EP1206487A2 - Ligands recepteurs du gene notch, et utilisations associees - Google Patents

Ligands recepteurs du gene notch, et utilisations associees

Info

Publication number
EP1206487A2
EP1206487A2 EP00957528A EP00957528A EP1206487A2 EP 1206487 A2 EP1206487 A2 EP 1206487A2 EP 00957528 A EP00957528 A EP 00957528A EP 00957528 A EP00957528 A EP 00957528A EP 1206487 A2 EP1206487 A2 EP 1206487A2
Authority
EP
European Patent Office
Prior art keywords
seq
amino acids
polynucleotide
polypeptide
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00957528A
Other languages
German (de)
English (en)
Inventor
Chan Vivien
Michael Rohan
Lewis T. Williams
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.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP1206487A2 publication Critical patent/EP1206487A2/fr
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/02Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to receptors and ligands consisting of human Notch gene products, and uses of these receptors, ligands and derivatives thereof to modulate cell-cell interaction in biological processes and conditions including angiogenesis and cancer.
  • Notch pathway is involved in cell fate determination and differentiation, and signaling through the Notch pathway receptors is an evolutionarily conserved mechanism for cell-cell interaction.
  • Notch proteins of which four have been identified in humans (Notch 1, Notch 2, Notch 3 and Notch 4), are a family of closely related transmembrane receptors.
  • Notch 4 is expressed specifically in endothelial cells (Shiratoshi, Y., Genes Cells 2:213-224, 1997; Uyttendaele, H., Development 122:2251- 2259, 1996) and may play an important role in angiogenesis.
  • Notch When Notch is activated by a ligand, its intracellular domain is proteolytically cleaved and transported to the nucleus, along with CSL (CBF-l/Su(H)/Lag-l/RBP-J K ) transcription factor to activate transcription of downstream effectors. The resulting effector can repress the transcriptional activity of other genes encoding transcription factors for entry into terminal differentiation.
  • CSL CBF-l/Su(H)/Lag-l/RBP-J K
  • the ligands that interact with the extracellular portion of Notch include Delta, Serrate, and Jagged; the ligands also are transmembrane proteins.
  • Adjacent cells of identical lineage can follow separate pathways of differentiation as a result of the Notch pathway.
  • Sample pathways of differentiation include axis formation, cartilage formation, and somite formation.
  • a Notch receptor is expressed on the cell surface of a "suppressed" cell, and interacts with a Notch ligand located on the cell surface of a dominating cell. After ligand interaction with a Notch receptor, the intracellular domain of the Notch receptor is cleaved and transported to the nucleus, where it forms a complex and affects gene transcription. (Lendahl, U., BwEssays 20:103-107, 1998.)
  • ligands including Delta and Serrate have been identified and studied.
  • the corresponding genes in mammals include Dll-1 (Delta- 1) and Dll-3 (Delta-3), as well as Jag-1 and Jag-2.
  • Mutations and decreased expression of Delta are related to phenotypic changes, and a translocation in the human Notch 1 locus (TAN-1) has been found in T-cell acute lymphoblastic leukemia/lymphoma (Ellisen et al., Cell 66:649-661, 1991).
  • Jagged 1 Mutations in the human Jagged 1 gene are associated with Alagille syndrome, which involves abnormal development of liver, heart, skeleton, eye, and face. Alagille patients also exhibit valvular and arterial stenonis and high incidence of intracranial hemorrhage. Four separate mutations, all frameshifts, have been identified in patients with the syndrome. (Li, L. et al., Nature Genetics 7(5:243-251, 1997; Oda, T. et al., Nature Genetics 7(5:235-242, 1997.) The mutations are likely to interfere with the ability of Jagged 1 to interact with Notch, thereby affecting the differentiation of cells whose fate would otherwise be determined by interaction of Notch with functional Jagged 1. Mice rendered genetically deficient for Jagged 1 exhibit defects in vascular development (Xue et al., Hum. Mol. Gen. 5:723, 1999). These results are consistent with the hypothesis that Jagged 1 is also involved in vascular development and integrity.
  • CADASIL cerebral autosomal arteriopathy with subcortical infarction and leukoencephalopathy.
  • the present invention relates to nucleotide sequences of human Notch ligand genes, and amino acid sequences of the encoded proteins, as well as derivatives and fragments thereof wherein the derivatives and fragments exhibit biological activity such as binding to Notch receptors.
  • the invention also relates to methods of modulating angiogenesis by using the Notch ligands of the invention, and derivatives and fragments thereof.
  • the invention further relates to modulation of endothelial cell proliferation using polynucleotides encoding all of part of the Notch ligands of the invention, such as antisense oligonucleotides that can target nucleic acid encoding the Notch ligand.
  • the invention still further relates to methods of modulating the development and maturation of T-cells and other cells of the immune system, thereby regulating cell-mediated immunity and antibody responses to alleviate conditions such as rheumatoid arthritis.
  • Notch ligands of the invention include those encoded by the 3md3 gene and the 2hdl gene.
  • the invention relates to an isolated nucleic acid molecule comprising a polynucleotide selected from the group consisting of:
  • the invention also relates to an isolated nucleic acid molecule consisting of a nucleic acid comprising 50-1752 contiguous nucleotides from the coding region of SEQ ID NO: 1.
  • the invention further relates to an isolated nucleic acid molecule comprising a polynucleotide encoding a polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has an amino acid sequence selected from the group consisting of:
  • the invention still further relates to an isolated polypeptide comprising amino acids at least 95% identical to amino acids selected from the group consisting of:
  • the invention also relates to a complex comprising a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
  • the invention further relates to a complex comprising a fragment of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and a Dishevelled protein wherein said fragment is capable of forming a complex with said Dishevelled protein.
  • Figure 1 is an alignment of the amino acid sequences of novel human Notch ligand, 3md3, of the present invention (SEQ ID NO:l), the mouse ortholog Delta-3 (3721842_md3), a human Delta polypeptide (w80813_hd3), and human sequence hdeltalp.
  • Figure 2 is a protein phylogenic analysis based on the sequences of Figure 1, and shows that 3md3 is more closely related to murine Delta 3 than is the human sequence W80813_hd3 or human sequence hdeltalp.
  • Figure 3 is an alignment of the nucleotide sequences of 3md3 (SEQ ID NO:2), mouse dll3, hdelta, and W80813.
  • Figure 4 is a polynucleotide phylogenic analysis based on the sequences of Figure 3.
  • Figure 5 provides the polynucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO:2) sequences of 3md3.
  • Figure 6 provides the polynucleotide (SEQ ID NO:3) and amino acid
  • Figure 7 is a diagram representing the interaction between the Notch receptor and the Notch ligands.
  • the intracellular domain of Notch Upon binding to Delta or Jagged (Notch ligands), the intracellular domain of Notch is cleaved by a presenilin-dependent pathway and translocates to the nucleus along with DNA-binding protein, CSL, to activate transcription of downstream genes.
  • the intracellular domain of Notch also downregulates the Jun-Kinase (JNK) pathway through a CSL independent mechanism (Zecchini, V. et al., Biol. 9:460-499, 1999).
  • Modulators of the pathway includes Kuzbanian, a disintegrin and metalloproteinase (ADAM) that positively regulate Notch signaling, and Fringe, a putative glycosyltransferase that inhibits Jagged signaling.
  • ADAM disintegrin and metalloproteinase
  • Fringe a putative glycosyltransferase that inhibits Jagged signaling.
  • the present invention relates to nucleotide sequences of Notch ligands, the proteins encoded by the nucleotide sequences, and uses of the polynucleotides, proteins, fragments thereof, and antibodies specific for the proteins and polypeptides.
  • Notch pathway In human disease, All of the components of the pathway have yet to be identified, but among those identified to date, mutations that affect their interaction with each other can lead to a variety of syndromes and pathological conditions.
  • a role for Notch- Jagged interaction in angiogenesis has been reported, based on studies with a model for angiogenesis, which measures bovine microvascular endothelial cell invasion into a collagen gel, through formation of a network of capillary-like tubes. (Zimrin, A. et al., Jour. Biol. Chem. 277:32499-32502, 1996.) Growth factors such as FGF and VEGF can be added to determine the effect of Notch and Notch ligands on growth factor-induced endothelial cell behavior and differentiation.
  • the Notch pathway is also implicated in the development and maturation of T cells, as described in Radtke, F. et al., Immunity 70:547-558, 1999.
  • the Notch ligands of the invention are therefore useful candidates for modulating the immune system, including determining the fate of T cells, thereby affecting antibody production and/or cell-mediated immunity.
  • the Notch ligands of the present invention can be used to further elucidate the role of the Notch pathway in human development and disease.
  • the ligand gene referred to as 3md3 displays a pattern of tissue expression that overlaps with the endothelial cell-specific gene Notch 4. This has important implications for the use of 3md3 in modulating angiogenesis, such as preventing cancer-related angiogenesis to stop tumor growth and for therapeutic angiogenesis, e.g., to induce blood vessel formation in ischemia.
  • a mouse mammary tumor virus (MMTV) oncogene, int-3 encodes the intracellular signaling domain of Notch 4.
  • the MMTV may target angiogenesis as a component of its tumor promoting activity.
  • 3md3 is therefore a candidate for regulating Notch 4-mediated angiogenesis. Without being bound by a particular mechanism, applicants believe that by modulating the interaction between Notch ligand 3md3 and its receptors, the growth and differentiation of microvascular endothelial cells can be regulated.
  • the role of the 3md3 gene product in angiogenesis can be determined using a model of angiogenesis, for example as described in more detail in the Examples.
  • GenBank protein ID 3721842 (D113, GenBank protein ID 3721842). It is expressed in the heart, kidney, skeletal muscle and kidney as a 4.9 kb message.
  • the nucleotide sequence for the full-length coding region (1752 basepairs) is disclosed in SEQ ID NO: l, and the encoded amino acid sequence (583 amino acids) is disclosed in SEQ ID NO:2.
  • the mouse D113 gene is predominantly expressed in the neuroectoderm and paraxial mesoderm during embryogenesis (Dunwoodie, S.L. et al., Development 124:3065-3076, 1997).
  • the cDNA consists of 2243 basepairs, encoding a protein of 585 amino acids.
  • Figure 1 compares the polynucleotide sequences of 3md3 with murine D113.
  • Figure 1 also compares the sequence of a human Delta polynucleotide sequence disclosed in WO 98 45434-A1 (W80813). An examination of the alignments in Figure 1 indicates that the novel sequence of the invention, 3md3, is more closely related to the mouse Delta 3 sequence than is the human Delta sequence.
  • 3md3 is the human ortholog of the mouse D113 gene.
  • the tissue expression of 3md3 overlaps with that of the endothelial-specific gene Notch 4, suggesting that it may be a functional ligand for Notch 4.
  • the present invention provides new methods and materials for modulating angiogenesis and immune responses using products of 3md3.
  • 2hdl This gene contains a region that is identical to the DSL (Delta/Serrate/Lag) domain in human Delta 1, followed by a divergent sequence of 24 amino acids and a stop codon.
  • the DSL domain is a region of homology common to the known Notch ligand and is involved in receptor binding (Fitzgerald and Greenwald, Development 727:4275-4282, 1995).
  • SEQ ID NO:3 represents the coding region of 2hdl
  • SEQ ID NO:4 represents the translation of the coding region.
  • the invention relates to the specific polynucleotide sequences disclosed in SEQ ID NO:l and 3, and to additional embodiments described herein.
  • the polynucleotides of the invention also include polynucleotides having sequence similarity or sequence identity to SEQ ID NO:l or 3.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in lXSSC.
  • Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate).
  • Hybridization methods and conditions are well known in the art, see, e.g., U.S. Patent No. 5,707,829.
  • Nucleic acids that are substantially identical to the provided polynucleotide sequences e.g., allelic variants, genetically altered versions of the gene, etc., bind to one of the provided polynucleotide sequences (SEQ ID NOs:l and 3) under stringent hybridization conditions.
  • probes particularly labeled probes of DNA sequences, one of skill can isolate homologous or related genes.
  • the source of homologous genes can be any species, e.g., primate species, particularly human; rodents, such as rats and mice; canines, felines, bovines, ovines, equines, yeast, nematodes, etc.
  • hybridization is performed using at least 15 contiguous nucleotides (nt) of at least one of SEQ ID NOs:l and 3. That is, when at least 15 contiguous nt of one of the disclosed SEQ ID NOs. is used as a probe, the probe will preferentially hybridize with a nucleic acid comprising the complementary sequence, allowing the identification and retrieval of the nucleic acids that uniquely hybridize to the selected probe.
  • Probes of more than 15 nt can be used, e.g., probes of from about 18 nt to about 100 nt, but 15 nt represents sufficient sequence for unique identification.
  • the polynucleotides of the invention also include naturally occurring variants of the nucleotide sequences (e.g., degenerate variants, allelic variants). Variants of the polynucleotides of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions. For example, by using appropriate wash conditions, variants of the polynucleotides of the invention can be identified where the allelic variant exhibits at most about 25-30% base pair (bp) mismatches relative to the selected polynucleotide probe. In general, allelic variants contain 15-25% bp mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2%) bp mismatches, as well as a single bp mismatch.
  • allelic variants contain 15-25% bp mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2%) bp mismatches
  • the invention also encompasses homologs corcesponding to the polynucleotides of SEQ ID NOs: l and 3, where the source of homologous genes can be any mammalian species, e.g., primate species, particularly human; rodents, such as rats; canines, felines, bovines, ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs generally have substantial sequence similarity, e.g., at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences.
  • Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc.
  • a reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared.
  • Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., J. Mol. Biol. 275:403-10 (1990).
  • a preferred method of calculating percent identity is the Smith- Waterman algorithm, using the following. Global DNA sequence identity is greater than 65% as determined by the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.
  • the subject nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active gene product and/or are useful in the methods disclosed herein (e.g., in diagnosis, as a unique identifier of a differentially expressed gene of interest, etc.).
  • cDNA as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide of the invention.
  • a genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It can also include the 3' and 5' untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region.
  • the genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence.
  • the genomic DNA flanking the coding region, either 3' and 5', or internal regulatory sequences as sometimes found in introns contains sequences required for proper tissue, stage-specific, or disease-state specific expression.
  • the nucleic acid compositions of the subject invention can encode all or a part of the subject polypeptides. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc.
  • Isolated polynucleotides and polynucleotide fragments of the invention comprise at least about 10, about 15, about 20, about 35, about 50, about 100, about 150 to about 200, about 250 to about 300, or about 350 contiguous nt selected from the polynucleotide sequences as shown in SEQ ID NOs: l and 3.
  • fragments are exemplary only, and include all intervening sizes, such as 11, 12, 13, etc.; 51, 52, 53, etc.; 151, 152, 153, etc., and so on.
  • fragments will be of at least 15 nt, usually at least 18 nt or 25 nt, and up to at least about 50 contiguous nt in length or more.
  • the polynucleotide molecules comprise a contiguous sequence of at least 12 nt selected from the group consisting of the polynucleotides shown in SEQ ID NOs: l and 3.
  • Preferred fragments are those which contain an antigenic determinant, and/or which are functionally active. "Functionally active" fragments include those with adhesive properties. Preferred fragments of 3md3 and/or 2hdl include the full- length protein, an extracellular domain with a transmembrane region, and fragments of the proteins that are homologous or functionally equivalent to the adhesive fragments of Drosophila Delta mediating heterotypic (amino acids 1-230) or homotypic (32-320) interactions. Such fragments can be expressed independently or as fusion proteins. Fusion proteins may include myc-, HA-, or His6- tags. Fusion proteins may contain the Fc domain of human IgG.
  • WO 97/06571 Derivatives of Delta proteins, and assays for measuring the biological activity of the derivatives, are disclosed in WO 97/06571, which is incorporated by reference.
  • WO 97/06571 also discloses therapeutics, including antisense compositions, based on or derived from polynucleotides encoding Delta proteins. Such methods and compositions are applicable to the novel ligands of the invention. Truncations of Notch ligands are disclosed in U.S. Patent No. 5,648,464, which is incorporated by reference.
  • Probes specific to the polynucleotides of the invention can be generated using the polynucleotide sequences disclosed in SEQ ID NOs:l and 3.
  • the probes are preferably at least about a 12, 15, 16, 18, 20, 22, 24, or 25 nt fragment of a corresponding contiguous sequence of SEQ ID NOs:l and 3, and can be less than 2, 1, 0.5, 0.1, or 0.05 kb in length.
  • the probes can be synthesized chemically or can be generated from longer polynucleotides using restriction enzymes.
  • the probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag.
  • polynucleotides of the subject invention are isolated and obtained in substantial purity, generally as other than an intact chromosome.
  • the polynucleotides either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant," e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
  • the polynucleotides of the invention can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the polynucleotides can be regulated by their own or by other regulatory sequences known in the art.
  • the polynucleotides of the invention can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA- coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.
  • the subject nucleic acid compositions can be used, for example, to produce polypeptides, as probes for the detection of mRNA of the invention in biological samples (e.g., extracts of human cells), to generate additional copies of the polynucleotides, to generate ribozymes or antisense oligonucleotides, and as single stranded DNA probes or as triple-strand forming oligonucleotides.
  • the probes described herein can be used to, for example, determine the presence or absence of the polynucleotide sequences as shown in SEQ ID NOs: l and 3 or variants thereof in a sample. These and other uses are described in more detail below.
  • the invention provides polypeptide fragments of the disclosed protein.
  • Polypeptide fragments of the invention can comprise at least 8, 10, 12, 15, 18, 19, 20, 25, 50, 75, 100, 125, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 560, 570 or 580 contiguous amino acids from SEQ ID NO:2.
  • Also included are all intermediate length fragments in this range, such as 101, 102, 103, etc.; 170, 171, 172, etc.; and 600, 601, 601, etc.
  • the specific lengths listed herein are exemplary only and not limiting.
  • the invention also provides fragments of at least 81 contiguous amino acids from SEQ ID NO:4.
  • Variants of the protein and polypeptides disclosed herein can also occur. Variants can be naturally or non-naturally occurring. Naturally occurring variants are found in humans or other species and comprise amino acid sequences which are substantially identical to the amino acid sequence shown in SEQ ID NO:2 or 4. Species homologs of the protein can be obtained using subgenomic polynucleotides of the invention, as described below, to make suitable probes or primers to screening cDNA expression libraries from other species, such as mice, monkeys, yeast, or bacteria, identifying cDNAs which encode homologs of the protein, and expressing the cDNAs as is known in the art.
  • Non-naturally occurring variants which retain substantially the same biological activities as naturally occurring protein variants, specifically the four transmembrane configuration and the interaction with other cell surface proteins, are also included here.
  • naturally or non-naturally occurring variants have amino acid sequences which are at least 85%, 90%>, or 95% identical to the amino acid sequence shown in SEQ ID NO:2 or 4. More preferably, the molecules are at least 98% or 99%o identical. Percent identity is determined using any method known in the art.
  • a non-limiting example is the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
  • the Smith- Waterman homology search algorithm is taught in Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489.
  • ammo acid changes in the protem vanants disclosed herein are conservative ammo acid changes, i e , substitutions of similarly charged or uncharged amino acids
  • a conservative amino acid change involves substitution of one of a family of ammo acids which are related in their side chains
  • Naturally occurnng ammo acids are generally divided into four families acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, vahne, leucine, lsoleucme, prohne, phenylalanme, methionme, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, senne, threonme, tyrosme) amino acids Phenylalanme, tryptophan, and tyros
  • Vanants of the 3md3 and/or 2hdl proteins disclosed herein include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties
  • Covalent vanants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-termmal residue, as is known in the art Vanants also include allelic vanants, species vanants, and muteins Truncations or deletions of regions which do not affect functional activity of the proteins are also vanants
  • mutants is a group of polypeptides in which neutral amino acids, such as sennes, are substituted for cysteine residues which do not participate in disulfide bonds These mutants may be stable over a broader temperature range than native secreted proteins See Mark et al , US Patent 4,959,314
  • amino acid changes in the 3md3 and/or 2hdl protein or polypeptide vanants are conservative amino acid changes, i e , substitutions of similarly charged or uncharged ammo acids
  • a conservative ammo acid change involves substitution of one of a family of amino acids which are related in their side chains
  • Naturally occurnng ammo acids are generally divided into four families acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. It is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological properties of the resulting secreted protein or polypeptide variant.
  • Properties and functions of 3md3 and/or 2hdl protein or polypeptide variants are of the same type as a protein comprising the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:2 or 4, respectively, although the properties and functions of variants can differ in degree.
  • 3md3 and/or 2hdl protein variants include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties.
  • 3md3 and or 2hdl protein variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect the differential expression of the 3md3 and/or 2hdl genes are also variants.
  • Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art. It will be recognized in the art that some amino acid sequence of the
  • 3md3 and/or 2hdl proteins of the invention can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there are critical areas on the protein which determine activity. In general, it is possible to replace residues that form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein. The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 5(57:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors.
  • the polypeptides of the present invention may include one or more ammo acid substitutions, deletions or additions, either from natural mutations or human manipulation
  • the invention further includes vanations of the 3md3 and/or 2hdl polypeptides which show comparable expression patterns or which include antige c regions
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al., "Deciphenng the Message in Protein Sequences: Tolerance to Ammo Acid Substitutions," Science 247:1306-1310 (1990).
  • substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids The latter results in proteins with reduced positive charge to improve the charactenstics of the disclosed protein. The prevention of aggregation is highly desirable.
  • Amino acids in the 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 alanme-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanme mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as binding to a natural or synthetic binding partner. 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) and de Vos et al. Science 255:306-312 (1992)).
  • Fusion proteins comprising proteins or polypeptide fragments of 3md3 and/or 2hdl can also be constructed. Fusion proteins are useful for generating antibodies against amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with a protein of the invention or which interfere with its biological function. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can also be used for this purpose. Such methods are well known in the art and can also be used as drug screens.
  • Fusion proteins comprising a signal sequence and/or a transmembrane domain of 3md3 and/or 2hdl or a fragment thereof can be used to target other protein domains to cellular locations in which the domains are not normally found, such as bound to a cellular membrane or secreted extracellularly.
  • a fusion protein comprises two protein segments fused together by means of a peptide bond.
  • Amino acid sequences for use in fusion proteins of the invention can be utilize the amino acid sequence shown in SEQ ID NO:2 or 4 or can be prepared from biologically active variants of SEQ ID NO:2 or 4, such as those described above.
  • the first protein segment can include of a full-length 3md3 and/or 2hdl.
  • first protein segments can consist of at least 8, 10, 12, 15, 18, 19, 20, 25, 50, 75, 100, 125, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 560, 570 or 580 contiguous amino acids from SEQ ID NO:2.
  • the second protein segment can be a full-length protein or a polypeptide fragment.
  • Proteins commonly used in fusion protein construction include ⁇ - galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags can be used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • DBD Lex a DNA binding domain
  • GAL4 GAL4 DNA binding domain
  • HSV herpes simplex virus
  • fusions can be made, for example, by covalently linking two protein segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises a coding sequence of SEQ ID NO:l or 3 in proper reading frame with a nucleotide encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art.
  • kits for constructing fusion proteins are available from companies that supply research labs with tools for experiments, including, for example, Promega Co ⁇ oration (Madison, WI), Stratagene (La Jolla, CA), Clontech (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
  • 3md3 is expressed in human microvascular endothelial cells and can be extracted from these cells or from other human cells, such as recombinant cells comprising SEQ ID NO:l using standard biochemical methods. These methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, crystallization, electrofocusing, and preparative gel electrophoresis.
  • the isolated and purified protein or polypeptide is separated from other compounds which normally associate with the protein or polypeptide in a cell, such as other proteins, carbohydrates, lipids, or subcellular organelles.
  • a preparation of isolated and purified protein or polypeptide is at least 80%> pure; preferably, the preparations are 90%>, 95%>, or 99% pure. Purity of the preparations can be assessed by any means known in the art. For example, the purity of a preparation can be assessed by examining electrophoretograms of protein or polypeptide preparations at several pH values and at several polyacrylamide concentrations, as is known in the art.
  • Proteins, fusion proteins, or polypeptides of the invention can be produced by recombinant DNA methods.
  • a coding sequence of the nucleotide sequence shown in SEQ ID NO: l or 3 can be expressed in prokaryotic or eukaryotic host cells using expression systems known in the art. These expression systems include bacterial, yeast, insect, and mammalian cells. The resulting expressed 3md3 and/or 2hdl proteins can then be purified from the culture medium or from extracts of the cultured cells using purification procedures known in the art.
  • cell-free medium can be diluted with sodium acetate and contacted with a cation exchange resin, followed by hydrophobic interaction chromatography.
  • the desired protein or polypeptide is typically greater than 95%o pure. Further purification can be undertaken, using, for example, any of the techniques listed above.
  • 3md3 and/or 2hdl protein or polypeptide of the invention can also be expressed in cultured host cells in a form which will facilitate purification.
  • a protein or polypeptide can be expressed as a fusion protein comprising, for example, maltose binding protein, glutathione-S-transferase, or thioredoxin, and purified using a commercially available kit. Kits for expression and purification of such fusion proteins are available from companies such as New England BioLabs, Pharmacia, and Invitrogen. Proteins, fusion proteins, or polypeptides can also be tagged with an epitope, such as a "Flag" epitope (Kodak), and purified using an antibody which specifically binds to that epitope.
  • an epitope such as a "Flag" epitope (Kodak)
  • transgenic animals such as cows, goats, pigs, or sheep.
  • Female transgenic animals can then produce proteins, polypeptides, or fusion proteins of the invention in their milk.
  • Methods for constructing such animals are known and widely used in the art.
  • synthetic chemical methods such as solid phase peptide synthesis, can be used to synthesize a secreted protein or polypeptide.
  • General means for the production of peptides, analogs or derivatives are outlined in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins — A Survey of Recent Developments, B. Weinstein, ed. (1983).
  • Substitution of D-amino acids for the normal L-stereoisomer can be carried out to increase the half-life of the molecule. Variants can be similarly produced.
  • Isolated genes corresponding to the cDNA sequences disclosed herein are also provided. Standard molecular biology methods can be used to isolate the corresponding genes using the cDNA sequences provided herein. These methods include preparation of probes or primers from the nucleotide sequence shown in SEQ ID NO:l or 3 for use in identifying or amplifying the genes from human genomic libraries or other sources of human genomic DNA.
  • Polynucleotide molecules of the invention can also be used as primers to obtain additional copies of the polynucleotides, using polynucleotide amplification methods.
  • Polynucleotide molecules can be propagated in vectors and cell lines using techniques well known in the art.
  • Polynucleotide molecules can be on linear or circular molecules. They can be on autonomously replicating molecules or on molecules without replication sequences. They can be regulated by their own or by other regulatory sequences, as is known in the art.
  • Polynucleotide molecules comprising the coding sequences disclosed herein can be used in a polynucleotide construct, such as a DNA or RNA construct.
  • Polynucleotide molecules of the invention can be used, for example, in an expression construct to express all or a portion of a secreted protein, variant, fusion protein, or single-chain antibody in a host cell.
  • An expression construct comprises a promoter which is functional in a chosen host cell. The skilled artisan can readily select an appropriate promoter from the large number of cell type-specific promoters known and used in the art.
  • the expression construct can also contain a transcription terminator which is functional in the host cell.
  • the expression construct comprises a polynucleotide segment which encodes all or a portion of the desired protein. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter.
  • the expression construct can be linear or circular and can contain sequences, if
  • An expression construct can be introduced into a host cell.
  • the host cell comprising the expression construct can be any suitable prokaryotic or eukaryotic cell.
  • Expression systems in bacteria include those described in Chang et al, Nature (1978) 275: 615; Goeddel et al, Nature (1979) 257: 544; Goeddel et al, Nucleic Acids Res. (1980) 8: 4057; EP 36,776; U.S. 4,551,433; deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25; and Siebenlist et al., Cell (1980) 20: 269.
  • Expression systems in yeast include those described in Hinnen et ai, Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J. Bacteriol. (1983) 755: 163; Kurtz et al, Mol. Cell. Biol. (1986) 6: 142; Kunze et al, J Basic Microbiol (1985) 25: 141; Gleeson et al, J. Gen. Microbiol. (1986) 752: 3459, Roggenkamp et al, Mol. Gen. Genet. (1986) 202 :302); Das et al, J Bacteriol (1984) 158: 1165; De Louvencourt et al, J. Bacteriol.
  • Mammalian expression can be accomplished as described in Dijkema et al, EMBO J. (1985) 4: 761 ; Gorman et al., Proc. Natl Acad. Sci. USA (1982b) 79: 6777; Boshart et al, Cell (1985) 47: 521; and U.S. 4,399,216. Other features of mammalian expression can be facilitated as described in Ham and Wallace, Meth Enz. (1979) 55: 44; Barnes and Sato, Anal. Biochem. (1980) 702: 255; U.S. 4,767,704; U.S. 4,657,866; U.S. 4,927,762; U.S.
  • Expression constructs can be introduced into host cells using any technique known in the art. These techniques include transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and calcium phosphate- mediated transfection.
  • Expression of an endogenous gene encoding a protein of the invention can also be manipulated by introducing by homologous recombination a DNA construct comprising a transcription unit in frame with the endogenous gene, to form a homologously recombinant cell comprising the transcription unit.
  • the transcription unit comprises a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the new transcription unit can be used to turn the endogenous gene on or off as desired. This method of affecting endogenous gene expression is taught in U.S. Patent 5,641,670.
  • the targeting sequence is a segment of at least 10, 12, 15, 20, or 50 contiguous nucleotides from the nucleotide sequence shown in SEQ ID NO: l or 3.
  • the transcription unit is located upstream to a coding sequence of the endogenous gene.
  • the exogenous regulatory sequence directs transcription of the coding sequence of the endogenous gene.
  • polynucleotides e.g., a polynucleotide having a sequence of one of SEQ ID NOs:l and 3
  • the corresponding cDNA, or the full-length gene is used to express a partial or complete gene product.
  • Constructs of polynucleotides having sequences of SEQ ID NOs:l and 3 can be generated synthetically.
  • single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides is described by, e.g., Stemmer et al., Gene (Amsterdam) (1995) 7 ⁇ 54(7):49-53.
  • assembly PCR the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides
  • DNA shuffling Stepmmer, Nature (1994) 570:389-391).
  • polynucleotide constructs are purified using standard recombinant DNA techniques as described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • the gene product encoded by a polynucleotide of the invention is expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems.
  • Vectors, host cells and methods for obtaining expression in same are well known in the art. Suitable vectors and host cells are described in U.S. Patent No. 5,654,173.
  • Polynucleotide molecules comprising a polynucleotide sequence provided herein are generally propagated by placing the molecule in a vector.
  • Viral and non-viral vectors are used, including plasmids.
  • the choice of plasmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence.
  • Other vectors are suitable for expression in cells in culture.
  • Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. Methods for preparation of vectors comprising a desired sequence are well known in the art.
  • polynucleotides set forth in SEQ ID NOs:l and 3 or their corresponding full-length polynucleotides are linked to regulatory sequences as appropriate to obtain the desired expression properties. These can include promoters (attached either at the 5' end of the sense strand or at the 3' end of the antisense strand), enhancers, terminators, operators, repressors, and inducers.
  • the promoters can be regulated or constitutive. In some situations it may be desirable to use conditionally active promoters, such as tissue-specific or developmental stage-specific promoters. These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be used.
  • the resulting replicated nucleic acid, RNA, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism.
  • the product is recovered by any appropriate means known in the art.
  • Expression of a gene corresponding to SEQ ID NO:l or 3 can be regulated in the cell to which the gene is native.
  • an endogenous gene of a cell can be regulated by an exogenous regulatory sequence as disclosed in U.S. Patent No. 5,641,670.
  • Expression products of a polynucleotide of the invention can be prepared and used for raising antibodies for experimental, diagnostic, and therapeutic purposes.
  • antibodies can be generated using fusion proteins or peptide sequences derived from the corresponding protein.
  • the polynucleotide or related cDNA is expressed as described above, and antibodies are prepared. These antibodies are specific to an epitope on the polypeptide encoded by the polynucleotide, and can precipitate or bind to the corresponding native protein in a cell or tissue preparation or in a cell-free extract of an in vitro expression system.
  • Methods for production of monoclonal and polyclonal antibodies that specifically bind a selected antigen are well known in the art. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. Epitopes that involve non-contiguous amino acids may require a longer polypeptide, e.g., at least 15, 25, or 50 amino acids.
  • Antibodies that specifically bind to human polypeptides encoded by the provided polynucleotides should provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays.
  • antibodies that specifically bind poly- peptides of the invention do not bind to other proteins in immunochemical assays at detectable levels and can immunoprecipitate the specific polypeptide from solution.
  • the invention also contemplates naturally occurring antibodies specific for a polypeptide of the invention.
  • serum antibodies to a polypeptide of the invention in a human population can be purified by methods well known in the art, e.g., by passing antiserum over a column to which the corresponding selected polypeptide or fusion protein is bound. The bound antibodies can then be eluted from the column, for example using a buffer with a high salt concentration.
  • the invention also contemplates genetically engineered antibodies, antibody derivatives (e.g., single chain antibodies, antibody fragments (e.g., Fab, etc.)), according to methods well known in the art.
  • antibody derivatives e.g., single chain antibodies, antibody fragments (e.g., Fab, etc.)
  • humanized anti-3md3 or 2hdl monoclonal antibodies are provided.
  • the phrase "humanized antibody” refers to an antibody derived from a non-human antibody, typically a mouse monoclonal antibody.
  • a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.
  • chimeric antibody refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions.
  • humanized antibodies are far less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a human such as, e.g., use as radiation sensitizers for the treatment of neoplastic disease or use in methods to reduce the side effects of, e.g., cancer therapy.
  • Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as “humanizing”), or, alternatively, (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”).
  • CDRs complementarity determining regions
  • complementarity determining region refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. See, e.g., Chothia et al., J. Mol. Biol. 79(5:901-917 (1987); Kabat et al., U.S. Dept. of Health and Human Services NIH Publication No. 91-3242 (1991).
  • constant region refers to the portion of the antibody molecule that confers effector functions. In the present invention, mouse constant regions are substituted by human constant regions.
  • the constant regions of the subject humanized antibodies are derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu.
  • One method of humanizing antibodies comprises aligning the non- human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody.
  • Such humanized antibodies may be further derivatized to facilitate uptake and clearance, e.g., via Ashwell receptors. See, e.g., U.S. Patent Nos. 5,530,101 and 5,585,089 which patents are incorporated herein by reference.
  • Humanized antibodies to 3md3 and/or 2hdl can also be produced using transgenic animals that are engineered to contain human immunoglobulin loci.
  • transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci.
  • WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin-encoding loci are substituted or inactivated.
  • WO 96/30498 discloses the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule.
  • WO 94/02602 discloses non- human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci.
  • U.S. Patent No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy claims, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions.
  • an immune response can be produced to a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
  • Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735.
  • This publication discloses monoclonal antibodies against a variety of antigenic molecules including IL-6, IL-8, TNF , human CD4, L-selectin, gp39, and tetanus toxin.
  • the monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.
  • WO 96/33735 discloses that monoclonal antibodies against IL-8, derived from immune cells of transgenic mice immunized with IL-8, blocked IL-8-induced functions of neutrophils. Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096.
  • 3md3 and/or 2hdl polypeptides of the invention and variants thereof are used to immunize a transgenic animal as described above.
  • Monoclonal antibodies are made using methods known in the art, and the specificity of the antibodies is tested using corresponding isolated 3md3 and/or 2hdl polypeptides.
  • Polynucleotide arrays are created by spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellose, etc.) in a two-dimensional matrix or array having bound probes.
  • the probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions.
  • Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away.
  • arrays can be used to detect differential expression of a polynucleotide between a test cell and control cell (e.g., cancer cells and normal cells).
  • high expression of a particular message in a cancer cell can indicate a cancer specific gene product.
  • arrays are further described in, for example, Pappalarado et al., Sem. Radiation Oncol (1998) 5:217; and Ramsay, Nature Biotechnol. (1998) 7(5:40.
  • the polynucleotides of the invention can also be used to detect differences in expression levels between two cells, e.g., as a method to identify abnormal or diseased tissue in a human.
  • the expression of a gene corresponding to a specific polynucleotide is compared between a first tissue that is suspected of being diseased and a second, normal tissue of the human.
  • the tissue suspected of being abnormal or diseased can be derived from a different tissue type of the human, but preferably it is derived from the same tissue type; for example an intestinal polyp or other abnormal growth should be compared with normal intestinal tissue.
  • the normal tissue can be the same tissue as that of the test sample, or any normal tissue of the patient, especially those that express the polynucleotide-related gene of interest (e.g., brain, thymus, testis, heart, prostate, placenta, spleen, small intestine, skeletal muscle, pancreas, breast, ovary, lung, and the mucosal lining of the colon).
  • a difference between the polynucleotide-related gene, mRNA, or protein in the two tissues which are compared, for example in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicates a change in the gene, or a gene which regulates it, in the tissue of the human that was suspected of being diseased.
  • a genetic predisposition to Notch pathway-related disease in a human can also be detected by comparing expression levels of an mRNA or protein corresponding to a polynucleotide of the invention in a fetal tissue, with levels in normal fetal tissue.
  • Fetal tissues that are used for this purpose include, but are not limited to, amniotic fluid, chorionic villi, blood, and the blastomere of an in vitro- fertilized embryo.
  • the comparable normal polynucleotide-related gene is obtained from any tissue.
  • the mRNA or protein is obtained from a normal tissue of a human in which the polynucleotide-related gene is expressed. Differences such as alterations in the nucleotide sequence or size of the same product of the fetal polynucleotide-related gene or mRNA, or alterations in the molecular weight, amino acid sequence, or relative abundance of fetal protein, can indicate a germline mutation in the polynucleotide- related gene of the fetus, which indicates a genetic predisposition to disease.
  • diagnostic, prognostic, and other methods of the invention based on differential expression involve detection of a level or amount of a gene product, particularly a differentially expressed gene product, in a test sample obtained from a patient suspected of having or being susceptible to a disease (e.g., breast cancer, lung cancer, colon cancer and/or metastatic forms thereof), and comparing the detected levels to those levels found in normal cells (e.g., cells substantially unaffected by cancer) and/or other control cells (e.g., to differentiate a cancerous cell from a cell affected by dysplasia).
  • the severity of the disease can be assessed by comparing the detected levels of a differentially expressed gene product with those levels detected in samples representing the levels of differentially gene product associated with varying degrees of severity of disease.
  • diagnosis herein is not necessarily meant to exclude “prognostic” or “prognosis,” but rather is used as a matter of convenience.
  • the term "differentially expressed gene” is generally intended to encompass a polynucleotide that can, for example, include an open reading frame encoding a gene product (e.g., a polypeptide), and/or introns of such genes and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction.
  • the gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
  • a difference in expression level associated with a decrease in expression level of at least about 25%, usually at least about 50%) to 75%, more usually at least about 90% or more is indicative of a differentially expressed gene of interest, i.e., a gene that is underexpressed or down- regulated in the test sample relative to a control sample.
  • a difference in expression level associated with an increase in expression of at least about 25%, usually at least about 50%> to 75%, more usually at least about 90% and can be at least about 1 '/2-fold, usually at least about 2-fold to about 10-fold, or about 100-fold to about 1, 000- fold increase relative to a control sample is indicative of a differentially expressed gene of interest, i.e., an overexpressed or up-regulated gene.
  • “Differentially expressed polynucleotide” as used herein means a nucleic acid molecule (RNA or DNA) comprising a sequence that represents a differentially expressed gene, e.g., the differentially expressed polynucleotide comprises a sequence (e.g., an open reading frame encoding a gene product) that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample.
  • RNA or DNA nucleic acid molecule
  • the differentially expressed polynucleotide comprises a sequence (e.g., an open reading frame encoding a gene product) that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample.
  • “Differentially expressed polynucleotides” is also meant to encompass fragments of the disclosed polynucleotides, e.g., fragments retaining biological activity, as well as nucleic acids homologous, substantially similar, or substantially identical (e.g., having about 90%> sequence identity) to the disclosed polynucleotides.
  • Diagnosis generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, as well as the prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy).
  • sample or “biological sample” as used throughout here are generally meant to refer to samples of biological fluids or tissues, particularly samples obtained from tissues, especially from cells of the type associated with the disease for which the diagnostic application is designed, and the like. “Samples” is also meant to encompass derivatives and fractions of such samples (e.g., cell lysates). Where the sample is solid tissue, the cells of the tissue can be dissociated or tissue sections can be analyzed. Methods of the subject invention useful in diagnosis or prognosis typically involve comparison of the abundance of a selected differentially expressed gene product in a sample of interest with that of a control to determine any relative differences in the expression of the gene product, where the difference can be measured qualitatively and/or quantitatively.
  • Quantitation can be accomplished, for example, by comparing the level of expression product detected in the sample with the amounts of product present in a standard curve. A comparison can be made visually; by using a technique such as densitometry, with or without computerized assistance; by preparing a representative library of cDNA clones of mRNA isolated from a test sample, sequencing the clones in the library to determine that number of cDNA clones corresponding to the same gene product, and analyzing the number of clones corresponding to that same gene product relative to the number of clones of the same gene product in a control sample; or by using an array to detect relative levels of hybridization to a selected sequence or set of sequences, and comparing the hybridization pattern to that of a control.
  • diagnostic assays of the invention involve detection of a gene product of the polynucleotide sequence (e.g., mRNA or polypeptide) that corresponds to a sequence of SEQ ID NOs: l and 3.
  • the patient from whom the sample is obtained can be apparently healthy, susceptible to disease (e.g., as determined by family history or exposure to certain environmental factors), or can already be identified as having a condition in which altered expression of a gene product of the invention is implicated.
  • Diagnosis can be determined based on detected gene product expression levels of a gene product encoded by at least one of the polynucleotides having a sequence set forth in SEQ ID NOs: l and 3, and can involve detection of expression of genes corresponding to all of SEQ ID NOs:l and 3 and/or additional sequences that can serve as additional diagnostic markers and/or reference sequences.
  • the assay preferably involves detection of a gene product encoded by a gene corresponding to a polynucleotide that is differentially expressed in cancer.
  • detectable labels include fluorochromes, (e.g., fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2 ' , 7 ' -dimethoxy-4 ' , 5 ' -dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)), radioactive labels, (e.g., 32 P, 35 S, 3 H, etc.), and the like.
  • the detectable label can be used in connection with the various embodiments of the diagnostic methods of the invention.
  • Suitable detectable labels include flu
  • Reagents specific for the polynucleotides and polypeptides of the invention can be supplied in a kit for detecting the presence of an expression product in a biological sample.
  • the kit can also contain buffers or labeling components, as well as instructions for using the reagents to detect and quantify expression products in the biological sample. Exemplary embodiments of the diagnostic methods of the invention are described below in more detail.
  • the test sample is assayed for the level of a differentially expressed polypeptide.
  • Diagnosis can be accomplished using any of a number of methods to determine the absence or presence or altered amounts of the differentially expressed polypeptide in the test sample.
  • detection can utilize staining of cells or histological sections with labeled antibodies, performed in accordance with conventional methods. Cells can be permeabilized to stain cytoplasmic molecules.
  • antibodies that specifically bind a differentially expressed polypeptide of the invention are added to a sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes.
  • the antibody can be detectably labeled for direct detection (e.g., using radioisotopes, enzymes, fluorescers, chemiluminescers, and the like), or can be used in conjunction with a second stage antibody or reagent to detect binding (e.g., biotin with horseradish peroxidase-conjugated avidin, a secondary antibody conjugated to a fluorescent compound, e.g., fluorescein, rhodamine, Texas red, etc.).
  • the absence or presence of antibody binding can be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.
  • any suitable alternative methods of qualitative or quantitative detection of levels or amounts of differentially expressed polypeptide can be used, for example ELISA, western blot, immunoprecipitation, radioimmunoassay, etc. mRNA detection.
  • the diagnostic methods of the invention can also or alternatively involve detection of mRNA encoded by the 3md3 and/or 2hdl gene. Any suitable qualitative or quantitative methods known in the art for detecting specific mRNAs can be used.
  • mRNA can be detected by, for example, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA.
  • One of skill in the art can readily use these methods to determine differences in the size or amount of mRNA transcripts between two samples.
  • mRNA expression levels in a sample can also be determined by generation of a library of expressed sequence tags (ESTs) from the sample, where the EST library is representative of sequences present in the sample (Adams, et al., (1991) Science 252:1651). Enumeration of the relative representation of ESTs within the library can be used to approximate the relative representation of the gene transcript within the starting sample. The results of EST analysis of a test sample can then be compared to EST analysis of a reference sample to determine the relative expression levels of a selected polynucleotide, particularly a polynucleotide corresponding to one or more of the differentially expressed genes described herein.
  • ESTs expressed sequence tags
  • gene expression in a test sample can be performed using serial analysis of gene expression (SAGE) methodology (e.g., Velculescu et al., Science (1995) 270:484), differential display methodology (see, e.g., U.S. Patent Nos. 5,776,683 and 5,807,680), or DNA microarray methodology.
  • SAGE serial analysis of gene expression
  • differential display methodology see, e.g., U.S. Patent Nos. 5,776,683 and 5,807,680
  • DNA microarray methodology e.g., RNA microarray methodology.
  • gene expression can be analyzed using hybridization analysis. Oligonucleotides or cDNA can be used to selectively identify or capture DNA or RNA of specific sequence composition, and the amount of RNA or cDNA hybridized to a known capture sequence determined qualitatively or quantitatively, to provide information about the relative representation of a particular message within the pool of cellular messages in a sample.
  • the diagnostic methods of the invention can focus on the expression of a single differentially expressed gene represented by SEQ ID NO:l or 3.
  • the diagnostic method can involve detecting a differentially expressed gene, or a polymorphism of such a gene (e.g., a polymorphism in a coding region or control region), that is associated with disease.
  • Disease-associated polymorphisms can include deletion or truncation of the gene, mutations that alter expression level and/or affect binding specificity, such as interaction of the encoded protein with Notch receptor.
  • a number of methods are available for analyzing nucleic acids for the presence of a specific sequence, e.g., a disease associated polymorphism.
  • genomic DNA is used directly.
  • the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis.
  • Cells that express a differentially expressed gene can be used as a source of mRNA, which can be assayed directly or reverse transcribed into cDNA for analysis.
  • the nucleic acid can be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis, and a detectable label can be included in the amplification reaction (e.g., using a detectably labeled primer or detectably labeled oligonucleotides) to facilitate detection.
  • PCR polymerase chain reaction
  • the amplified or cloned sample nucleic acid can be analyzed by one of a number of methods known in the art.
  • the nucleic acid can be sequenced by dideoxy or other methods, and the sequence of bases compared to a selected sequence, e.g., to a wild-type sequence.
  • Hybridization with the polymorphic or variant sequence can also be used to determine its presence in a sample (e.g., by Southern blot, dot blot, etc.).
  • 5,445,934, or in WO 95/35505 can also be used as a means of identifying polymorphic or variant sequences associated with disease.
  • Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • SSCP Single strand conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • a polymorphism creates or destroys a recognition site for a restriction endonuclease
  • the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.
  • Screening for mutations in a gene can be based on the functional or antigenic characteristics of the protein. Protein truncation assays are useful in detecting deletions that can affect the biological activity of the protein. Various immunoassays designed to detect polymorphisms in proteins can be used in screening. Where many diverse genetic mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools. The activity of the encoded protein can be determined by comparison with the wild-type protein.
  • Polypeptides encoded by SEQ ID NOs:l and 3 or by the corresponding full length genes can be used to screen peptide libraries to identify binding partners, such as ligands and receptors, from among the encoded polypeptides.
  • Peptide libraries can be synthesized according to methods known in the art (see, e.g., U.S. Patent No. 5,010,175, and WO 91/17823). Screening methods may include, but are not limited to, yeast two-hybrid screens, protein co-immunoprecipitation assays, and lambda gtl l expression library screening.
  • Agonists or antagonists of the polypeptides of the invention can be screened using assay conditions that ideally resemble the conditions under which the native activity, i.e. interaction between Notch receptor and Notch ligand, is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native ligand can require concentrations equal to or greater than the native concentration, while inhibitors capable of binding irreversibly to the ligand can be added in concentrations on the order of the native concentration. In a non-limiting example, a peptide analog or antagonist would be tested for its ability to affect interaction between a ligand of the invention and a Notch receptor.
  • compositions of the invention can comprise polypeptides, antibodies, or polynucleotides (including antisense nucleotides and ribozymes) of the claimed invention in a therapeutically effective amount.
  • therapeutically effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance.
  • an effective dose will generally be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the polynucleotide constructs in the individual to which it is administered.
  • a non-limiting example of a pharmaceutical composition is a composition that either enhances or diminishes binding of Notch to a ligand comprising a polypeptide of SEQ ID NO:2 and 4 or fragment thereof. Where the ligand/Notch interaction promotes a disease-related process, interference with the interaction would be the goal of the therapy.
  • compositions that restores normal function are appropriate.
  • it may be desirable to promote angiogenesis for example in wound healing and in therapeutic angiogenesis, for example to treat ischemia.
  • a pharmaceutical composition can also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • Liposomes are included within the definition of a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions of the invention can be (1) administered directly to the subject (e.g., as polynucleotides or polypeptides); or (2) delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy).
  • Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously or intramuscularly, myocardial, intratumoral, peritumoral, or to the interstitial space of a tissue.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule.
  • Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778.
  • Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.
  • nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, direct micro injection of the DNA into nuclei, and viral-mediated, such as adenovirus or alphavirus, all well known in the art.
  • disorders of proliferation can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme), agonist, or antagonist.
  • the therapeutic agent can be administered in conjunction with one or more other agents including but not limited to FGF, VEGF, angiopoietin, angiogenin, and thrombopoietin.
  • Administered "in conjunction” includes administration at the same time, or within 1 day, 12 hours, 6 hours, one hour, or less than one hour, as the other therapeutic agent(s).
  • compositions may be mixed for co-administration, or may be administered separately by the same or different routes.
  • the dose and the means of administration of the inventive pharmaceutical compositions are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors.
  • administration of polynucleotide therapeutic compositions agents of the invention includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • the therapeutic polynucleotide composition can contain an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nucleotides of the polynucleotides disclosed herein.
  • a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor.
  • arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor.
  • a tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor.
  • the antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition.
  • X-ray imaging is used to assist in certain of the above delivery methods.
  • Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues can also be used.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol (1993) 77:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 2(55:621; Wu et al., J. Biol. Chem. (1994) 2f59:542; Zenke et al., Proc. Natl. Acad. Sci.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA can also be used during a gene therapy protocol.
  • Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
  • the present invention also relates to antisense oligonucleotides designed to interfere with the normal function of 3md3 and/or 2hdl polynucleotides.
  • Any modifications or variations of the antisense molecule which are known in the art to be broadly applicable to antisense technology are included within the scope of the invention. Such modifications include preparation of phosphorus-containing linkages as disclosed in U.S. Patents 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361, 5,625,050 and 5,958,773.
  • the antisense compounds of the invention can include modified bases as disclosed in 5,958,773 and patents disclosed therein.
  • the antisense oligonucleotides of the invention can also be modified by chemically linking the oligonucleotide to one or more moieties or conjugates to enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide.
  • moieties or conjugates include lipids such as cholesterol, cholic acid, thioether, aliphatic chains, phospholipids, polyamines, polyethylene glycol (PEG), palmityl moieties, and others as disclosed in, for example, U.S. Patents 5,514,758, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,597,696 and 5,958,773.
  • Chimeric antisense oligonucleotides are also within the scope of the invention, and can be prepared from the present inventive oligonucleotides using the methods described in, for example, U.S. Patents 5,013,830, 5,149,797, 5,403,711, 5,491,133, 5,565,350, 5,652,355, 5,700,922 and 5,958,773.
  • the antisense molecule preferably is targeted to an accessible, or exposed, portion of the target RNA molecule.
  • the current approach to inhibition using antisense is via experimentation.
  • mRNA levels in the cell can be measured routinely in treated and control cells by reverse transcription of the mRNA and assaying the cDNA levels.
  • the biological effect can be determined routinely by measuring cell growth or viability as is known in the art.
  • antisense oligonucleotides can be added to cultures of endothelial cells, and the effect on endothelial cell invasion can be determined, in order to identify antisense compositions useful for in vivo use.
  • RNA from treated and control cells should be reverse-transcribed and the resulting cDNA populations analyzed. (Branch, A. D., T.I.B.S. 25:45-50, 1998.)
  • the therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 7:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 7:185; and Kaplitt, Nature Genetics (1994) (5:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5, 219,740; WO 93/11230; WO 93/10218; U.S. Patent No. 4,777,127; GB Patent No.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR- 1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/1 1984 and WO 95/00655).
  • AAV adeno-associated virus
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 5:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. 2(54:16985 (1989)); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. 74:2411 (1994), and in Woffendin, Proc. Natl. Acad. Sci. (1994) 97:11581-11585.
  • non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 97(24):11581 (1994).
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Patent No. 5,206,152 and WO 92/11033).
  • Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Patent No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Patent No. 5,206,152 and WO 92/11033).
  • the first ligand is a 583 amino acid protein encoded by SEQ ID NO: l .
  • the sequences are shown in Figure 5, and the ligand is referred to as 3md3.
  • the ligand is referred to as 3md3.
  • 3md3 is putatively identified as the human ortholog of mouse Delta 3 ( Figure 1). Although a patent publication has identified a human "Delta 3" (WO 98 45434-A1), comparison of its alignment to mouse Delta 3, with 3md3 alignment to mouse Delta 3, indicates that 3md3 bears closer resemblance based on both amino acid ( Figure 1) and polynucleotide ( Figure 3) comparisons.
  • 3md3 The expression of 3md3 in human tissues was investigated using an EST AI363919 (Image Clone No. 2016425) as a probe.
  • the results from the Northern blot indicate that 3md3 was expressed as a 4.9 kb message in heart, kidney, skeletal muscle, and liver.
  • Table 1 shows the results of in situ hybridization of the 3md3 probe to normal and cancer tissue samples. Each result represents the data from six separate arrays.
  • 3md3 is expressed at a higher level in the following types of cancer compared to the corresponding normal tissue: adrenal, lung, pancreas, and thyroid.
  • 3md3 is expressed at a lower level in the following types of cancer compared to the corresponding normal tissue: colon, esophagus, liver, prostate, and stomach.
  • the second ligand is a protein encoded by SEQ ID NO:3.
  • the sequences are shown in Figure 6, and the ligand is referred to as 2hdl .
  • Adrenal cortex-derived bovine microvascular endothelial (BME) cells are grown in an ⁇ -modified minimal essential medium (Life Technologies, Inc. AG, Basel, Switzerland), supplemented with 15%> heat-inactivated donor calf serum (DCS, Life Technologies, Inc.), penicillin (110 units/ml), and streptomycin (110 ⁇ g/ml). BME cells are subcultured at a 1 :4 split ratio in 1.5% gelatin-coated tissue culture dishes or flasks (Falcon Labware, Becton Dickinson Company, Lincoln Park, NJ). The in vitro angiogenesis assay is performed as described (Montesano, R.
  • BME cells are seeded at 5.0-7.5 x 10 4 cells/well in 500 ⁇ l of ⁇ -modified minimal essential medium, 5% DCS (donor calf serum). Prior to reaching confluence (less than 3 days), DCS is further reduced to 2%, and cells are treated with recombinant human FGF-2, recombinant human VEGF, and antisense oligonucleotides. Oligonucleotides are added to the cells 2 h before cytokines on the first day of treatment.
  • HMEC human microvascular endothelial cells
  • mRNA expression levels are assayed by preparing probes for Northern blots as described above.
  • bFGF-treated HMEC are prepared by incubation with bFGF at 10 mg/ml for 2 hours.
  • VEGF-treated HMEC are prepared by incubation with 20 ng/ml VEGF for 2 hours. Following incubation with the respective growth factor, the cells are washed and lysis buffer added for RNA preparation.
  • HMEC treated as above can also be exposed to one or more antisense oligomers based on SEQ ID NO:l or 3, using methods described by Zimrin, A. et al., J. Biol. Chem. 277:32499-32502, 1996.
  • EXAMPLE 4 EFFECT OF 3MD3 EXPRESSION ON TUMOR INDUCED ANGIOGENESIS ,V VIVO
  • Adenoviruses encoding (i) the extracellular domain of 3md3, and (ii) full length 3md3 are injected into the retroperitoneal adipose tissue alone or in combination with adenoviruses encoding VEGF or bFGF using methods described by Magovem, C.J. et al., Hum. Gene Ther. 5:215, 1997, and U.S. Patent No. 5,869,037. Neovascularization of the adipose tissue is assessed on day 20 and day 30 post-injection by histological evaluation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Dermatology (AREA)
  • Immunology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Cardiology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des polynucléotides codant pour les ligands récepteurs de Notch, des polypeptides codés, ainsi que des anticorps spécifiques de ces polypeptides. Elle concerne également des procédés et compositions destinés à accroître ou inhiber l'angiogenèse de même qu'à moduler des réponses immunes.
EP00957528A 1999-08-19 2000-08-17 Ligands recepteurs du gene notch, et utilisations associees Ceased EP1206487A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14993499P 1999-08-19 1999-08-19
US149934P 1999-08-19
PCT/US2000/022609 WO2001012664A2 (fr) 1999-08-19 2000-08-17 Ligands recepteurs du gene notch, et utilisations associees

Publications (1)

Publication Number Publication Date
EP1206487A2 true EP1206487A2 (fr) 2002-05-22

Family

ID=22532424

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00957528A Ceased EP1206487A2 (fr) 1999-08-19 2000-08-17 Ligands recepteurs du gene notch, et utilisations associees

Country Status (4)

Country Link
EP (1) EP1206487A2 (fr)
JP (1) JP2003507029A (fr)
AU (1) AU6913100A (fr)
WO (1) WO2001012664A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1446424A2 (fr) * 2001-11-14 2004-08-18 Lorantis Limited Composition comprenant des inhibiteurs de la voie signalation de notch pour la modulation du systeme immunitaire
WO2005014854A1 (fr) 2003-08-08 2005-02-17 Licentia, Ltd. Substances et methodes pour le depistage, le diagnostic et le traitement du cancer colorectal
DK3342786T3 (en) 2010-01-29 2021-09-27 Chugai Pharmaceutical Co Ltd Anti-dll3-antistof
CN104520324A (zh) 2012-02-24 2015-04-15 施特姆森特Rx股份有限公司 Dll3调节剂及其使用方法
HUE043851T2 (hu) 2013-02-22 2019-09-30 Abbvie Stemcentrx Llc Anti-DLL3-antitest-PBD konjugátumok és alkalmazásuk
EP3892294A1 (fr) 2013-08-28 2021-10-13 AbbVie Stemcentrx LLC Procédés et compositions de conjugaison d'anticorps spécifique à un site
US10308721B2 (en) 2014-02-21 2019-06-04 Abbvie Stemcentrx Llc Anti-DLL3 antibodies and drug conjugates for use in melanoma
JP7314146B2 (ja) 2017-12-28 2023-07-25 中外製薬株式会社 細胞傷害誘導治療剤
KR102505383B1 (ko) 2020-03-31 2023-03-02 추가이 세이야쿠 가부시키가이샤 Dll3 표적 다중 특이성 항원 결합 분자 및 그의 사용

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5786158A (en) * 1992-04-30 1998-07-28 Yale University Therapeutic and diagnostic methods and compositions based on notch proteins and nucleic acids
ATE342358T1 (de) * 1997-04-04 2006-11-15 Millennium Biotherapeutics Inc Neue menschliche delta3-zusammensetzung und deren therapeutische und diagnostische verwendungen
CA2288218C (fr) * 1997-05-14 2013-03-12 Asahi Kasei Kogyo Kabushiki Kaisha Nouvel inhibiteur de differentiation
JP4139508B2 (ja) * 1998-02-19 2008-08-27 旭化成株式会社 ヒトデルター3

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0112664A2 *

Also Published As

Publication number Publication date
WO2001012664A3 (fr) 2002-01-10
JP2003507029A (ja) 2003-02-25
AU6913100A (en) 2001-03-13
WO2001012664A2 (fr) 2001-02-22

Similar Documents

Publication Publication Date Title
US7279554B2 (en) Notch receptor ligands and uses thereof
AU700915B2 (en) Lung cancer marker
JP2009000114A (ja) 腫瘍治療のための組成物及び方法
KR20090111307A (ko) 전립선 특이적 전사물 및 전립선암의 치료 및 진단에 사용되는 이의 용도
US7442520B2 (en) Gene BRCC-3 and diagnostic and therapeutic uses thereof
US7253272B2 (en) Gene BRCC-2 and diagnostic and therapeutic uses thereof
US20030228317A1 (en) Gene BRCC-1 and diagnostic and therapeutic uses thereof
WO2001012664A2 (fr) Ligands recepteurs du gene notch, et utilisations associees
US5726018A (en) Nucleic acid based assays to detect a novel mammalian protein associated with uncontrolled cell division
JP2004533206A (ja) 化学療法のための標的としてのガン関連遺伝子
JP2012143242A (ja) 乳癌において差次的に発現される遺伝子
US8236928B2 (en) Genes differentially expressed in breast cancer
US20020061552A1 (en) Mammalian dishevelled-associated proteins
US20040235104A1 (en) Human SEF molecule and uses therefor
JP2003519480A5 (fr)
US7309783B2 (en) Mammalian early developmental regulator gene
US20030186232A1 (en) Human and non-human primate homologues of Nkd protein, nucleic acid sequences encoding, and uses thereof
US20040161775A1 (en) Gene SHINC-1 and diagnostic and therapeutic uses thereof
CA2245340A1 (fr) Proteine associee au traf4

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020312

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: WILLIAMS, LEWIS, T.

Inventor name: VIVIEN, CHAN

Inventor name: ROHAN, MICHAEL

17Q First examination report despatched

Effective date: 20050222

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: NOVARTIS VACCINES AND DIAGNOSTICS, INC.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20100715