization. Brainiac-5 may also be employed to treat, prevent, and/or diagnose sepsis. Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5, can be used in the treatment of infectious agents.
  • Brainiac-5 polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5 may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated by Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5.
  • viruses include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), He ⁇ esviridae (such as, Cytomegalovirus, He ⁇ es Simplex, He ⁇ es Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
  • Orthomyxoviridae e.g., Influenza A, Influenza B, and parainfluenza
  • Papiloma virus Papovaviridae
  • Parvoviridae Picornaviridae
  • Poxviridae such as Smallpox or Vaccinia
  • Reoviridae e.g., Rotavirus
  • Retroviridae HTLV-I, HTLV-II, Lentivirus
  • Togaviridae e.g., Rubivirus
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiolhtis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
  • arthritis bronchiolhtis
  • respiratory syncytial virus e.g., respiratory syncy
  • Brainiac-5 polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5 can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases.
  • Brainiac-5 polynucleotides, polypeptides, or agonists are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B).
  • Brainiac-5 polynucleotides, polypeptides, or agonists are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines.
  • Brainiac-5 polynucleotides, polypeptides, or agonists are used to treat, prevent, and/or diagnose AIDS.
  • Brainiac-5 polypeptides, agonists, and/or antagonists are used to treat, prevent, and/or diagnose patients with cryptosporidiosis.
  • bacterial or fungal agents that can cause disease or symptoms and that can be treated by Brainiac-5 polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Bo ⁇ elia (e.g., Bo ⁇ elia burgdorferi, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.
  • Actinomycetales e.g., Corynebacterium, Mycobacterium, Norcardi
  • Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Se ⁇ atia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gono ⁇ hea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp
  • bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
  • Brainiac-5 polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5 can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases.
  • Brainiac-5 polynucleotides, polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.
  • parasitic agents causing disease or symptoms that can be treated by Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5 include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale).
  • Brainiac-5 polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5 can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases.
  • Brainiac-5 polynucleotides, polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose malaria.
  • the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing Brainiac-5 polypeptides or anti-Brainiac-5 antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, such as, for example, cells expressing Brainiac-5 receptor.
  • Brainiac-5 polypeptides or anti-Brainiac-5 antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
  • the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., Brainiac-5 polypeptides or anti-Brainiac-5 antibodies) that are associated with heterologous polypeptides or nucleic acids.
  • polypeptides of the invention e.g., Brainiac-5 polypeptides or anti-Brainiac-5 antibodies
  • the invention provides a method for delivering a therapeutic protein into the targeted cell.
  • the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
  • a single stranded nucleic acid e.g., antisense or ribozymes
  • double stranded nucleic acid e.g., DNA that
  • the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., Brainiac-5 polypeptides or anti-Brainiac-5 antibodies) in association with toxins or cytotoxic prodrugs.
  • polypeptides of the invention e.g., Brainiac-5 polypeptides or anti-Brainiac-5 antibodies
  • toxin compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
  • Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
  • radioisotopes known in the art
  • compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
  • Toxin also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213 Bi.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
  • cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
  • Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
  • An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5, is osteomyelitis.
  • Brainiac-5 polypeptide or polypeptides, or agonists of Brainiac-5 is endocarditis.
  • treatment using Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5 could either be by administering an effective amount of Brainiac-5 polypeptide to the patient, or by removing cells from the patient, supplying the cells with Brainiac-5 polynucleotide, and returning the engineered cells to the patient (ex vivo therapy).
  • the Brainiac-5 polypeptide or polynucleotide can be used as an adjuvant in a vaccine to raise an immune response against infectious disease.
  • Additional preferred embodiments of the invention include, but are not limited to, the use of Brainiac-5 and functional agonists thereof, in the following applications:
  • an animal e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human
  • boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
  • an animal including, but not limited to, those listed above, and also including transgenic animals
  • an animal incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobuhn molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741.
  • the antagonists may be employed for instance to inhibit Brainiac-5-mediated chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain auto-immune and chronic inflammatory and infective diseases.
  • auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes.
  • the antagonists may also be employed to treat, prevent, and/or diagnose infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitment and activation of mononuclear phagocytes. They may also be employed to treat, prevent, and/or diagnose idiopathic hyper-eosinophilic syndrome by preventing eosinophil production and migration.
  • Endotoxic shock may also be treated by the antagonists by preventing the migration of macrophages and their production of the Brainiac-5 polypeptides of the present invention.
  • the antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall.
  • the antagonists may also be employed to treat, prevent, and/or diagnose histamine-mediated allergic reactions and immunological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and basophil degranulation and release of histamine.
  • IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema may also be treated.
  • the antagonists may also be employed to treat, prevent, and/or diagnose chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung. Antagonists may also be employed to treat, prevent, and/or diagnose rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies.
  • the antagonists may be employed to interfere with the deleterious cascades attributed primarily to IL-1 and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be employed to prevent inflammation.
  • the antagonists may also be employed to inhibit prostaglandin-independent fever induced by Brainiac-5.
  • the antagonists may also be employed to treat, prevent, and/or diagnose cases of bone ma ⁇ ow failure, for example, aplastic anemia and myelodysplastic syndrome.
  • the antagonists may also be employed to treat, prevent, and/or diagnose asthma and allergy by preventing eosinophil accumulation in the lung.
  • the antagonists may also be employed to treat, prevent, and/or diagnose subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung.
  • the antagonists may also be employed to treat, prevent, and/or diagnose lymphomas (e.g., one or more of the extensive, but not limiting, list of lymphomas provided herein). All of the above described applications as they may apply to veterinary medicine. Moreover, all applications described herein may also apply to veterinary medicine.
  • Antibodies against Brainiac-5 may be employed to bind to and inhibit Brainiac-5 activity to treat, prevent, and/or diagnose ARDS, by preventing infiltration of neutrophils into the lung after injury.
  • the antagonists and antagonists of the instant may be employed in a composition with a pharmaceutically acceptable ca ⁇ ier, e.g., as described hereinafter.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof are used to treat, prevent, and/or diagnose various immune system-related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, may be an effective therapy in treating and/or preventing autoimmune disorders.
  • Brainiac-5 antagonists of the invention e.g., polypeptide fragments of Brainiac-5 and anti-Brainiac-5 antibodies
  • Such autoimmune disorders include, but are not limited to, autoimmune diseases such as, for example, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, glomerulonephritis, Multiple Sclerosis, Neuritis, Ophthalmia, Polyendocrinopathies, Pu ⁇ ura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • autoimmune diseases such as, for example, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, glomerulonephritis, Multiple Sclerosis, Neuritis, Ophthalmia, Polyendocrinopathies, Pu ⁇ ura,
  • autoimmune thyroiditis i.e., Hashimoto's thyroiditis
  • systemic lupus erhthematosus often characterized, e.g., by circulating and locally generated immune complexes
  • Goodpasture's syndrome often characterized, e.g., by anti-basement membrane antibodies
  • Pemphigus often characterized, e.g., by epidermal acantholytic antibodies
  • Receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia
  • Additional autoimmune disorders include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by
  • Additional autoimmune disorders include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to
  • the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, and/or diagnosed using anti-Brainiac-5 antibodies.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof may also be treated by Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof.
  • these molecules can be used to treat, prevent, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith.
  • Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof may also be used to modulate inflammation.
  • Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
  • cytokines e.g., TNF or IL-1.
  • the present invention is directed to a method for enhancing apoptosis induced by a Brainiac-5 polypeptide, which involves administering to a cell which expresses a Brainiac-5 receptor an effective amount of Brainiac-5, analog or an agonist capable of increasing Brainiac-5-mediated signaling.
  • Brainiac-5-mediated signaling is increased to treat, prevent, and/or diagnose a disease wherein decreased apoptosis or decreased cytokine and adhesion molecule expression is exhibited.
  • An agonist can include soluble forms of Brainiac-5 and monoclonal antibodies directed against the Brainiac-5 polypeptide.
  • the present invention is directed to a method for inhibiting apoptosis induced by a Brainiac-5 polypeptide, which involves administering to a cell which expresses the Brainiac-5 receptor an effective amount of an antagonist capable of decreasing Brainiac-5-mediated signaling.
  • an antagonist capable of decreasing Brainiac-5-mediated signaling.
  • Brainiac-5-mediated signaling is decreased to treat, prevent, and/or diagnose a disease wherein increased apoptosis or NF-kappaB expression is exhibited.
  • An antagonist can include soluble forms of Brainiac-5 and monoclonal antibodies directed against the Brainiac-5 polypeptide.
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
  • diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2 infection, he ⁇ esvirus infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, po
  • osteomyelodysplasia e.g., aplastic anemia, etc.
  • liver disease e.g., acute and chronic hepatitis, liver injury, and ci ⁇ hosis
  • autoimmune disease e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic pu ⁇ ura, Grave's disease, Hashimoto's thyroiditis, etc.
  • cardiomyopathy e.g., dilated cardiomyopathy
  • diabetes diabetic complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
  • Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e.g., wounds, burns, and bone fractures). Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen, anti-viral immune responses,. More generally, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response.
  • polynucleotides and/or polypeptides of the invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
  • polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders.
  • polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
  • the Brainiac-5 polypeptide composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment, prevention, and/or diagnosis with Brainiac-5 polypeptides alone), the site of delivery of the Brainiac-5 polypeptide composition, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the "effective amount" of Brainiac-5 polypeptide for pu ⁇ oses herein is thus determined by such considerations.
  • the total pharmaceutically effective amount of Brainiac-5 polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
  • the Brainiac-5 polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini- pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
  • Effective dosages of the compositions of the present invention to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity. Such determination is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • Bioexposure of an organism to Brainiac-5 polypeptide during therapy may also play an important role in determining a therapeutically and/or pharmacologically effective dosing regime. Variations of dosing such as repeated administrations of a relatively low dose of Brainiac-5 polypeptide for a relatively long period of time may have an effect which is therapeutically and/or pharmacologically distinguishable from that achieved with repeated administrations of a relatively high dose of Brainiac-5 for a relatively short period of time. See, for instance, the serum immunoglobuhn level experiments presented in Example 6. Using the equivalent surface area dosage conversion factors supplied by
  • doses of 0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate to effect doses of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7 micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively, in the human.
  • compositions containing the Brainiac-5 polypeptides of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable ca ⁇ ier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • pharmaceutically acceptable ca ⁇ ier means a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • "pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly humans.
  • suitable pharmaceutical ca ⁇ iers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred ca ⁇ ier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the Brainiac-5 polypeptide is also suitably administered by sustained-release systems.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-efhyl-L-glutamate (Sidman, LT., et al,
  • Sustained-release Brainiac-5 polypeptide compositions also include liposomally entrapped Brainiac-5 polypeptide. Liposomes containing
  • Brainiac-5 polypeptides are prepared by methods known in the art (DE 3,218,121; Epstein, et al, Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang, et al, Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324). Ordinarily, the liposomes are of the small (about 200- 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Brainiac-5 polypeptide therapy.
  • the Brainiac-5 polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable ca ⁇ ier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a pharmaceutically acceptable ca ⁇ ier i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.
  • the formulations are prepared by contacting the Brainiac-5 polypeptide uniformly and intimately with liquid ca ⁇ iers or finely divided solid ca ⁇ iers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the ca ⁇ ier is a parenteral ca ⁇ ier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpy ⁇ olidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorb
  • Brainiac-5 polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, ca ⁇ iers, or stabilizers will result in the formation of Brainiac-5 polypeptide salts.
  • Brainiac-5 polypeptide to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
  • Therapeutic Brainiac-5 polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • Brainiac-5 polypeptide ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Brainiac-5 polypeptide solution, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized Brainiac-5 polypeptide using bacteriostatic water- for-injection (WFI).
  • WFI bacteriostatic water- for-injection
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
  • compositions of the invention may be administered alone or in combination with other adjuvants.
  • Adjuvants that may be administered with the compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.). QS21 (Genentech, Inc.), BCG, and MPL.
  • compositions of the invention are administered in combination with alum.
  • compositions of the invention are administered in combination with QS-21.
  • compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.
  • Vaccines that may be administered with the compositions of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially.
  • MMR measles, mumps, rubella
  • polio varicella
  • tetanus/diptheria hepatitis A
  • hepatitis B haemophilus influenzae B
  • cholera yellow fever
  • Japanese encephalitis polio
  • compositions of the invention may be administered alone or in combination with other therapeutic agents, including but not limited to, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anti- inflammatories, conventional immunotherapeutic agents and cytokines. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concu ⁇ ently; or sequentially.
  • Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • compositions of the invention are administered in combination with members of the TNF family.
  • TNF, TNF-related or TNF-like molecules that may be administered with the compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (Inte ⁇ iational Publication No. WO 96/14328), AIM-I (International Publication No.
  • WO 98/32856 discloses TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International
  • compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
  • Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, RETRO VIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine).
  • Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz).
  • Protease inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir).
  • antiretroviral agents nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.
  • compositions of the invention may be administered in combination with anti-opportunistic infection agents.
  • Anti-opportunistic agents that may be administered in combination with the compositions of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM,
  • PYRAZINAMIDETM ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMINETM, LEUCOVORINTM, NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM (sargramostim/GM-CSF).
  • compositions of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat, prevent, and/or diagnose an opportunistic Pneumocystis carinii pneumonia infection.
  • compositions of the invention are used in any combination with
  • compositions of the invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection.
  • compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection.
  • compositions of the invention are used in any combination with FLUCONAZOLETM, ITRA ⁇ ONAZOLETM, and or KETOCONAZOLETM to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection.
  • compositions of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic he ⁇ es simplex virus type I and/or type II infection.
  • compositions of the invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection.
  • compositions of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.
  • compositions of the invention are administered in combination with an antiviral agent.
  • Antiviral agents that may be administered with the compositions of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
  • the compositions of the invention are administered in combination with an antibiotic agent.
  • Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim- sulfamthoxazole, and vancomycin.
  • Nonspecific immunosuppressive agents that may be administered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells.
  • compositions of the invention are administered in combination with immunosuppressants.
  • Immunosuppressants preparations that may be administered with the compositions of the invention include, but are not limited to, ORTHOCLONETM (OKT3), SANDIMMUNETM/NEORALTM/SANGDYATM (cyclosporin), PROGRAFTM (tacrolimus), CELLCEPTTM (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNETM (sirolimus).
  • immunosuppressants may be used to prevent rejection of organ or bone ma ⁇ ow transplantation.
  • compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.
  • Intravenous immune globulin preparations that may be administered with the compositions of the invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, and GAMIMUNETM.
  • compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
  • compositions of the invention are administered alone or in combination with an anti -inflammatory agent.
  • Anti- inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti- inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e- acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, par
  • compositions of the invention are administered in combination with a chemotherapeutic agent.
  • Chemotherapeutic agents that may be administered with the compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6- thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.
  • compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP.
  • CHOP cyclophosphamide, doxorubicin, vincristine, and prednisone
  • compositions of the invention are administered in combination with Rituximab.
  • compositions of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP.
  • the compositions of the invention are administered in combination with cytokines.
  • Cytokines that may be administered with the compositions of the invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta.
  • compositions of the invention may be administered with any interleukin, including, but not limited to, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22.
  • interleukin including, but not limited to, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22.
  • compositions of the invention are administered with a chemokine.
  • compositions of the invention are administered with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein-4.
  • the compositions of the invention are administered with chemokine beta-8.
  • compositions of the invention are administered in combination with an IL-4 antagonist.
  • IL-4 antagonists that may be administered with the compositions of the invention include, but are not limited to: soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor without transducing the biological signal elicited by IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do not transduce the biological signal elicited by IL-4.
  • the antibodies employed according to this method are monoclonal antibodies (including antibody fragments, such as, for example, those described herein).
  • compositions of the invention are administered in combination with hematopoietic growth factors.
  • Hematopoietic growth factors that may be administered with the compositions of the invention include, but are not limited to, LEUKINETM (SARGRAMOSTIMTM) and NEUPOGENTM (FILGRASTIMTM).
  • compositions of the invention are administered in combination with fibroblast growth factors.
  • Fibroblast growth factors that may be administered with the compositions of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF- 10, FGF-11, FGF- 12, FGF- 13, FGF- 14, and FGF- 15.
  • compositions of the invention may be administered alone or in combination with other therapeutic regimens, including but not limited to, radiation therapy.
  • Such combinatorial therapy may be administered sequentially and/or concomitantly.
  • the invention also provides a method of screening compounds to identify those which enhance or block the action of Brainiac-5 polypeptides on cells, such as its interaction with Brainiac-5 polypeptide-binding molecules such as receptor molecules.
  • An agonist is a compound which increases the natural biological functions of Brainiac-5 polypeptides or which functions in a manner similar to Brainiac-5 polypeptides, while antagonists decrease or eliminate such functions.
  • the invention provides a method for identifying a receptor protein or other ligand-binding protein which binds specifically to a Brainiac-5 polypeptide.
  • a cellular compartment such as a membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds Brainiac-5 polypeptides.
  • the preparation is incubated with labeled Brainiac-5 polypeptide and complexes of Brainiac-5 polypeptide bound to the receptor or other binding protein are isolated and characterized according to routine methods known in the art.
  • the Brainiac-5 polypeptide may be bound to a solid support so that binding molecules solubilized from cells are bound to the column and then eluted and characterized according to routine methods.
  • a cellular compartment such as a membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds Brainiac-5 polypeptide, such as a molecule of a signaling or regulatory pathway modulated by Brainiac-5 polypeptide.
  • the preparation is incubated with labeled Brainiac-5 polypeptide in the absence or the presence of a candidate molecule which may be a Brainiac-5 polypeptide agonist or antagonist.
  • the ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand.
  • Molecules which bind gratuitously, i.e., without inducing the effects of Brainiac-5 polypeptide on binding the Brainiac-5 polypeptide-binding molecule, are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same as or closely related to Brainiac-5 polypeptide are agonists.
  • Brainiac-5 polypeptide-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of Brainiac-5 polypeptides or molecules that elicit the same effects as Brainiac-5 polypeptide.
  • Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
  • Brainiac-5 polypeptide antagonists are competitive assay that combines Brainiac-5 polypeptides and a potential antagonist with membrane-bound Brainiac-5 polypeptide receptor molecules or recombinant Brainiac-5 polypeptide receptor molecules under appropriate conditions for a competitive inhibition assay.
  • Brainiac-5 polypeptides can be labeled, such as by radioactivity, such that the number of Brainiac-5 polypeptide molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity.
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing Brainiac-5 polypeptide-induced activities, thereby preventing the action of Brainiac-5 polypeptides by excluding Brainiac-5 polypeptides from binding.
  • Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation.
  • Antisense techniques are discussed in a number of studies (for example, Okano, J. Neurochem. 56:560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.” CRC Press, Boca Raton, FL (1988)).
  • Triple helix formation is discussed in a number of studies, as well (for instance, Lee, et al, Nucleic Acids Research 10-1573 (1979); Cooney, et al, Science 241:456 (1988); Dervan, et al, Science 251: 1360 (1991)).
  • the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA ohgonucleotide of from about 10 to 40 base pairs in length.
  • a DNA ohgonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of a Brainiac-5 polypeptide.
  • the antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a Brainiac-5 polypeptide.
  • the ohgonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of Brainiac-5 polypeptides.
  • the Brainiac-5 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the Brainiac-5 antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells.
  • Expression of the sequence encoding Brainiac-5, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells.
  • Such promoters can be inducible or constitutive.
  • Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the he ⁇ es thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445 (1981), the regulatory sequences of the metahothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a Brainiac-5 gene.
  • absolute complementarity although preferred, is not required.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a Brainiac-5 RNA it may contain and still form a stable duplex (or triplex as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • 5' untranslated sequence up to and including the AUG initiation codon should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
  • ohgonucleotides complementary to either the 5'- or 3'- non- translated, non- coding regions of Brainiac-5 shown in Figures 1A-B could be used in an antisense approach to inhibit translation of endogenous Brainiac-5 mRNA.
  • Ohgonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense ohgonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of Brainiac-5 mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably ohgonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the ohgonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • the polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • the ohgonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the ohgonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the ohgonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense ohgonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine,
  • the antisense ohgonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense ohgonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense ohgonucleotide is an a-anomeric ohgonucleotide.
  • An a-anomeric ohgonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)).
  • the ohgonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131- 6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327- 330 (1997)).
  • Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate ohgonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209 (1988))
  • methylphosphonate ohgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451 (1988)), etc. While antisense nucleotides complementary to the Brainiac-5 coding region sequence could be used, those complementary to the transcribed untranslated region are most prefe ⁇ ed.
  • Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy Brainiac-5 mRNAs, the use of hammerhead ribozymes is prefe ⁇ ed. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • hammerhead ribozymes The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the Brainiac-5 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the invention can be composed of modified ohgonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express Brainiac-5 in vivo.
  • DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
  • a prefe ⁇ ed method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous Brainiac-5 messages and inhibit translation.
  • ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Endogenous gene expression can also be reduced by inactivating or "knocking out" the Brainiac-5 gene and/or its promoter using targeted homologous recombination.
  • endogenous gene expression can also be reduced by inactivating or "knocking out" the Brainiac-5 gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is inco ⁇ orated by reference herein in its entirety).
  • a mutant, nonfunctional polynucleotide of the invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo.
  • techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene.
  • antagonists according to the present invention include soluble forms of Brainiac-5.
  • Such soluble forms of the Brainiac-5 which may be naturally occurring or synthetic, antagonize Brainiac-5-mediated signaling by competing with native Brainiac-5 for binding to Brainiac-5 receptors and/or by forming a multimer that may or may not be capable of binding the receptor, but which is incapable of inducing signal transduction.
  • these antagonists inhibit Brainiac-5-mediated stimulation of lymphocyte (e.g., Dendritic cell, monocyte, macrophage, T cell, and/or B-cell) proliferation, differentiation, and/or activation.
  • Antagonists of the present invention also include antibodies specific for TNF-family ligands (e.g., CD30) and Brainiac-5-Fc fusion proteins.
  • Polyclonal and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed in Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO 94/09137 and are preferably specific to (i.e., bind uniquely to polypeptides of the invention having the amino acid sequence of SEQ ID NO:2.
  • antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') fragments) which are capable of binding an antigen.
  • Fab, Fab' and F(ab') fragments lack the Fc fragment intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med., 24:316-325 (1983)).
  • antibodies according to the present invention are mAbs.
  • Such mAbs can be prepared using hybridoma technology (Kohler and Millstein,
  • Proteins and other compounds which bind the Brainiac-5 domains are also candidate agonists and antagonists according to the present invention.
  • binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
  • yeast two-hybrid system Fields and Song, Nature 340:245-246 (1989)
  • a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell 72:223-232 (1993)).
  • Such compounds are good candidate agonists and antagonists of the present invention.
  • screening techniques include the use of cells which express the polypeptide of the present invention (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, 246:181-296 (1989).
  • potential agonists or antagonists may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, e.g., signal transduction may be measured to determine whether the potential antagonist or agonist is effective.
  • Agonists according to the present invention include naturally occu ⁇ ing and synthetic compounds such as, for example, TNF family ligand peptide fragments, transforming growth factor, neurotransmitters (such as glutamate, dopamine, N- methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells and antimetabolites.
  • Preferred agonists include chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and -amyloid peptide. (Science 267:1457-1458 (1995)).
  • Preferred agonists are fragments of Brainiac-5 polypeptides of the invention which stimulate lymphocyte (e.g., B cell) proliferation, differentiation and/or activation.
  • Further preferred agonists include polyclonal and monoclonal antibodies raised against the Brainiac-5 polypeptides of the invention, or a fragment thereof.
  • Such agonist antibodies raised against a T ⁇ F-family receptor are disclosed in Tartaglia et al., Proc. Natl. Acad. Sci. USA 88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem. 267:4304- 4307(1992). See, also, PCT Application WO 94/09137.
  • immunoregulatory molecules such as, for example, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IF ⁇ -gamma and T ⁇ F-alpha, may be used as agonists of Brainiac-5 polypeptides of the invention which stimulate lymphocyte proliferation, differentiation and/or activation.
  • cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc.
  • the cells are genetically engineered in vitro using
  • ⁇ recombinant D ⁇ A techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
  • the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
  • the cells can be inco ⁇ orated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
  • genetically engineered fibroblasts can be implanted as part of a skin graft
  • genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
  • the cells to be administered are non- autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • the activity of Brainiac-5 polypeptide can be reduced using a "dominant negative.”
  • constructs which encode defective Brainiac-5 polypeptide such as, for example, mutants lacking all or a portion of a conserved domain, can be used in gene therapy approaches to diminish the activity of Brainiac-5 on appropriate target cells.
  • nucleotide sequences that direct host cell expression of Brainiac-5 polypeptide in which all or a portion of a conserved domain is altered or missing can be introduced into monocytic cells or other cells or tissues (either by in vivo or ex vivo gene therapy methods described herein or otherwise known in the art).
  • targeted homologous recombination can be utilized to introduce such deletions or mutations into the subject's endogenous Brainiac-5 gene in monocytes.
  • the engineered cells will express non-functional Brainiac-5 polypeptides (i.e., a ligand (e.g., multimer) that may be capable of binding, but which is incapable of inducing signal transduction).
  • the agonists and antagonists may be employed in a composition with a pharmaceutically acceptable ca ⁇ ier, e.g., as described above.
  • the nucleic acid molecules of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymo ⁇ hisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in co ⁇ elating those sequences with genes associated with disease.
  • the cDNA herein disclosed is used to clone genomic DNA of a Brainiac-5 gene.
  • genomic DNA then is used for in situ chromosome mapping using well known techniques for this pu ⁇ ose.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with probes from the cDNA as short as 50 or 60 bp (for a review of this technique, see Verma, et al, Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988)).
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, on the World Wide Web (McKusick, V.
  • Example 1 Expression and Purification of "His-tagged" Brainiac-5 in E. coli
  • the novel pHE4 series of bacterial expression vectors in particular, the pHE4- 5 vector may be used for bacterial expression in this example.
  • pHE4-5/MPIFD23 vector plasmid DNA contains an insert which encodes another ORF. The construct was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, on September 30, 1997 and given Accession No. 209311.
  • the pHE4-5 bacterial expression vector includes a neomycin phosphotransferase gene for selection, an E. coli origin of replication, a T5 phage promoter sequence, two lac operator sequences, a Shine-Delgarno sequence, and the lactose operon repressor gene (laclq). These elements are a ⁇ anged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues (i.e., a "6 X His tag”) covalently linked to the amino terminus of that polypeptide.
  • the promoter and operator sequences of the pHE4-5 vector were made synthetically. Synthetic production of nucleic acid sequences is well known in the art (CLONETECH 95/96 Catalog, pages 215-216, CLONETECH, 1020 East Meadow Circle, Palo Alto, CA 94303).
  • the DNA sequence encoding the desired portion of the Brainiac-5 polypeptide is amplified from the deposited cDNA clone using PCR ohgonucleotide primers which anneal to the amino terminal sequences of the desired portion of the Brainiac-5 polypeptide and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pHE4-5 vector are added to the 5' and 3' primer sequences, respectively.
  • the 5' primer has the sequence 5' CAA
  • the 3' primer has the sequence 5' GTA CGC AAG CTT GGA GTC CCA TTG GAA GGG 3' (SEQ ID NO: 12) containing the underlined Hin dill restriction site followed by 18 nucleotides complementary to the 3' end of the coding sequence of the Brainiac-5 DNA sequence shown in Figures 1A and IB (SEQ ID NO:l).
  • the amplified Brainiac-5 DNA fragment and the vector pHE4-5 are digested with Bam HI and Hin dill and the digested DNAs are then ligated together. Insertion of the Brainiac-5 DNA into the restricted pHE4-5 vector places the Brainiac-5 polypeptide coding region downstream from the IPTG-inducible promoter and in- frame with an initiating AUG and the six histidine codons.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pR ⁇ P4, which expresses the lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing Brainiac-5 polypeptide, is available commercially (QIAGEN, Inc., supra). Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl-beta-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor.
  • IPTG Isopropyl-beta-D-thiogalactopyranoside
  • the cells are then sti ⁇ ed for 3-4 hours at 4°C in 6M guanidine-HCl, pH 8.
  • the cell debris is removed by centrifugation, and the supernatant containing the Brainiac-5 polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA”) affinity resin column (QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist, 1995, QIAGEN, Inc., supra).
  • the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the Brainiac-5 polypeptide is eluted with 6 M guanidine-HCl, pH 5.
  • the purified polypeptide is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl.
  • PBS phosphate-buffered saline
  • the protein can be successfully refolded while immobilized on the Ni-NTA column.
  • the recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors.
  • the renaturation should be performed over a period of 1.5 hours or more.
  • the proteins can be eluted by the addition of 250 0 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.
  • the purified protein is stored at 4°C or frozen at
  • the following alternative method may be used to purify Brainiac-5 polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless 5 otherwise specified, all of the following steps are conducted at 4-10°C.
  • the cell culture Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10°C and the cells are harvested by continuous centrifugation at 15,000 ⁇ (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate o amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM ⁇ DTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
  • the cells ware then lysed by passing the solution through a microfluidizer (Microfuidics, Co ⁇ . or APV Gaulin, Inc.) twice at 4000-6000 psi.
  • the homogenate is 5 then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 x g for 15 min.
  • the resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM ⁇ DTA, pH 7.4.
  • the resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 x g centrifugation for 15 min., the pellet is discarded and the Brainiac-5 polypeptide-containing supernatant is incubated at 4°C overnight to allow further GuHCl extraction.
  • guanidine hydrochloride (GuHCl)
  • the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous sti ⁇ ing.
  • the refolded diluted protein solution is kept at 4°C without mixing for 12 hours prior to further purification steps.
  • a previously prepared tangential filtration unit equipped with 0.16 micrometer membrane filter with appropriate surface area e.g., Filtron
  • 40 mM sodium acetate, pH 6.0 is employed.
  • the filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).
  • the column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner.
  • the absorbance at 280 mm of the effluent is continuously monitored.
  • Fractions are collected and further analyzed by SDS-PAGE. Fractions containing the Brainiac-5 polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.
  • the CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A 280 monitoring of the effluent. Fractions containing the Brainiac-5 polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
  • Brainiac-5 polypeptide exhibits greater than 95% purity after the above refolding and purification steps. No major contaminant bands are observed from Commassie blue stained 16% SDS-PAGE gel when 5 micrograms of purified protein is loaded. The purified protein is also tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
  • Example 2 Cloning and Expression of Brainiac-5 polypeptide in a Baculovirus Expression System
  • the plasmid shuttle vector pA2 GP is used to insert the cloned DNA encoding the mature protein, lacking its naturally associated secretory signal (leader) sequence, into a baculovirus to express a Brainiac-5 polypeptide, using a baculovirus leader and standard methods as described by Summers and colleagues (A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987)).
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as Bam HI, Xba I and Asp 718.
  • the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.
  • Many other baculovirus vectors could be used in place of the vector above, such as pA2, 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, by Luckow and colleagues (Virology 170:31-39 (1989)).
  • the cDNA sequence encoding the Brainiac-5 polypeptide in the deposited clone is amplified using PCR ohgonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • the 5' primer has the sequence 5'-CGC GGA TCC GCC ATC ATG GTG GCA GAG GAC TTC GAG C-3' (SEQ ID NO: 13) containing the underlined Bam HI restriction enzyme site followed by 19 nucleotides of the sequence of the Brainiac-5 polypeptide shown in SEQ ID NO:2, beginning with the currently known N-terminus.
  • the 3' primer has the sequence 5'-CAC TTA GGT ACC GGA GTC CCA TTG GAA GGG-3' (SEQ ID NO: 14) containing the underlined Asp 718 restriction site followed by 18 nucleotides complementary to the carboxy-terminal sequence in Figures 1A and IB.
  • the amplified fragment is isolated from a 1 % agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with Bam HI and Asp 718 and again is purified on a 1% agarose gel. This fragment is designated herein FI.
  • the plasmid is digested with the restriction enzymes Bam HI and Asp 718 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”. Fragment FI and the dephosphorylated plasmid VI are ligated together with
  • E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria are identified that contain the plasmid with the human Brainiac-5 gene by digesting DNA from individual colonies using Bam HI and Asp 718 and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pA2GPBrainiac-5.
  • plasmid pA2GPBrainiac-5 Five ⁇ g of the plasmid pA2GPBrainiac-5 is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method described by
  • the plate is then incubated for 5 hours at 27°C.
  • the transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27°C for four days.
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith (supra). An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of o 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., Gaithersburg, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf).
  • a micropipettor e.g., Eppendorf
  • the agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ⁇ l of Grace's medium and the 5 suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supematants of these culture dishes are harvested and then they are stored at 4°C.
  • the recombinant virus is called V-Brainiac-5.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the 0 recombinant baculovirus V-Brainiac-5 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 from Life Technologies Inc., Rockville, MD). After 42 hours, 5 ⁇ Ci of 5 S-methionine and 5 ⁇ Ci 35 S-cysteine (available from Amersham) are added.
  • the cells are further incubated for 5 16 hours and then 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).
  • Example 3 Cloning and Expression of Brainiac-5 in Mammalian Cells
  • a typical mammalian expression vector contains the 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, HTLV-I, HIV-1 and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • 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 CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected 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
  • the mammalian cells are ⁇ grown in selective medium and the cells with the highest resistance are selected.
  • the expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al, Mol. Cel. 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 Bam HI, Xba I and Asp 718, 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.
  • the expression plasmid, pBrainiac-5HA is made by cloning a portion of the cDNA encoding the mature form of the Brainiac-5 polypeptide 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 eukaryotic 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) followed by a termination codon and polyadenylation signal a ⁇ anged 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.
  • HA hemagglutinin fragment
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson and colleagues (Cell 37:767 (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.
  • a DNA fragment encoding the Brainiac-5 polypeptide is cloned into the polylinker region of the vector so that recombinant polypeptide expression is directed by the CMV promoter.
  • the plasmid construction strategy is as follows. The Brainiac-5 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of Brainiac-5 in E. coli. Suitable primers include the following, which are used in this example.
  • the 5' primer containing the underlined Bam HI site, a Kozak sequence (in italics), an AUG start codon, and 16 nucleotides of the 5' coding region of the Brainiac-5 polypeptide, has the following sequence: 5' CGC GGA TCC GCCATC ATG GTG GCA GAG GAC TTC GAG C 3' (SEQ ID NO: 15).
  • the 3' primer containing the underlined Asp 718 and 17 of nucleotides complementary to the 3' coding sequence immediately before the stop codon, has the following sequence: 5'-CAC TTA GGT ACC GGA GTC CCA TTG GAA GGG-3' (SEQ ID NO: 16).
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Bam HI and Asp 718 and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, 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 fragment encoding the complete Brainiac-5 polypeptide
  • COS cells are transfected with an expression vector, as described above, using DEAE-dextran, as described, for instance, by Sambrook and coworkers (Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989)). Cells are incubated under conditions for expression of Brainiac-5 by the vector.
  • Brainiac-5-HA fusion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow and colleagues (Antibodies: A Laboratory Manual, 2nd Ed. ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S- cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson and colleagues (supra).
  • Polypeptides 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 Brainiac-5 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.
  • the amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Mol. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV; Boshart, et al, Cell 41:521-530 (1985)).
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: Bam HI, Xba I, and Asp 718. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • Other high efficiency promoters can also be used for the expression, e.g., the human ⁇ -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 Brainiac-5 polypeptide in a regulated way in mammalian cells (Gossen, M., and Bujard, H. 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 the restriction enzymes Bam HI and Asp 118 and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete Brainiac-5 polypeptide is amplified using PCR ohgonucleotide primers co ⁇ esponding to the 5' and 3' sequences of the desired portion of the gene.
  • the 5' primer contains a Bam HI restriction site, a Kozak sequence, an AUG start codon, and 16 nucleotides of the 5' coding region of the
  • the 3' primer contains an Asp 718 restriction site, and 17 of nucleotides complementary to the 3' coding sequence immediately before the stop codon, and is shown in SEQ ID NO: 16.
  • the amplified fragment is digested with the endonucleases Bam HI and Asp 718 and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB 101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary cells lacking an active DHFR gene are used for transfection.
  • Five ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSVneo using lipofectin (Feigner, et al, supra).
  • the plasmid pSV2-neo 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 mg/ml G418.

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Abstract

La présente invention concerne de nouveaux polypeptides Brainiac-5 qui font partie de la famille des Brainiac. L'invention concerne, en particulier, des molécules d'acide nucléique isolées codant les polypeptides Brainiac-5 humains ; ainsi que des vecteurs, des cellules hôtes et des procédés de recombinaison permettant de produire ces derniers ; des procédés de criblage permettant d'identifier des agonistes et des antagonistes des activités de Brainiac-5 ; des procédés de diagnostic permettant de détecter des affections du système immunitaire et du système nerveux et des procédés thérapeutiques permettant de traiter, de prévenir et/ou de diagnostiquer les affections du système immunitaire et du système nerveux.
EP99966502A 1998-12-23 1999-12-20 Brainiac-5 humains Withdrawn EP1140120A4 (fr)

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WO2011082345A2 (fr) 2009-12-30 2011-07-07 Brigham Young University Compositions et procédés de gestion du cancer à l'aide d'anticorps de liaison aux enzymes des voies de récupération nucléotidique et à leurs complexes
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AU6778998A (en) * 1997-03-28 1998-10-22 Human Genome Sciences, Inc. Human muscle derived growth factor - cardiac and pancreatic protein (capp) and gene
WO1999031116A1 (fr) * 1997-12-18 1999-06-24 Human Genome Sciences, Inc. Dendriac et brainiac-3 humains
KR100543857B1 (ko) * 1999-09-01 2006-01-23 제넨테크, 인크. 혈관신생 및 심혈관형성의 촉진 또는 억제

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 1271 bp, 27 September 1999 (1999-09-27) "Human brainiac-3 cDNA" retrieved from EBI Database accession no. AAX87194 XP002234111 & WO 99 31116 A (HUMAN GENOME SCI INC) 24 June 1999 (1999-06-24) *
DATABASE EMBL [Online] 13339 bp, mRNA, Drosophila megalogaster, 9 January 1996 (1996-01-09) "Drosophila megalogaster putative neurogenic secreted signaling protein (brn) mRNA, complete cds." retrieved from NCBI Database accession no. U41449 XP002228623 *
DATABASE EMBL [Online] 228516 bp, DNA, Homo sapiens, 4 August 1999 (1999-08-04) "Homo sapiens chromosome 16 clone CTA-277H1, working draft sequence" retrieved from EBI Database accession no. AC008594 XP002228626 *
DATABASE EMBL [Online] 2684 bp, 25 January 2001 (2001-01-25) "Human PRO4397 nucleotide sequence SEQ ID NŸ41" retrieved from EBI Database accession no. AAC58117 XP002234112 & WO 00 53750 A (GENENTECH INC) 14 September 2000 (2000-09-14) *
DATABASE EMBL [Online] 3054 bp, mRNA, Homo sapiens, 14 January 1998 (1998-01-14) "UDP-galactose-2-acetamido-2-deoxy-D-gluco se-3-beta-galactosyltransferase" retrieved from CNBI Database accession no. Y15014 XP002228624 *
DATABASE EMBL [Online] 352 aa, 27 September 1999 (1999-09-27) "Human brainiac-3" retrieved from EBI Database accession no. AAY06462 XP002228627 -& WO 99 31116 A (HUMAN GENOME SCI INC) 24 June 1999 (1999-06-24) *
DATABASE EMBL [Online] 397 aa, Human, 18 January 1999 (1999-01-18) "Human cardiac and pancreatic protein (CAPP)" retrieved from EBI Database accession no. AAW80212 XP002228625 -& WO 98 44112 A (HUMAN GENOME SCI INC) 8 October 1998 (1998-10-08) *
DATABASE EMBL [Online] 402 aa, 25 January 2001 (2001-01-25) "Human PRO4397 protein sequence SEQ ID NŸ42; " retrieved from EBI Database accession no. AAB24035 XP002228628 -& WO 00 53750 A (GENENTECH INC) 14 September 2000 (2000-09-14) *
GOODE S ET AL: "BRAINIAC AND FRINGE ARE SIMILAR PIONEER PROTEINS THAT IMPART SPECIFICITY TO NOTCH SIGNALING DURING DROSOPHILA DEVELOPMENT" COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, NEW YORK, NY, US, vol. 62, 1997, pages 177-184, XP001041794 *
See also references of WO0039136A2 *

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