EP1266000A1 - Chordin-like-2 molekule und deren verwendungen - Google Patents

Chordin-like-2 molekule und deren verwendungen

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Publication number
EP1266000A1
EP1266000A1 EP01913290A EP01913290A EP1266000A1 EP 1266000 A1 EP1266000 A1 EP 1266000A1 EP 01913290 A EP01913290 A EP 01913290A EP 01913290 A EP01913290 A EP 01913290A EP 1266000 A1 EP1266000 A1 EP 1266000A1
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Prior art keywords
polypeptide
seq
chl2
amino acid
set forth
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French (fr)
Inventor
Ke Zhang
Cam Linh
Naoki Nakayama
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Amgen Inc
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Amgen Inc
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates to Chordin-Like-2 (CHL2) polypeptides and nucleic acid molecules encoding the same.
  • the invention also relates to selective binding agents, vectors, host cells, and methods for producing CHL2 polypeptides.
  • the invention further relates to pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, and/or prevention of diseases, disorders, and conditions associated with CHL2 polypeptides.
  • CHL2 is structurally related to the bone morphogenetic protein (BMP) inhibitor known as chordin (CHD), (Sasai et al., 1994, Cell 19:119-90), or short gastrulation (SOG; Francois, et al., 1994, Genes Dev. 8:2602-16).
  • BMP bone morphogenetic protein
  • CHD chordin
  • SOG short gastrulation
  • the CHL2 gene is believed to be a member of CHD/SOG family.
  • Bone morphogenetic protein is a member of the transforming growth factor-beta family, which was originally identified as a factor promoting bone formation from a cartridge implant (Wozney et al., 1988, Science 242:1528- 34; Celeste et al., 1990, Proc Nat. Acad. Sci. USA 87:9843-47). BMP is also known to play an essential role during the early embryogenesis of the frog, the fly, and in mammals. The precise concentration of active BMP seems to be important for the specification of particular cell types (Dale et al, 1992, Development 115:573-85; Dosch et al, 199 Development 124:2325-34). An activity gradient of BMP2/4 is observed in, for example, Xenopus embryos in which the lowest expression is detected at the dorsal tip and the highest expression at the ventral tip
  • BMP concentration at specific sites of tissue development suggests a role for BMP in organogenesis.
  • Control of BMP expression is achieved by either localized expression of the BMP gene products or through the influence of the BMP inhibitor chordin (CFfD) (Sasai et al, 1994, Cell 79:779-90)
  • CHD/SOG is a large secreted protein produced from the Spemann's organizer, the master-controlling region for the dorsoventral axis specification at the gastrulation stage of Xenopus embryogenesis.
  • CHD/SOG functions as a dorsalization factor, as does Noggin (Smith and Harland, 1992, Cell 70:829-40), which is also secreted from the organizer.
  • the Drosophila SOG has a transmembrane domain at its amino-terminus, suggesting that it may be a type II transmembrane protein (Francois et al, 1994, Genes Dev. 8:2602-16).
  • CHD/SOG is known to bind to one of the ventralizing factors, BMP4
  • TLD metalloprotease - Tolloid
  • BMP1 BMP1 - that cleaves CHD/SOG to liberate either, or both, the first (CRl) and last (CR4) CR motifs
  • BMP4 itself only migrates a short distance and seems to act essentially on the cell autonomously (Jones et al, 1996, Curr. Biol. 6:1468-75).
  • BMP4 inhibitors Noggin and CHD/SOG appear to exert a long-range effect, thereby forming an activity gradient of BMP4.
  • BMPs also play important roles outside of early embryogenesis, for example in the organogenesis of lung, gut, kidney, skin, heart and teeth, as well as in the later stages of embryogenesis (Hogan, 1996, Genes Dev. 10:1580-94). Some BMPs are expressed in a very localized fashion while others are expressed widely in a tissue. The importance of the localized action of BMP for organogenesis has been supported by transgenic mouse experiments using constructs by which BMP concentration is artificially elevated throughout the target tissue.
  • BMP4 is expressed in the distal tips of epithelium in the developing lung, and when overexpressed with the surfactant protein C promoter, the development of a small lung in which the structural organization (i.e., branching) has been severely disrupted is observed (Bellusci et al, 1996, Development 122:1693-702). Since the putative BMP-activity gradient could also be disrupted by the transgene expression, BMPs expressed widely in the tissue could also play a role in the determination of the structural organization of a tissue.
  • Noggin is another BMP2/4 inhibitor secreted from Spemann's organizer (Zimmerman et al, 1996, Cell 86:599-606).
  • the biological role of Noggin and its mode of action are similar to CHD/SOG in Xenopus.
  • Noggin null-mutant mice have shown a bone phenotype (hyperplasia of chondrocytes) instead of an early embryonic phenotype (McMahon et al, 1998, Genes Dev. 12:1438-52; Brunet et al, 1998, Science 280:1455-57). This suggests that CHL2 or even CHD might have a non-dispensable function in the later stage of embryogenesis.
  • the present invention relates to novel CHL2 nucleic acid molecules and encoded polypeptides.
  • the invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • the invention also provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, the nucleotide sequence of the DNA insert in either ATCC Deposit Nos. PTA- 1479 or PTA-1480, or (a);
  • the invention further provides for an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acid substitution, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; (b) a nucleotide sequence encoding a polypeptide as set forth in either
  • nucleotide sequence encoding a polypeptide as set forth in either SEQ LD NO: 2 or SEQ ID NO: 5 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C-terminal truncation, and N-terminal truncation, wherein the encoded polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;
  • the present invention provides for an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5; and
  • the invention also provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:
  • the invention further provides for an isolated polypeptide comprising the amino acid sequence selected from the group consisting of:
  • polypeptide (e) the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, C- terminal truncation, and N-terminal truncation, wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO : 2 or SEQ ID NO : 5.
  • the invention still further provides for an isolated polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 5 with at least one conservative amino acid substitution selected from the group consisting of: leucine or methionine at position 2; methionine at position 5; lysine at position 6; alanine at position 7; isoleucine at position 8; phenylalanine at position 14; leucine at position 15; threonine at position 23; leucine at position 25; valine at position 27; glutamic acid at position 30; tyrosine at position 32; methionine at position 34; glutamine at position 36; lysine at position 39; alanine at position 41; threonine at position 45; valine at position 55; valine at position 59; asparagine at position 60; proline at position 66; asparagine at position 68; serine or threonine at position 72; valine at position 74; arginine at position 75; arginine at position
  • leucine at position 334 asparagine at position 337; valine at position 339; leucine at position 340; serine at position 342; phenylalanine at position 344; glutamic acid at position 349; isoleucine or valine at position 354; methionine at position 356; valine at position 366; methionine or valine at position 368; isoleucine at position 371; leucine at position 375; leucine at position 376; glutamine at position 377; phenylalanine at position 381; asparagine at position 383; isoleucine at position 384; leucine at position 393; arginine at position 395; valine at position 398; alanine or valine at position 399; tyrosine at position 403; asparagine at position 406; isoleucine at position 409; alanine or valine at position 415; isoleucine at position 417; and leucine at
  • the present invention also provides for an expression vector comprising the isolated nucleic acid molecules as set forth herein, recombinant host cells comprising the recombinant nucleic acid molecules as set forth herein, and a method of producing a CHL2 polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced.
  • a transgenic non-human animal comprising a nucleic acid molecule encoding a CHL2 polypeptide is also encompassed by the invention.
  • the CHL2 nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of a CHL2 polypeptide, which may include increased circulating levels.
  • the CHL2 nucleic acid molecules are introduced into the animal in a manner that prevents expression of endogenous CHL2 polypeptide (i.e., generates a transgenic animal possessing a CHL2 polypeptide gene knockout).
  • the transgenic non-human animal is preferably a mammal, and more preferably a rodent, such as a rat or a mouse.
  • CHL2 polypeptides of the present invention are also provided.
  • selective binding agents such as antibodies and peptides capable of specifically binding the CHL2 polypeptides of the invention.
  • Such antibodies and peptides may be agonistic or antagonistic.
  • compositions comprising the nucleotides, polypeptides, or selective binding agents of the invention and one or more pharmaceutically acceptable formulation agents are also encompassed by the invention.
  • the pharmaceutical compositions are used to provide therapeutically effective amounts of the nucleotides or polypeptides of the present invention.
  • the invention is also directed to methods of using the polypeptides, nucleic acid molecules, and selective binding agents.
  • the CHL2 polypeptides and nucleic acid molecules of the present invention may be used to treat, prevent, ameliorate, and/or detect diseases and disorders, including those recited herein.
  • the present invention also provides a method of assaying test molecules to identify a test molecule that binds to a CHL2 polypeptide.
  • the method comprises contacting a CHL2 polypeptide with a test molecule to determine the extent of binding of the test molecule to the polypeptide.
  • the method further comprises determining whether such test molecules are agonists or antagonists of a CHL2 polypeptide.
  • the present invention further provides a method of testing the impact of molecules on the expression of CHL2 polypeptide or on the activity of CHL2 polypeptide.
  • Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of a CHL2 polypeptide are also encompassed by the invention.
  • One method comprises administering to an animal a nucleic acid molecule encoding a CHL2 polypeptide.
  • a nucleic acid molecule comprising elements that regulate or modulate the expression of a CHL2 polypeptide may be administered. Examples of these methods include gene therapy, cell therapy, and anti-sense therapy as further described herein.
  • the CHL2 polypeptides may be used for identifying receptors thereof ("CHL2 polypeptide receptors").
  • CHL2 polypeptide receptors Various forms of "expression cloning" have been extensively used to clone receptors for protein ligands. See, e.g., Simonsen and Lodish, 1994, Trends Pharmacol Sci. 15:437-41 and Tartaglia et al, 1995, Cell 83:1263-71.
  • the isolation of a CHL2 polypeptide receptor is useful for identifying or developing novel agonists and antagonists of the CHL2 polypeptide signaling pathway.
  • Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor-selective binding agents (such as antibodies and derivatives thereof), small molecules, and antisense oligonucleotides, any of which can be used for treating one or more disease or disorder, including those disclosed herein.
  • Figures 1A-1C illustrate the nucleotide sequence of the murine CHL2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of murine CHL2 polypeptide (SEQ ED NO: 2). The predicted signal sequence is indicated (underline);
  • FIG 2 illustrates an amino acid sequence alignment of murine CHL2 polypeptide (mouse CHL2; SEQ ID NO: 2) and murine chordin (Af069501; SEQ ID NO: 7);
  • Figures 3A-3C illustrate the nucleotide sequence of the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of human CHL2 polypeptide (SEQ ID NO: 5). The predicted signal sequence is indicated (underline):
  • Figure 4 illustrates an amino acid sequence alignment of human chordin (huCHD; SEQ ID NO: 8), human CHL1 polypeptide (huCHL; SEQ ED NO: 9), and human CHL2 polypeptide (huCHL2; SEQ ID NO: 5);
  • Figure 5 illustrates a schematic representation of murine chordin (Chordin), CHL1 polypeptide, and CHL2 polypeptide.
  • Pro-collagen repeats CR; homologous CR domains are indicated by gray boxes
  • signal peptides SP
  • putative BMPI/Tolloid cleavage sites *
  • sites of amino acid sequence variation in CHL1 dE and d5 are indicated;
  • Figure 6 illustrates the expression of murine CHL2 mRNA as detected by in situ hybridization in E17.5 mouse hip joint (panels A and B) and costal-chondral articulation (panel C; showing signal in articular chondrocytes on both sides of the articulation);
  • Figure 7 illustrates the expression of murine CHL2 mRNA as detected by in situ hybridization in normal adult vertebral articulation (panels A, B, and C; showing signal in articular chondrocytes at the surface of the articular cartilage on both sides of the zygapophyseal or facet joint between the processes of adjacent vertebrae) and the fibrocartilage of the anulus f ⁇ brosus of a vertebral disc (panels A, B, and C; showing signal in articular chondrocytes at the surface of the articular cartilage on both sides of the zygapophyseal or facet joint between the processes of adjacent vertebrae) and the fibrocartilage of the anulus f ⁇ brosus of a vertebral disc (panels A, B, and C; showing signal in articular chondrocytes at the surface of the articular cartilage on both sides of the zygapophyseal or facet joint between the processes of adjacent vertebrae
  • Figure 8 illustrates the expression of murine CHL2 mRNA as detected by in situ hybridization in El 8.5 mouse sternum and placenta and normal adult mouse uterus, colon, and small intestine;
  • Figure 9 illustrates the secondary axis-forming activity of murine CHL2 polypeptide
  • Figure 10 illustrates the effect of CHL2 polypeptide on the BMP4-dependent generation of CD34 + /CD31 + erythro-myeloid progenitor cells (R7) and CD34 " /CD31+ cells (R3);
  • Figure 11 illustrates the effect of CHL2 polypeptide on the BMP2-dependent induction of alkaline phosphatase in C2C12 myoblastic cells
  • Figure 12 illustrates the results of Western blot analysis using mCHL2-FLAG proteins.
  • mCHL2-FLAG was mixed with BMP5 or activin A, treated with an anti-mCHL2 antiserum, and precipitated with protein A agarose beads.
  • mCHL2-FLAG was mixed with BMP2, BMP4, BMP5, BMP6, GDF5 (BMP 14), or activin A, treated with an anti-mCHL2 antiserum, and precipitated with protein A agarose beads.
  • CHL2 gene or “CHL2 nucleic acid molecule” or “CHL2 polynucleotide” refer to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ED NO: 4, a nucleotide sequence encoding the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, a nucleotide sequence of the DNA insert in either ATCC Deposit Nos. PTA-1479 or PTA-1480, and nucleic acid molecules as defined herein.
  • CHL2 polypeptide allelic variant refers to one of several possible naturally occurring alternate forms of a gene occupying a . given locus on a chromosome of an organism or a population of organisms.
  • CHL2 polypeptide splice variant refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences in an RNA transcript of CHL2 polypeptide amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • isolated nucleic acid molecule refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "isolated nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • nucleic acid sequence refers to a DNA or RNA sequence.
  • the term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy- methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-
  • vector is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.
  • expression vector refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • operably linked is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function.
  • flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence.
  • a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
  • a flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • the term "host cell” is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.
  • CHL2 polypeptide refers to a polypeptide comprising the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5 and related polypeptides.
  • Related polypeptides include CHL2 polypeptide fragments, CHL2 polypeptide orthologs, CHL2 polypeptide variants, and CHL2 polypeptide derivatives, which possess at least one activity of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ED NO: 5.
  • CHL2 polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino-temiinal methionine residue, depending on the method by which they are prepared.
  • CHL2 polypeptide fragment refers to a polypeptide that comprises a truncation at the amino-terminus (with or without a leader sequence) and/or a truncation at the carboxyl-terminus of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • CHL2 polypeptide fragment also refers to amino-terminal and/or carboxyl-teraiinal truncations of CHL2 polypeptide orthologs, CHL2 polypeptide derivatives, or CHL2 polypeptide variants, or to amino-terminal and/or carboxyl-termmal truncations of the polypeptides encoded by CHL2 polypeptide allelic variants or CHL2 polypeptide splice variants.
  • CHL2 polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity.
  • Membrane-bound forms of a CHL2 polypeptide are also contemplated by the present invention.
  • truncations and/or deletions comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids.
  • the polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acids.
  • Such CHL2 polypeptide fragments may optionally comprise an amino-terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to CHL2 polypeptides.
  • CHL2 polypeptide ortholog refers to a polypeptide from another species that corresponds to CHL2 polypeptide amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • mouse and human CHL2 polypeptides are considered orthologs of each other.
  • CHL2 polypeptide variants refers to CHL2 polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or CHL2 polypeptide fragments), and/or additions (such as internal additions and/or CHL2 fusion polypeptides) as compared to the CHL2 polypeptide amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 (with or without a leader sequence).
  • Variants may be naturally occurring (e.g., CHL2 polypeptide allelic variants, CHL2 polypeptide orthologs, and CHL2 polypeptide splice variants) or artificially constructed.
  • Such CHL2 polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4.
  • the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.
  • CHL2 polypeptide derivatives refers to the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ED NO: 5, CHL2 polypeptide fragments, CHL2 polypeptide orthologs, or CHL2 polypeptide variants, as defined herein, that have been chemically modified.
  • CHL2 polypeptide derivatives also refers to the polypeptides encoded by CHL2 polypeptide allelic variants or CHL2 polypeptide splice variants, as defined herein, that have been chemically modified.
  • mature CHL2 polypeptide refers to a CHL2 polypeptide lacking a leader sequence.
  • a mature CHL2 polypeptide may also include other modifications such as proteolytic processing of the amino-terminus (with or without a leader sequence) and/or the carboxyl-terminus, cleavage of a smaller polypeptide from a larger precursor, N-linked and/or O-linked glycosylation, and the like.
  • Exemplary mature CHL2 polypeptides are depicted by the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 6.
  • CHL2 fusion polypeptide refers to a fusion of one or more amino acids (such as a heterologous protein or peptide) at the amino- or carboxyl- terminus of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5, CHL2 polypeptide fragments, CHL2 polypeptide orthologs, CHL2 polypeptide variants, or CHL2 derivatives, as defined herein.
  • CHL2 fusion polypeptide also refers to a fusion of one or more amino acids at the amino- or carboxyl-terminus of the polypeptide encoded by CHL2 polypeptide allelic variants or CHL2 polypeptide splice variants, as defined herein.
  • biologically active CHL2 polypeptides refers to CHL2 polypeptides having at least one activity characteristic of the polypeptide comprising the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5.
  • a CHL2 polypeptide may be active as an immunogen; that is, the CHL2 polypeptide contains at least one epitope to which antibodies may be raised.
  • isolated polypeptide refers to a polypeptide of the present invention that (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated polypeptide” is linked in nature, (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (4) does not occur in nature.
  • the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences.
  • Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”).
  • similarity is a related concept, but in contrast to “identity,”
  • similarity refers to a measure of relatedness which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non- conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.
  • non-naturally occurring or “non-native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man.
  • non-naturally occurring or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
  • phrases “effective amount” and “therapeutically effective amount” each refer to the amount of a CHL2 polypeptide or CHL2 nucleic acid molecule used to support an observable level of one or more biological activities of the CHL2 polypeptides as set forth herein.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of the CHL2 polypeptide, CHL2 nucleic acid molecule, or CHL2 selective binding agent as a pharmaceutical composition.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a selective binding agent such as an antibody
  • An antigen may have one or more epitopes.
  • selective binding agent refers to a molecule or molecules having specificity for a CHL2 polypeptide.
  • specific and specificity refer to the ability of the selective binding agents to bind to human CHL2 polypeptides and not to bind to human non-CHL2 polypeptides. It will be appreciated, however, that the selective binding agents may also bind orthologs of the polypeptide as set forth in either SEQ ED NO: 2 or SEQ ID NO: 5, that is, interspecies versions thereof, such as mouse and rat CHL2 polypeptides.
  • transduction is used to refer to the transfer of genes from one bacterium to another, usually by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses.
  • transfection is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52:456; Sambrook et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al, Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al, 1981, Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of any of SEQ ID NO: 1 or SEQ ED NO: 4, and include sequences which are complementary to any of the above nucleotide sequences.
  • Related nucleic acid molecules also include a nucleotide sequence encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide in either SEQ ID NO: 2 or SEQ ED NO: 5.
  • Such related CHL2 polypeptides may comprise, for example, an addition and/or a deletion of one or more N-linked or O-linked glycosylation sites or an addition and/or a deletion of one or more cysteine residues.
  • nucleic acid molecules also include fragments of CHL2 nucleic acid molecules which encode a polypeptide of at least about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than 200 amino acid residues of the CHL2 polypeptide of any of SEQ ED NO: 2 or SEQ ID NO: 5.
  • CHL2 nucleic acid molecules also include those molecules which comprise nucleotide sequences which hybridize under moderately or highly stringent conditions as defined herein with the fully complementary sequence of the CHL2 nucleic acid molecule of any of SEQ ID NO: 1 or SEQ ID NO: 4, or of a molecule encoding a polypeptide, which polypeptide comprises the amino acid sequence as shown in either SEQ ID NO: 2 or SEQ ID NO: 5, or of a nucleic acid fragment as defined herein, or of a nucleic acid fragment encoding a polypeptide as defined herein.
  • Hybridization probes may be prepared using the CHL2 sequences provided herein to screen cDNA, genomic or synthetic DNA libraries for related sequences.
  • Regions of the DNA and/or amino acid sequence of CHL2 polypeptide that exhibit significant identity to known sequences are readily determined using sequence alignment algorithms as described herein and those regions may be used to design probes for screening.
  • the term "highly stringent conditions” refers to those conditions that are designed to permit hybridization of DNA strands whose sequences are highly complementary, and to exclude hybridization of significantly mismatched DNAs. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide.
  • highly stringent conditions for hybridization and washing are 0.015 M sodium CHL2oride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium CHL2oride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al, Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited).
  • More stringent conditions may also be used - however, the rate of hybridization will be affected.
  • Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1 % sodium pyrophosphate, 0.1 % sodium dodecylsulfate, NaDodSO 4 , (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used.
  • moderately stringent conditions refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under “highly stringent conditions” is able to form.
  • typical “moderately stringent conditions” are 0.015 M sodium CHL2oride, 0.0015 M sodium citrate at 50-65°C or 0.015 M sodium CHL2oride, 0.0015 M sodium citrate, and 20%> formamide at 37-50°C.
  • “moderately stringent conditions” of 50°C in 0.015 M sodium ion will allow about a 21% mismatch.
  • Tm 2°C per A-T base pair + 4°C per G-C base pair *The sodium ion concentration in 6X salt sodium citrate (SSC) is IM.
  • SSC 6X salt sodium citrate
  • nucleic acid molecules comprise or consist df a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or comprise or consist essentially of a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ED NO: 5.
  • the nucleotide sequences are about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or the nucleotide sequences encode a polypeptide that is about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the polypeptide sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • Related nucleic acid molecules encode polypeptides possessing at least one activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • CHL2 polypeptides having functional and chemical characteristics similar to those of CHL2 polypeptides.
  • substantial modifications in the functional and/or chemical characteristics of CHL2 polypeptides may be accomplished by selecting substitutions in the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5 that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scaiming mutagenesis.”
  • amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • substituted residues may be introduced into regions of the human CHL2 polypeptide that are homologous with non-human CHL2 polypeptides, or into the non-homologous regions of the molecule.
  • hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • the hydropathic indices are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8) cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4) threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6) histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine ( ⁇
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al, 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (- 0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1-3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • amino acid substitutions can be used to identify important residues of the CHL2 polypeptide, or to increase or decrease the affinity of the CHL2 polypeptides described herein.
  • amino acid substitutions are set forth in Table I.
  • a skilled artisan will be able to determine suitable variants of the polypeptide as set forth in either SEQ ED NO: 2 or SEQ ID NO: 5 using well- known techniques.
  • suitable areas of the molecule may be changed without destroying biological activity.
  • one skilled in the art may target areas not believed to be important for activity.
  • one skilled in the art may compare the amino acid sequence of a CHL2 polypeptide to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of CHL2 polypeptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each amino acid residue. The variants could be screened using activity assays known to those with skill in the art. Such variants could be used to gather information about suitable variants.
  • polypeptides or proteins which have a sequence identity of greater than 30%>, or similarity greater than 40%, often have similar structural topologies.
  • PDB protein structural database
  • CHL2 polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the amino acid sequence set forth in either SEQ ED NO: 2 or SEQ ED NO: 5.
  • CHL2 polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline.
  • substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain.
  • substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain.
  • rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
  • Additional preferred CHL2 variants include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) as compared to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ED NO: 5.
  • Cysteine variants are useful when CHL2 polypeptides must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • nucleic acid molecules comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 1
  • polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ED NO: 5, or a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ED NO: 5 with at least one amino acid deletion and wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • nucleic acid molecules also comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 wherein the polypeptide has a carboxyl- and/or amino-terminal truncation and further wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
  • nucleic acid molecules also comprise or consist of a nucleotide sequence encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selected from the group consisting of amino acid substitutions, amino acid insertions, amino acid deletions, carboxyl-terminal truncations, and amino-terminal truncations and wherein the polypeptide has an activity of the polypeptide set forth in either SEQ ID NO : 2 or SEQ ED NO : 5.
  • polypeptide comprising the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide, may be fused to a homologous polypeptide to form a homodimer or to a heterologous polypeptide to form a heterodimer.
  • Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow for the detection and/or isolation of a CHL2 fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from the polypeptide comprising the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ED NO: 5, or other CHL2 polypeptide.
  • Fusions can be made either at the amino-terminus or at the carboxyl- terminus of the polypeptide comprising the amino acid sequence set forth in either SEQ ED NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide. Fusions may be direct with no linker or adapter molecule or may be through a linker or adapter molecule.
  • a linker or adapter molecule may be one or more amino acid residues, typically from about 20 to about 50 amino acid residues.
  • a linker or adapter molecule may also be designed with a cleavage site for a DNA restriction endonuclease or for a protease to allow for the separation of the fused moieties. It will be appreciated that once constructed, the fusion polypeptides can be derivatized according to the methods described herein.
  • the polypeptide comprising the amino acid sequence of any of SEQ ID NO: 2 OR SEQ ID NO: 5, or other CHL2 polypeptide is fused to one or more domains of an Fc region of human IgG.
  • Antibodies comprise two functionally independent parts, a variable domain known as "Fab,” that binds an antigen, and a constant domain known as "Fc,” that is involved in effector functions such as complement activation and attack by phagocytic cells.
  • Fab variable domain known as "Fab”
  • Fc constant domain
  • An Fc has a long serum half-life, whereas an Fab is short-lived.
  • an Fc domain When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation, and perhaps even placental transfer. Id. Table II summarizes the use of certain Fc fusions known in the art.
  • a human IgG hinge, CH2, and CH3 region may be fused at either the amino-terminus or carboxyl-terminus of the CHL2 polypeptides using methods known to the skilled artisan, hi another example, a human IgG hinge, CH2, and CH3 region may be fused at either the amino-terminus or carboxyl- terminus of a CHL2 polypeptide fragment (e.g., the predicted extracellular portion of CHL2 polypeptide).
  • the resulting CHL2 fusion polypeptide may be purified by use of a Protein A affinity column.
  • Peptides and proteins fused to an Fc region have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart.
  • a fusion to an Fc region allows for dimerization/multimerization of the fusion polypeptide.
  • the Fc region may be a naturally occurring Fc region, or may be altered to improve certain qualities, such as therapeutic qualities, circulation time, or reduced aggregation.
  • Identity and similarity of related nucleic acid molecules and polypeptides are readily calculated by known methods. Such methods include, but are not limited to those described in Computational Molecular Biology (A.M.
  • Preferred methods to determine identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are described in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J. Mol. Biol. 215:403-10).
  • GCG program package including GAP (Devereux et al, 1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J. Mol. Biol. 215:403-10).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al, BLAST Manual (NCB NLM NTH, Bethesda, MD); Altschul et al, 1990, supra).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in a preferred embodiment, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the claimed polypeptide.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, WI
  • two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span," as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3X the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 0.1X the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • polypeptide sequence comparison A standard comparison matrix is also used by the algorithm (see Dayhoff et al, 5 Atlas of Protein Sequence and Structure (Supp. 3 1978)(PAM250 comparison matrix); Henikoff et al, 1992, Proc. Natl. Acad. Sci USA 89:10915-19 (BLOSUM 62 comparison matrix)).
  • Preferred parameters for polypeptide sequence comparison include the following:
  • the GAP program is useful with the above parameters.
  • the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.
  • Preferred parameters for nucleic acid molecule sequence comparison include the following:
  • the GAP program is also useful with the above parameters.
  • the aforementioned parameters are the default parameters for nucleic acid molecule comparisons.
  • gap opening penalties may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9, September, 1997.
  • the particular choices to be made will be apparent to those of skill in the art and will depend on the specific comparison to be made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of a CHL2 polypeptide can readily be obtained in a variety of ways including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA.
  • a gene encoding the amino acid sequence of a CHL2 polypeptide may be used as a probe to identify orthologs or related genes from the same species.
  • the probes or primers may be used to screen cDNA libraries from various tissue sources believed to express the CHL2 polypeptide.
  • part or all of a nucleic acid molecule having the sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4 may be used to screen a genomic library to identify and isolate a gene encoding the amino acid sequence of a CHL2 polypeptide.
  • conditions of moderate or high stringency will be employed for screening to minimize the number of false positives obtained from the screening.
  • Nucleic acid molecules encoding the amino acid sequence of CHL2 polypeptides may also be identified by expression cloning which employs the detection of positive clones based upon a property of the expressed protein.
  • nucleic acid libraries are screened by the binding an antibody or other binding partner (e.g., receptor or ligand) to cloned proteins that are expressed and displayed on a host cell surface.
  • the antibody or binding partner is modified with a detectable label to identify those cells expressing the desired clone.
  • Recombinant expression techniques conducted in accordance with the descriptions set forth below may be followed to produce these polynucleotides and to express the encoded polypeptides.
  • a nucleic acid sequence that encodes the amino acid sequence of a CHL2 polypeptide into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers.
  • a polynucleotide encoding the amino acid sequence of a CHL2 polypeptide can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the encoded CHL2 polypeptide may be produced in large amounts.
  • PCR polymerase chain reaction
  • cDNA is prepared from poly(A)+RNA or total RNA using the enzyme reverse transcriptase.
  • Two primers typically complementary to two separate regions of cDNA encoding the amino acid sequence of a CHL2 polypeptide, are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.
  • a polymerase such as Taq polymerase
  • Another means of preparing a nucleic acid molecule encoding the amino acid sequence of a CHL2 polypeptide is chemical synthesis using methods well known to the skilled artisan such as those described by Engels et al, 1989, Angew. Chem. Intl.
  • nucleic acid variants contain codons which have been altered for optimal expression of a CHL2 polypeptide in a given host cell. Particular codon alterations will depend upon the CHL2 polypeptide and host cell selected for expression.
  • Codon optimization can be carried out by a variety of methods, for example, by selecting codons which are preferred for use in highly expressed genes in a given host cell.
  • Computer algorithms which incorporate codon frequency tables such as "Eco_high.Cod” for codon preference of highly expressed bacterial genes may be used and are provided by the University of Wisconsin Package Version 9.0 (Genetics Computer Group, Madison, WI).
  • Other useful codon frequency tables include "Celegans_high.cod,” “Celegans_low.cod,” “Drosophilaj ⁇ gh.cod,”
  • nucleic acid molecules encoding CHL2 polypeptide variants may be produced using site directed mutagenesis, PCR amplification, or other appropriate methods, where the primer(s) have the desired point mutations (see
  • a nucleic acid molecule encoding the amino acid sequence of a CHL2 polypeptide is inserted into an appropriate expression vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • a nucleic acid molecule encoding the amino acid sequence of a CHL2 polypeptide may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend in part on whether a CHL2 polypeptide is to be post-translationally modified (e.g., glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable.
  • yeast, insect, or mammalian host cells are preferable.
  • expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secreti
  • the vector may contain a "tag"-encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the CHL2 polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another "tag” such as FLAG, HA (hemaglutinin influenza virus), or myc for which commercially available antibodies exist.
  • This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the CHL2 polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix.
  • the tag can subsequently be removed from the purified CHL2 polypeptide by various means such as using certain peptidases for cleavage.
  • Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, or the flanking sequences may be native sequences which normally function to regulate CHL2 polypeptide expression.
  • flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.
  • Flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art.
  • flanking sequences useful herein - other than the CHL2 gene flanking sequences - will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases.
  • the full nucleotide sequence of a flanking sequence may be known.
  • the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
  • flanking sequence may be obtained using PCR and/or by screening a genomic library with a suitable oligonucleotide and/or flanking sequence fragment from the same or another species.
  • flanking sequence may be not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen ® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for the optimal expression of a CHL2 polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).
  • a transcription termination sequence is typically located 3' of the end of a polypeptide coding region and serves to terminate transcription.
  • a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells. Other selection genes may be used to amplify the gene that will be expressed.
  • Amplification is the process wherein genes that are in greater demand for the production of a protein critical for growth are reiterated in tandem within the cliromosomes of successive generations of recombinant cells.
  • suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase.
  • DHFR dihydrofolate reductase
  • the mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to the amplification of both the selection gene and the DNA that encodes a CHL2 polypeptide. As a result, increased quantities of CHL2 polypeptide are synthesized from the amplified DNA.
  • a ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of a CHL2 polypeptide to be expressed.
  • the Shine- Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content).
  • Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth herein and used in a prokaryotic vector.
  • a leader, or signal, sequence may be used to direct a CHL2 polypeptide out of the host cell.
  • a nucleotide sequence encoding the signal sequence is positioned in the coding region of a CHL2 nucleic acid molecule, or directly at the 5' end of a CHL2 polypeptide coding region.
  • Many signal sequences have been identified, and any of those that are functional in the selected host cell may be used in conjunction with a CHL2 nucleic acid molecule. Therefore, a signal sequence may be homologous (naturally occurring) or heterologous to the CHL2 nucleic acid molecule. Additionally, a signal sequence may be chemically synthesized using methods described herein.
  • the signal sequence may be a component of the vector, or it may be a part of a CHL2 nucleic acid molecule that is inserted into the vector.
  • nucleotide sequence encoding a native CHL2 polypeptide signal sequence joined to a CHL2 polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence joined to a CHL2 polypeptide coding region.
  • the heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • yeast secretion the native CHL2 polypeptide signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders.
  • the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed.
  • the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino-terminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired CHL2 polypeptide, if the enzyme cuts at such area within the mature polypeptide.
  • transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is particularly true where a polypeptide is produced in eukaryotic host cells, especially mammalian host cells.
  • the introns used may be naturally occurring within the CHL2 gene especially where the gene used is a full-length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the gene (as for most cDNAs), the intron may be obtained from another source.
  • the position of the intron with respect to flanking sequences and the CHL2 gene is generally important, as the intron must be transcribed to be effective.
  • the preferred position for the intron is 3' to the transcription start site and 5' to the poly-A transcription termination sequence.
  • the intron or introns will be located on one side or the other (i.e., 5' or 3') of the cDNA such that it does not interrupt the coding sequence.
  • Any intron from any source including viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell into which it is inserted.
  • synthetic introns may be used to practice this invention, provided that it is compatible with the host cell into which it is inserted.
  • more than one intron may be used in the vector.
  • the expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the CHL2 polypeptide. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
  • Constitutive promoters initiate continual gene product production; that is, there is little or no control over gene expression.
  • a large number of promoters, recognized by a variety of potential host cells, are well known.
  • a suitable promoter is operably linked to the DNA encoding CHL2 polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.
  • the native CHL2 promoter sequence may be used to direct amplification and/or expression of a CHL2 nucleic acid molecule.
  • a heterologous promoter is preferred, however, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter, and if it is compatible with the host cell system that has been selected for use.
  • Promoters suitable for use with prokaryotic hosts include the beta- lactamase and lactose promoter systems; alkaline phosphatase; a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence, using linkers or adapters as needed to supply any useful restriction sites.
  • Suitable promoters for use with yeast hosts are also well known in the art.
  • Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • Other suitable mammalian promoters
  • Additional promoters which may be of interest in controlling CHL2 gene expression include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-46; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-40; Kollias et al, 1986, Cell 46:89-94); the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-12); the myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234:1372- 78).
  • Enhancers are cis-acting elements of DNA, usually about 10- 300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus will be used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a CHL2 nucleic acid molecule, it is typically located at a site
  • Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
  • Preferred vectors for practicing this invention are those which are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, pCP I, pCR3, and pcDNA3.1 (hivitrogen, San Diego, CA), pBS ⁇ (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No.
  • plasmids such as Bluescript plasmid derivatives (a high copy number ColEl -based phagemid, Stratagene Cloning Systems, La Jolla CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPOTM TA
  • Cloning ® Kit PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, CA
  • mammalian, yeast or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).
  • the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector for a CHL2 polypeptide into a selected host cell may be accomplished by well known methods including methods such as transfection, infection, calcium CHL2oride, electroporation, microinjection, Hpofection, DEAE-dextran method, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al, supra.
  • Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast, insect, or vertebrate cell).
  • the host cell when cultured under appropriate conditions, synthesizes a CHL2 polypeptide which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
  • suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO), CHO DHFR(-) cells (Uriaub et al, 1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney (HEK) 293 or 293T cells, or 3T3 cells.
  • CHO Chinese hamster ovary cells
  • CHO DHFR(-) cells Uriaub et al, 1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20
  • HEK human embryonic kidney
  • suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line.
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • Other suitable mammalian cell lines include but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.
  • Each of these cell lines is known by and available to those skilled in the art of protein expression.
  • bacterial cells suitable for the present invention are bacterial cells.
  • E. coli e.g., HBIOI, DH5 ⁇ , DH10, and MC1061
  • B. subtilis Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the like may also be employed in this method.
  • Many strains of yeast cells known to those skilled in the art are also available as host cells for the expression of the polypeptides of the present invention.
  • Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia astoris.
  • insect cell systems may be utilized in the methods of the present invention. Such systems are described, for example, in Kitts et al, 1993, Biotechniques, 14:810-17; Lucklow, 1993, Curr. Opin.
  • Preferred insect cells are Sf-9 and Hi5 (Invitrogen).
  • transgenic animals to express glycosylated CHL2 polypeptides.
  • a transgenic milk-producing animal a cow or goat, for example
  • plants to produce CHL2 polypeptides, however, in general, the glycosylation occurring in plants is different from that produced in mammalian cells, and may result in a glycosylated product which is not suitable for human therapeutic use.
  • Host cells comprising a CHL2 polypeptide expression vector may be cultured using standard media well known to the skilled artisan.
  • the media will usually contain all nutrients necessary for the growth and survival of the cells.
  • Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and/or Terrific Broth (TB).
  • Suitable media for culturing eukaryotic cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which may be supplemented with serum and/or growth factors as necessary for the particular cell line being cultured.
  • RPMI 1640 Roswell Park Memorial Institute medium 1640
  • MEM Minimal Essential Medium
  • DMEM Dulbecco's Modified Eagle Medium
  • an antibiotic or other compound useful for selective growth of transfected or transformed cells is added as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • Other compounds for selective growth include ampicillin, tetracycline, and neomycin.
  • the amount of a CHL2 polypeptide produced by a host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel elecfrophoresis, High Performance Liquid Chromatography (HPLC) separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays. If a CHL2 polypeptide has been designed to be secreted from the host cells, the majority of polypeptide may be found in the cell culture medium.
  • the CHL2 polypeptide is not secreted from the host cells, it will be present in the cytoplasm and/or the nucleus (for eukaryotic host cells) or in the cytosol (for gram-negative bacteria host cells).
  • the CHL2 polypeptide situated in the host cell cytoplasm and/or nucleus for eukaryotic host cells
  • the cytosol for gram-negative bacteria host cells
  • the intracellular material can be extracted from the host cell using any standard technique known to the skilled artisan.
  • the host cells can be lysed to release the contents of the periplasm/cytoplasm by French press, homogenization, and/or sonication followed by centrifugation.
  • a CHL2 polypeptide has formed inclusion bodies in the cytosol, the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material can then be treated at pH extremes or with a chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • the solubilized CHL2 polypeptide can then be analyzed using gel electrophoresis, immunoprecipitation, or the like. If it is desired to isolate the CHL2 polypeptide, isolation may be accomplished using standard methods such as those described herein and in Marston et al, 1990, Meth. Enz., 182:264-75.
  • a CHL2 polypeptide may not be biologically active upon isolation.
  • Various methods for "refolding" or converting the polypeptide to its tertiary structure and generating disulfide linkages can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridges.
  • Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric CHL2oride, dithiothreitol(DTT)/dithiane DTT, and 2-2- mercaptoethanol(bME)/dithio-b(ME).
  • a cosolvent may be used or may be needed to increase the efficiency of the refolding, and the more common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.
  • polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate.
  • the polypeptide may be further isolated from the supernatant using methods such as those described herein.
  • CHL2 polypeptide The purification of a CHL2 polypeptide from solution can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (CHL2 polypeptide/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carlsbad, CA) at either its carboxyl- or amino-terminus, it may be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag.
  • CHL2 polypeptide/hexaHis a tag that it contains a tag such as Hexahistidine (CHL2 polypeptide/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carlsbad, CA) at either its carboxyl- or amino-terminus
  • polyhistidine binds with great affinity and specificity to nickel.
  • an affinity column of nickel such as the Qiagen ® nickel columns
  • CHL2 polypeptide/polyHis See, e.g., Current
  • CHL2 polypeptides may be purified through the use of a monoclonal antibody that is capable of specifically recognizing and binding to a CHL2 polypeptide.
  • Suitable procedures for purification include, without limitation, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (including native gel electrophoresis) followed by gel elution, and preparative isoelecfric focusing ("Isoprime” machine/teclmique, Hoefer Scientific, San Francisco, CA).
  • two or more purification techniques may be combined to achieve increased purity.
  • CHL2 polypeptides may also be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Memfield et al, 1963, J Am. Chem. Soc. 85:2149; Houghten et al, 1985, Proc Natl Acad. Sci. USA 82:5132; and Stewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical Co. 1984). Such polypeptides may be synthesized with or without a methionine on the amino-terminus. Chemically synthesized CHL2 polypeptides may be oxidized using methods set forth in these references to form disulfide bridges.
  • Chemically synthesized CHL2 polypeptides are expected to have comparable biological activity to the corresponding CHL2 polypeptides produced recombinantly or purified from natural sources, and thus may be used interchangeably with a recombinant or natural CHL2 polypeptide.
  • CHL2 polypeptide Another means of obtaining CHL2 polypeptide is via purification from biological samples such as source tissues and/or fluids in which the CHL2 polypeptide is naturally found. Such purification can be conducted using methods for protein purification as described herein. The presence of the CFEL2 polypeptide during purification may be monitored, for example, using an antibody prepared against recombinantly produced CHL2 polypeptide or peptide fragments thereof.
  • a number of additional methods for producing nucleic acids and polypeptides are known in the art, and the methods can be used to produce polypeptides having specificity for CHL2 polypeptide. See, e.g., Roberts et al, 1997, Proc. Natl. Acad. Sci. U.S.A.
  • oligonucleotides capable of carrying out a specific biological function.
  • the procedure involves generating a heterogeneous pool of oligonucleotides, each having a 5' randomized sequence, a central preselected sequence, and a 3' randomized sequence.
  • the resulting heterogeneous pool is introduced into a population of cells that do not exhibit the desired biological function. Subpopulations of the cells are then screened for those that exhibit a predetermined biological function.
  • oligonucleotides capable of carrying out the desired biological function are isolated.
  • U.S. Patent Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describe processes for producing peptides or polypeptides. This is done by producing stochastic genes or fragments thereof, and then introducing these genes into host cells which produce one or more proteins encoded by the stochastic genes. The host cells are then screened to identify those clones producing peptides or polypeptides having the desired activity.
  • RAGE-GD Random Activation of Gene Expression for Gene Discovery
  • the process involves the activation of endogenous gene expression or over-expression of a gene by in situ recombination methods. For example, expression of an endogenous gene is activated or increased by integrating a regulatory sequence into the target cell which is capable of activating expression of the gene by non- homologous or illegitimate recombination.
  • the target DNA is first subjected to radiation, and a genetic promoter inserted. The promoter eventually locates a break at the front of a gene, initiating transcription of the gene. This results in expression of the desired peptide or polypeptide.
  • CHL2 polypeptide expression libraries can also be used to create comprehensive CHL2 polypeptide expression libraries, which can subsequently be used for high throughput phenotypic screening in a variety of assays, such as biochemical assays, cellular assays, and whole organism assays (e.g., plant, mouse, etc.).
  • assays such as biochemical assays, cellular assays, and whole organism assays (e.g., plant, mouse, etc.).
  • nucleic acid and polypeptide molecules described herein may be produced by recombinant and other means.
  • selective binding agent refers to a molecule that has specificity for one or more CHL2 polypeptides.
  • Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable selective binding agents may be prepared using methods known in the art.
  • An exemplary CHL2 polypeptide selective binding agent of the present invention is capable of binding a certain portion of the CHL2 polypeptide thereby inhibiting the binding of the polypeptide to a CHL2 polypeptide receptor.
  • Selective binding agents such as antibodies and antibody fragments that bind CHL2 polypeptides are within the scope of the present invention.
  • the antibodies may be polyclonal including monospecif ⁇ c polyclonal; monoclonal (MAbs); recombinant; chimeric; humanized, such as CDR-grafted; human; single chain; and/or bispecific; as well as fragments; variants; or derivatives thereof.
  • Antibody fragments include those portions of the antibody that bind to an epitope on the CHL2 polypeptide. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
  • Polyclonal antibodies directed toward a CHL2 polypeptide generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of CHL2 polypeptide and an adjuvant. It may be useful to conjugate a CHL2 polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immimization, the animals are bled and the serum is assayed for anti-CHL2 antibody titer.
  • a carrier protein that is immunogenic in the species to be immunized
  • aggregating agents such as alum are used to enhance the immune response. After immimization, the animals are bled and the serum is assayed for anti-CHL2 antibody titer.
  • Monoclonal antibodies directed toward CHL2 polypeptides are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture.
  • suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et al, 1975, Nature 256:495-97 and the human B-cell hybridoma method (Kozbor, 1984, J Immunol. 133:3001; Brodeur et al, Monoclonal Antibody Production Techniques and Applications 51-63 (Marcel Dekker, Inc., 1987).
  • Also provided by the invention are hybridoma cell lines that produce monoclonal antibodies reactive with CHL2 polypeptides.
  • Monoclonal antibodies of the invention may be modified for use as therapeutics.
  • One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and/or light (L) chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass.
  • fragments of such antibodies so long as they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; Morrison et al,
  • a monoclonal antibody of the invention is a "humanized" antibody.
  • Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human.
  • Humanization can be performed, for example, using methods described in the art (Jones et al, 1986, Nature 321:522-25; Riechmann et al, 1998, Nature 332:323-27; Verhoeyen et al, 1988, Science 239:1534-36), by substituting at least a portion of a rodent complementarity-determining region (CDR) for the corresponding regions of a human antibody.
  • CDR rodent complementarity-determining region
  • human antibodies that bind CHL2 polypeptides are also encompassed by the invention.
  • transgenic animals e.g., mice
  • a CHL2 polypeptide antigen i.e., having at least 6 contiguous amino acids
  • a carrier i.e., having at least 6 contiguous amino acids
  • such transgenic animals are produced by incapacitating the endogenous loci encoding the heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof.
  • Partially modified animals that is those having less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications.
  • these transgenic animals produce antibodies with human (rather than, e.g., murine) amino acid sequences, including variable regions which are immunospecific for these antigens. See PCT App. Nos. PCT/US96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT App. Nos.
  • Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein. En an alternative embodiment, human antibodies can also be produced from phage-display libraries (Hoogenboom et al, 1991, J Mol. Biol 227:381; Marks et al, 1991, J. Mol. Biol. 222:581). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • PCT App. No. PCT/US98/17364 describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk- receptors using such an approach.
  • Chimeric, CDR grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
  • the anti-CHL2 antibodies of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) for the detection and quantitation of CHL2 polypeptides.
  • the antibodies will bind CHL2 polypeptides with an affinity that is appropriate for the assay method being employed.
  • anti-CHL2 antibodies may be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S,
  • a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase (Bayer, et al, 1990, Meth. Enz. 184:138-63).
  • Competitive binding assays rely on the ability of a labeled standard (e.g. , a
  • CHL2 polypeptide or an immunologically reactive portion thereof to compete with the test sample analyte (an CHL2 polypeptide) for binding with a limited amount of anti-CHL2 antibody.
  • the amount of a CHL2 polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies typically are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound.
  • Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and/or quantitated.
  • the test sample analyte is typically bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three- part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assays).
  • sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.
  • the selective binding agents are also useful for in vivo imaging.
  • An antibody labeled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed.
  • the antibody may be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • Selective binding agents of the invention, including antibodies may be used as therapeutics. These therapeutic agents are generally agonists or antagonists, in that they either enhance or reduce, respectively, at least one of the biological activities of a CHL2 polypeptide.
  • antagonist antibodies of the invention are antibodies or binding fragments thereof which are capable of specifically binding to a CHL2 polypeptide and which are capable of inhibiting or eliminating the functional activity of a CHL2 polypeptide in vivo or in vitro.
  • the selective binding agent e.g., an antagonist antibody
  • the selective binding agent will inhibit the functional activity of a CHL2 polypeptide by at least about 50%, and preferably by at least about 80%.
  • the selective binding agent may be an anti-CHL2 polypeptide antibody that is capable of interacting with a CHL2 polypeptide binding partner (a ligand or receptor) thereby inhibiting or eliminating CHL2 polypeptide activity in vitro or in vivo.
  • Selective binding agents including agonist and antagonist anti-CHL2 polypeptide antibodies, are identified by screening assays that are well known in the art.
  • the invention also relates to a kit comprising CHL2 selective binding agents (such as antibodies) and other reagents useful for detecting CHL2 polypeptide levels in biological samples.
  • CHL2 selective binding agents such as antibodies
  • Such reagents may include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • DNA microarray technology can be utilized in accordance with the present invention.
  • DNA microarrays are miniature, high- density arrays of nucleic acids positioned on a solid support, such as glass. Each cell or element within the array contains numerous copies of a single nucleic acid species that acts as a target for hybridization with a complementary nucleic acid sequence (e.g., mRNA).
  • mRNA is first extracted from a cell or tissue sample and then converted enzymatically to fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing.
  • the expression of discrete genes represented on the array is then visualized by quantitating the amount of labeled cDNA that is specifically bound to each target nucleic acid molecule.
  • This high throughput expression profiling has a broad range of applications with respect to the CHL2 molecules of the invention, including, but not limited to: the identification and validation of CHL2 disease-related genes as targets for therapeutics; molecular toxicology of related CHL2 molecules and inhibitors thereof; stratification of populations and generation of surrogate markers for clinical trials; and enhancing related CHL2 polypeptide small molecule drug discovery by aiding in the identification of selective compounds in high throughput screens.
  • CHL2 polypeptide derivatives may be prepared by one skilled in the art, given the disclosures described herein.
  • CHL2 polypeptide derivatives are modified in a manner that is different - either in the type or location of the molecules naturally attached to the polypeptide.
  • Derivatives may include molecules formed by the deletion of one or more naturally-attached chemical groups.
  • the polypeptide comprising the amino acid sequence of any of SEQ ED NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide may be modified by the covalent attachment of one or more polymers.
  • the polymer selected is typically water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Included within the scope of suitable polymers is a mixture of polymers.
  • the polymer will be pharmaceutically acceptable.
  • the polymers each may be of any molecular weight and may be branched or unbranched.
  • the polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa (the term "about” indicating that in preparations of a water-soluble polymer, some molecules will weigh more, some less, than the stated molecular weight).
  • the average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa and most preferably between about 20 kDa and about 35 kDa.
  • Suitable water-soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(C 1 -C 10 ), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol.
  • PEG polyethylene glycol
  • dextran such as low molecular weight dextran of, for example, about 6 kD
  • cellulose or other carb
  • bifunctional crosslinking molecules which may be used to prepare covalently attached CHL2 polypeptide multimers.
  • chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer molecule.
  • Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of: (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby the polypeptide comprising the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide, becomes attached to one or more polymer molecules, and (b) obtaining the reaction products.
  • the optimal reaction conditions will be determined based on known parameters and the desired result. For example, the larger the ratio of polymer molecules to protein, the greater the percentage of attached polymer molecule. In one embodiment, the
  • CHL2 polypeptide derivative may have a single polymer molecule moiety at the amino-terminus. See, e.g., U.S. Patent No. 5,234,784.
  • pegylation of a polypeptide may be specifically carried out using any of the pegylation reactions known in the art. Such reactions are described, for example, in the following references: Francis et al, 1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and 0401384; and U.S. Patent No. 4,179,337.
  • pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer) as described herein.
  • a selected polymer should have a single reactive ester group.
  • reductive alkylation a selected polymer should have a single reactive aldehyde group.
  • a reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water stable, or mono Ci- o alkoxy or aryloxy derivatives thereof (see U.S. Patent No. 5,252,714).
  • CHL2 polypeptides may be chemically coupled to biotin.
  • the biotin CHL2 polypeptide molecules are then allowed to bind to avidin, resulting in tetravalent avidin/biotin/CHL2 polypeptide molecules.
  • CHL2 polypeptides may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of 10.
  • DNP dinitrophenol
  • TNP trinitrophenol
  • conditions that may be alleviated or modulated by the administration of the present CHL2 polypeptide derivatives include those described herein for CHL2 polypeptides.
  • the CHL2 polypeptide derivatives disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half- life, as compared to the non-derivatized molecules.
  • non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which the genes encoding native CHL2 polypeptide have been disrupted (i.e., "knocked out") such that the level of expression of CHL2 polypeptide is significantly decreased or completely abolished.
  • Such animals may be prepared using techniques and methods such as those described in U.S. Patent No. 5,557,032.
  • the present invention further includes non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which either the native form of a CHL2 gene for that animal or a heterologous CHL2 gene is over-expressed by the animal, thereby creating a "transgenic" animal.
  • non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which either the native form of a CHL2 gene for that animal or a heterologous CHL2 gene is over-expressed by the animal, thereby creating a "transgenic" animal.
  • Such transgenic animals may be prepared using well known methods such as those described in U.S. Patent No 5,489,743 and PCT Pub. No. WO 94/28122.
  • the present invention further includes non-human animals in which the promoter for one or more of the CHL2 polypeptides of the present invention is either activated or inactivated (e.g., by using homologous recombination methods) to alter the level of expression of one or more of the native CHL2 polypeptides.
  • These non-human animals may be used for drug candidate screening. In such screening, the impact of a drug candidate on the animal may be measured. For example, drug candidates may decrease or increase the expression of the CHL2 gene.
  • the amount of CHL2 polypeptide that is produced may be measured after the exposure of the animal to the drug candidate. Additionally, in certain embodiments, one may detect the actual impact of the drug candidate on the animal.
  • over-expression of a particular gene may result in, or be associated with, a disease or pathological condition.
  • a drug candidate may test a drug candidate's ability to decrease expression of the gene or its ability to prevent or inhibit a pathological condition.
  • the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition.
  • CHL2 polypeptides may be identified using one or more screening assays, such as those described herein. Such molecules may be administered either in an ex vivo manner or in an in vivo manner by injection, or by oral delivery, implantation device, or the like.
  • Test molecule refers to a molecule that is under evaluation for the ability to modulate (i.e., increase or decrease) the activity of a CHL2 polypeptide. Most commonly, a test molecule will interact directly with a CHL2 polypeptide. However, it is also contemplated that a test molecule may also modulate CHL2 polypeptide activity indirectly, such as by affecting CHL2 gene expression, or by binding to a CHL2 polypeptide binding partner (e.g., receptor or ligand).
  • CHL2 polypeptide binding partner e.g., receptor or ligand
  • a test molecule will bind to a CHL2 polypeptide with an affinity constant of at least about 10 "6 M, preferably about 10 "8 M, more preferably about 10 "9 M, and even more preferably about 10 " ° M.
  • Methods for identifying compounds that interact with CHL2 polypeptides are encompassed by the present invention.
  • a CHL2 polypeptide is incubated with a test molecule under conditions that permit the interaction of the test molecule with a CHL2 polypeptide, and the extent of the interaction is measured.
  • the test molecule can be screened in a substantially purified form or in a crude mixture.
  • a CHL2 polypeptide agonist or antagonist may be a protein, peptide, carbohydrate, lipid, or small molecular weight molecule that interacts with CHL2 polypeptide to regulate its activity.
  • Molecules which regulate CHL2 polypeptide expression include nucleic acids which are complementary to nucleic acids encoding a CHL2 polypeptide, or are complementary to nucleic acids sequences which direct or control the expression of CHL2 polypeptide, and which act as anti-sense regulators of expression.
  • test molecule Once a test molecule has been identified as interacting with a CHL2 polypeptide, the molecule may be further evaluated for its ability to increase or decrease CHL2 polypeptide activity.
  • the measurement of the interaction of a test molecule with CHL2 polypeptide may be carried out in several formats, including cell-based binding assays, membrane binding assays, solution-phase assays, and immunoassays.
  • a test molecule is incubated with a CHL2 polypeptide for a specified period of time, and CHL2 polypeptide activity is determined by one or more assays for measuring biological activity.
  • test molecules with CHL2 polypeptides may also be assayed directly using polyclonal or monoclonal antibodies in an immunoassay.
  • modified forms of CHL2 polypeptides containing epitope tags as described herein may be used in solution and immunoassays.
  • a binding partner e.g. , a receptor or a ligand
  • in vitro assays may be used to measure the binding of a CHL2 polypeptide to the corresponding binding partner (such as a selective binding agent, receptor, or ligand).
  • a CHL2 polypeptide is immobilized in the wells of a microtiter plate.
  • Radiolabeled CHL2 polypeptide binding partner for example, iodinated CHL2 polypeptide binding partner
  • a test molecule can then be added either one at a time (in either order) or simultaneously to the wells. After incubation, the wells can be washed and counted for radioactivity, using a scintillation counter, to determine the extent to which the binding partner bound to the CHL2 polypeptide.
  • a molecule will be tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assays can be used for accuracy in the evaluation of the results.
  • An alternative to this method involves reversing the "positions" of the proteins, i.e., immobilizing CHL2 polypeptide binding partner to the microtiter plate wells, incubating with the test molecule and radiolabeled CHL2 polypeptide, and determining the extent of CHL2 polypeptide binding. See, e.g., Current Protocols in Molecular Biology, chap. 18 (Ausubel et al, eds., Green Publishers Inc. and Wiley and Sons 1995).
  • a CHL2 polypeptide or its binding partner may be conjugated to biotin, and the presence of biotinylated protein can then be detected using streptavidin linked to an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (AP), which can be detected colorometrically, or by fluorescent tagging of streptavidin.
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • An antibody directed to a CHL2 polypeptide or to a CHL2 polypeptide binding partner, and which is conjugated to biotin may also be used for purposes of detection following incubation of the complex with enzyme-linked streptavidin linked to AP or HRP.
  • a CHL2 polypeptide or a CHL2 polypeptide binding partner can also be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert solid phase substrates.
  • the substrate-protein complex can be placed in a solution containing the complementary protein and the test compound. After incubation, the beads can be precipitated by centrifugation, and the amount of binding between a CHL2 polypeptide and its binding partner can be assessed using the methods described herein.
  • the substrate-protein complex can be immobilized in a column with the test molecule and complementary protein passing through the column. The formation of a complex between a CHL2 polypeptide and its binding partner can then be assessed using any of the techniques described herein (e.g., radiolabelling or antibody binding).
  • Another in vitro assay that is useful for identifying a test molecule which increases or decreases the formation of a complex between a CHL2 polypeptide binding protein and a CHL2 polypeptide binding partner is a surface plasmon resonance detector system such as the BIAcore assay system (Pharmacia, Piscataway, NJ).
  • the BIAcore system is utilized as specified by the manufacturer.
  • This assay essentially involves the covalent binding of either CHL2 polypeptide or a CHL2 polypeptide binding partner to a dextran-coated sensor chip that is located in a detector.
  • the test compound and the other complementary protein can then be injected, either simultaneously or sequentially, into the chamber containing the sensor chip.
  • the amount of complementary protein that binds can be assessed based on the change in molecular mass that is physically associated with the dextran-coated side of the sensor chip, with the change in molecular mass being measured by the detector system.
  • test compounds it may be desirable to evaluate two or more test compounds together for their ability to increase or decrease the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner.
  • the assays set forth herein can be readily modified by adding such additional test compound(s) either simultaneously with, or subsequent to, the first test compound. The remainder of the steps in the assay are as set forth herein.
  • In vitro assays such as those described herein may be used advantageously to screen large numbers of compounds for an effect on the formation of a complex between a CHL2 polypeptide and CHL2 polypeptide binding partner.
  • the assays may be automated to screen compounds generated in phage display, synthetic peptide, and chemical synthesis libraries.
  • Compounds which increase or decrease the formation of a complex between a CHL2 polypeptide and a CHL2 polypeptide binding partner may also be screened in cell culture using cells and cell lines expressing either CHL2 polypeptide or CHL2 polypeptide binding partner.
  • Cells and cell lines may be obtained from any mammal, but preferably will be from human or other primate, canine, or rodent sources.
  • the binding of a CHL2 polypeptide to cells expressing CHL2 polypeptide binding partner at the surface is evaluated in the presence or absence of test molecules, and the extent of binding may be determined by, for example, flow cytometry using a biotinylated antibody to a CHL2 polypeptide binding partner.
  • Cell culture assays can be used advantageously to further evaluate compounds that score positive in protein binding assays described herein.
  • Cell cultures can also be used to screen the impact of a drug candidate.
  • drug candidates may decrease or increase the expression of the CHL2 gene.
  • the amount of CHL2 polypeptide or a CHL2 polypeptide fragment that is produced may be measured after exposure of the cell culture to the drug candidate, hi certain embodiments, one may detect the actual impact of the drug candidate on the cell culture.
  • the over- expression of a particular gene may have a particular impact on the cell culture. In such cases, one may test a drug candidate's ability to increase or decrease the expression of the gene or its ability to prevent or inhibit a particular impact on the cell culture.
  • the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition.
  • a drug candidate may test a drug candidate's ability to decrease the production of such a metabolic product in a cell culture.
  • the tat protein sequence (from HIV) can be used to internalize proteins into a cell. See, e.g., Falwell et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:664-68.
  • an 11 amino acid sequence (Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 1
  • FITC- constructs (FITC-labeled G-G-G-G- Y-G-R-K-K-R-R-Q-R-R-R; SEQ ED NO: 11), which penetrate tissues following intraperitoneal administration, are prepared, and the binding of such constructs to cells is detected by fluorescence-activated cell sorting (FACS) analysis.
  • FACS fluorescence-activated cell sorting
  • Cells treated with a tat- ⁇ -gal fusion protein will demonstrate ⁇ -gal activity.
  • expression of such a construct can be detected in a number of tissues, including liver, kidney, lung, heart, and brain tissue. It is believed that such constructs undergo some degree of unfolding in order to enter the cell, and as such, may require a refolding following entry into the cell.
  • the tat protein sequence may be used to internalize a desired polypeptide into a cell.
  • a CHL2 antagonist such as an anti-CHL2 selective binding agent, small molecule, soluble receptor, or antisense oligonucleotide
  • CHL2 molecule refers to both CHL2 nucleic acid molecules and CHL2 polypeptides as defined herein.
  • the CHL2 protein itself may also be internally administered to a cell using these procedures. See also,
  • nucleic acids encoding a CHL2 polypeptide can be used as a probe to identify cells described herein by screening the nucleic acids of the cells with such a probe.
  • CHL2 Polypeptide Compositions and Administration Therapeutic compositions are within the scope of the present invention.
  • Such CHL2 polypeptide pharmaceutical compositions may comprise a therapeutically effective amount of a CHL2 polypeptide or a CHL2 nucleic acid molecule in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Pharmaceutical compositions may comprise a therapeutically effective amount of one or more CHL2 polypeptide selective binding agents in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emuls
  • compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the CHL2 molecule.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection may be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute.
  • CHL2 polypeptide compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the CHL2 polypeptide product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the CHL2 polypeptide pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired CHL2 molecule in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a CHL2 molecule is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • an agent such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition may be formulated for inhalation.
  • CHL2 polypeptide may be formulated as a dry powder for inhalation.
  • CHL2 polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized.
  • Pulmonary administration is further described in PCT Pub. No. WO 94/20069, which describes the pulmonary delivery of chemically modified proteins. It is also contemplated that certain formulations may be administered orally.
  • CHL2 polypeptides that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the CHL2 polypeptide. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • Another pharmaceutical composition may involve an effective quantity of CHL2 polypeptides in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • CHL2 polypeptide pharmaceutical compositions will be evident to those skilled in the art, including formulations involving CHL2 polypeptides in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, e.g., PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
  • sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919 and European Patent No. 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, 1983, Biopolymers 22:547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al, 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech.
  • Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European Patent Nos. 036676, 088046, and 143949.
  • the CHL2 pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method may be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration may be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit may each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
  • the effective amount of a CHL2 pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the CHL2 molecule is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • a typical dosage may range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg up to about 100 mg/kg.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the CHL2 molecule in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose- response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition may be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • a CHL2 polypeptide can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the CHL2 polypeptide.
  • Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic.
  • the cells may be immortalized.
  • the cells may be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • CHL2 polypeptides such as stem cells, lymphocytes, red blood cells, chondrocytes, neurons, and the like. This can be accomplished by exposing the isolated cells to the polypeptide directly, where it is in a form that is permeable to the cell membrane.
  • Additional embodiments of the present invention relate to cells and methods (e.g., homologous recombination and/or other recombinant production methods) for both the in vitro production of therapeutic polypeptides and for the production and delivery of therapeutic polypeptides by gene therapy or cell therapy.
  • Homologous and other recombination methods may be used to modify a cell that contains a normally transcriptionally-silent CHL2 gene, or an under- expressed gene, and thereby produce a cell which expresses therapeutically efficacious amounts of CHL2 polypeptides.
  • Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes.
  • the DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to targeting DNA.
  • the targeting DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process. It is a general property of DNA that has been inserted into a cell to hybridize, and therefore, recombine with other pieces of endogenous DNA through shared homologous regions.
  • this complementary strand is attached to an oligonucleotide that contains a mutation or a different sequence or an additional nucleotide, it too is incorporated into the newly synthesized strand as a result of the recombination.
  • the proofreading function it is possible for the new sequence of DNA to serve as the template.
  • the transferred DNA is incorporated into the genome.
  • Attached to these pieces of targeting DNA are regions of DNA that may interact with or control the expression of a CHL2 polypeptide, e.g., flanking sequences.
  • a promoter/enhancer element, a suppressor, or an exogenous transcription modulatory element is inserted in the genome of the intended host cell in proximity and orientation sufficient to influence the transcription of DNA encoding the desired CHL2 polypeptide.
  • the control element controls a portion of the DNA present in the host cell genome.
  • the expression of the desired CHL2 polypeptide may be achieved not by transfection of DNA that encodes the CHL2 gene itself, but rather by the use of targeting DNA (containing regions of homology with the endogenous gene of interest) coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals for transcription of a CHL2 gene.
  • the expression of a desired targeted gene in a cell is altered via homologous recombination into the cellular genome at a preselected site, by the introduction of DNA which includes at least a regulatory sequence, an exon, and a splice donor site.
  • DNA which includes at least a regulatory sequence, an exon, and a splice donor site.
  • Altered gene expression encompasses activating (or causing to be expressed) a gene which is normally silent (unexpressed) in the cell as obtained, as well as increasing the expression of a gene which is not expressed at physiologically significant levels in the cell as obtained.
  • the embodiments further encompass changing the pattern of regulation or induction such that it is different from the pattern of regulation or induction that occurs in the cell as obtained, and reducing (including eliminating) the expression of a gene which is expressed in the cell as obtained.
  • homologous recombination can be used to increase, or cause, CHL2 polypeptide production from a cell's endogenous CHL2 gene involves first using homologous recombination to place a recombination sequence from a site-specific recombination system (e.g., Cre/loxP, FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol, 5:521-27; Sauer, 1993, Methods Enzymol, 225:890-900) upstream of (i.e., 5' to) the cell's endogenous genomic CHL2 polypeptide coding region.
  • a site-specific recombination system e.g., Cre/loxP, FLP/FRT
  • a plasmid containing a recombination site homologous to the site that was placed just upstream of the genomic CHL2 polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme.
  • This recombinase causes the plasmid to integrate, via the plasmid' s recombination site, into the recombination site located just upstream of the genomic CHL2 polypeptide coding region in the cell line (Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025-29; O'Gorman et al, 1991, Science 251:1351-55).
  • flanking sequences known to increase transcription e.g., enhancer/promoter, intron, translational enhancer
  • if properly positioned in this plasmid would integrate in such a manner as to create a new or modified transcriptional unit resulting in de novo or increased CHL2 polypeptide production from the cell's endogenous CHL2 gene.
  • a further method to use the cell line in which the site specific recombination sequence had been placed just upstream of the cell's endogenous genomic CHL2 polypeptide coding region is to use homologous recombination to introduce a second recombination site elsewhere in the cell line's genome.
  • the appropriate recombinase enzyme is then introduced into the two-recombination- site cell line, causing a recombination event (deletion, inversion, and translocation) (Sauer, 1994, Curr. Opin.
  • An additional approach for increasing, or causing, the expression of CHL2 polypeptide from a cell's endogenous CHL2 gene involves increasing, or causing, the expression of a gene or genes (e.g., transcription factors) and/or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner which results in de novo or increased CHL2 polypeptide production from the cell's endogenous CHL2 gene.
  • This method includes the introduction of a non-naturally occurring polypeptide (e.g., a polypeptide comprising a site specific DNA binding domain fused to a transcriptional factor domain) into the cell such that de novo or increased CHL2 polypeptide production from the cell's endogenous CHL2 gene results.
  • the present invention further relates to DNA constructs useful in the method of altering expression of a target gene.
  • the exemplary DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, and (d) an unpaired splice-donor site.
  • the targeting sequence in the DNA construct directs the integration of elements (a) - (d) into a target gene in a cell such that the elements (b) - (d) are operatively linked to sequences of the endogenous target gene.
  • the DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice- acceptor site, wherein the targeting sequence directs the integration of elements (a) - (f) such that the elements of (b) - (f) are operatively linked to the endogenous gene.
  • the targeting sequence is homologous to the preselected site in the cellular chromosomal DNA with which homologous recombination is to occur.
  • the exon is generally 3' of the regulatory sequence and the splice-donor site is 3' of the exon.
  • sequence of a particular gene is known, such as the nucleic acid sequence of CHL2 polypeptide presented herein
  • a piece of DNA that is complementary to a selected region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest.
  • This piece serves as a targeting sequence upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be incorporated into the newly synthesized daughter strand of DNA.
  • the present invention therefore, includes nucleotides encoding a CHL2 polypeptide, which nucleotides may be used as targeting sequences.
  • CHL2 polypeptide cell therapy e.g., the implantation of cells producing CHL2 polypeptides
  • This embodiment involves implanting cells capable of synthesizing and secreting a biologically active form of CHL2 polypeptide.
  • Such CHL2 polypeptide-producing cells can be cells that are natural producers of CHL2 polypeptides or may be recombinant cells whose ability to produce CHL2 polypeptides has been augmented by transformation with a gene encoding the desired CHL2 polypeptide or with a gene augmenting the expression of CHL2 polypeptide.
  • Such a modification may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion.
  • the natural cells producing CHL2 polypeptide be of human origin and produce human CHL2 polypeptide.
  • the recombinant cells producing CHL2 polypeptide be transformed with an expression vector containing a gene encoding a human CHL2 polypeptide.
  • Implanted cells may be encapsulated to avoid the infiltration of surrounding tissue.
  • Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that allow the release of CHL2 polypeptide, but that prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue.
  • the patient's own cells, transformed to produce CHL2 polypeptides ex vivo may be implanted directly into the patient without such encapsulation.
  • the devices provide for the delivery of the molecules from living cells to specific sites within a recipient.
  • U.S. Patent Nos. 4,892,538; 5,011,472; and 5,106,627 A system for encapsulating living cells is described in PCT Pub. No. WO 91/10425 (Aebischer et al). See also, PCT Pub. No. WO 91/10470 (Aebischer et al); Winn et al, 1991, Exper. Neurol 113:322-29; Aebischer et al,
  • CHL2 gene either genomic DNA, cDNA, and/or synthetic DNA
  • the promoter may be homologous or heterologous to the endogenous CHL2 gene, provided that it is active in the cell or tissue type into which the construct will be inserted.
  • Other components of the gene therapy DNA construct may optionally include DNA molecules designed for site-specific integration (e.g., endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or silencers, DNA molecules capable of providing a selective advantage over the parent cell, DNA molecules useful as labels to identify transformed cells, negative selection systems, cell specific binding agents (as, for example, for cell targeting), cell-specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
  • DNA molecules designed for site-specific integration e.g., endogenous sequences useful for homologous recombination
  • tissue-specific promoters e.g., enhancers or silencers
  • DNA molecules capable of providing a selective advantage over the parent cell DNA molecules useful as labels to identify transformed cells
  • negative selection systems e.g., cell specific binding agents (as, for example, for cell targeting), cell-specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
  • a gene therapy DNA construct can then be introduced into cells (either ex vtvo or in vivo) using viral or non-viral vectors.
  • One means for introducing the gene therapy DNA construct is by means of viral vectors as described herein.
  • Certain vectors, such as retroviral vectors will deliver the DNA construct to the chromosomal DNA of the cells, and the gene can integrate into the chromosomal DNA.
  • Other vectors will function as episomes, and the gene therapy DNA construct will remain in the cytoplasm.
  • regulatory elements can be included for the controlled expression of the CHL2 gene in the target cell. Such elements are turned on in response to an appropriate effector. In this way, a therapeutic polypeptide can be expressed when desired.
  • One conventional control means involves the use of small molecule dimerizers or rapalogs to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating a biological process, such as a DNA-binding protein or transcriptional activation protein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899). The dimerization of the proteins can be used to initiate transcription of the transgene.
  • An alternative regulation technology uses a method of storing proteins expressed from the gene of interest inside the cell as an aggregate or cluster.
  • the gene of interest is expressed as a fusion protein that includes a conditional aggregation domain that results in the retention of the aggregated protein in the endoplasmic reticulum.
  • the stored proteins are stable and inactive inside the cell.
  • the proteins can be released, however, by administering a drug (e.g., small molecule ligand) that removes the conditional aggregation domain and thereby specifically breaks apart the aggregates or clusters so that the proteins may be secreted from the cell. See Aridor et al, 2000, Science 287:816-17 and Rivera et al, 2000, Science 287:826-30.
  • Mifepristone (RU486) is used as a progesterone antagonist.
  • the binding of a modified progesterone receptor ligand-binding domain to the progesterone antagonist activates transcription by forming a dimer of two transcription factors that then pass into the nucleus to bind DNA.
  • the ligand-binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand.
  • the modified steroid hormone receptor system is further described in U.S. Patent No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and
  • ecdysone a fruit fly steroid hormone
  • ecdysone receptor cytoplasmic receptor
  • the receptor then translocates to the nucleus to bind a specific DNA response element (promoter from ecdysone-responsive gene).
  • the ecdysone receptor includes a transactivation domain, DNA-binding domain, and ligand-binding domain to initiate transcription.
  • the ecdysone system is further described in U.S. Patent No. 5,514,578 and PCT Pub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.
  • Another control means uses a positive tetracycline-controllable transactivator.
  • This system involves a mutated tet repressor protein DNA-binding domain (mutated tet R-4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline) linked to a polypeptide which activates transcription.
  • mutated tet repressor protein DNA-binding domain mutated tet R-4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline linked to a polypeptide which activates transcription.
  • mutated tet repressor protein DNA-binding domain mutated tet R-4 amino acid changes which resulted in a reverse tetra
  • In vivo gene therapy may be accomplished by introducing the gene encoding CHL2 polypeptide into cells via local injection of a CHL2 nucleic acid molecule or by other appropriate viral or non- viral delivery vectors. Hefti 1994, Neurobiology 25:1418-35.
  • a nucleic acid molecule encoding a CHL2 polypeptide may be contained in an adeno-associated virus (AAV) vector for delivery to the targeted cells (see, e.g., Johnson, PCT Pub. No. WO 95/34670; PCT App. No. PCT/US95/07178).
  • AAV adeno-associated virus
  • the recombinant AAV genome typically contains AAV inverted terminal repeats flanking a DNA sequence encoding a CHL2 polypeptide operably linked to functional promoter and polyadenylation sequences.
  • Alternative suitable viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma virus vectors.
  • U.S. Patent No. 5,672,344 describes an in vivo viral- mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • U.S. Patent No. 5,399,346 provides examples of a process for providing a patient with a therapeutic protein by the delivery of human cells which have been treated in vitro to insert a DNA segment encoding a therapeutic protein.
  • Nonviral delivery methods include, but are not limited to, liposome- mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene gun).
  • Gene therapy materials and methods may also include inducible promoters, tissue-specific enhancer- promoters, DNA sequences designed for site-specific integration, DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture.
  • inducible promoters tissue-specific enhancer- promoters
  • DNA sequences designed for site-specific integration DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture.
  • CHL2 gene therapy or cell therapy can further include the delivery of one or more additional polypeptide(s) in the same or a different cell(s).
  • additional polypeptide(s) in the same or a different cell(s).
  • Such cells may be separately introduced into the patient, or the cells may be contained in a single implantable device, such as the encapsulating membrane described above, or the cells may be separately modified by means of viral vectors.
  • a means to increase endogenous CHL2 polypeptide expression in a cell via gene therapy is to insert one or more enhancer elements into the CHL2 polypeptide promoter, where the enhancer elements can serve to increase transcriptional activity of the CHL2 gene.
  • the enhancer elements used will be selected based on the tissue in which one desires to activate the gene - enhancer elements known to confer promoter activation in that tissue will be selected. For example, if a gene encoding a CHL2 polypeptide is to be "turned on" in T-cells, the lck promoter enhancer element may be used.
  • the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing the CHL2 polypeptide promoter (and optionally, inserted into a vector and/or 5' and/or 3' flanking sequences) using standard cloning techniques.
  • This construct known as a "homologous recombination construct," can then be introduced into the desired cells either ex vivo or in vivo.
  • Gene therapy also can be used to decrease CHL2 polypeptide expression by modifying the nucleotide sequence of the endogenous promoter. Such modification is typically accomplished via homologous recombination methods.
  • a DNA molecule containing all or a portion of the promoter of the CHL2 gene selected for inactivation can be engineered to remove and/or replace pieces of the promoter that regulate transcription.
  • the TATA box and/or the binding site of a transcriptional activator of the promoter may be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity thereby repressing the transcription of the corresponding CHL2 gene.
  • the deletion of the TATA box or the transcription activator binding site in the promoter may be accomplished by generating a DNA construct comprising all or the relevant portion of the CHL2 polypeptide promoter (from the same or a related species as the CHL2 gene to be regulated) in which one or more of the TATA box and/or transcriptional activator binding site nucleotides are mutated via substitution, deletion and/or insertion of one or more nucleotides.
  • the TATA box and/or activator binding site has decreased activity or is rendered completely inactive.
  • This construct which also will typically contain at least about 500 bases of DNA that correspond to the native (endogenous) 5' and 3' DNA sequences adjacent to the promoter segment that has been modified, may be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described herein.
  • the integration of the construct into the genomic DNA of the cells will be via homologous recombination, where the 5' and 3' DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof can be used to treat, diagnose, ameliorate, or prevent a number of diseases, disorders, or conditions, including those recited herein.
  • CHL2 polypeptide agonists and antagonists include those molecules which regulate CHL2 polypeptide activity and either increase or decrease at least one activity of the mature form of the CHL2 polypeptide.
  • Agonists or antagonists may be co-factors, such as a protein, peptide, carbohydrate, lipid, or small molecular weight molecule, which interact with CHL2 polypeptide and thereby regulate its activity.
  • Potential polypeptide agonists or antagonists include antibodies that react with either soluble or membrane-bound forms of CHL2 polypeptides that comprise part or all of the extracellular domains of the said proteins.
  • Molecules that regulate CHL2 polypeptide expression typically include nucleic acids encoding CHL2 polypeptide that can act as anti-sense regulators of expression.
  • BMP2 and BMP4 are the major roles of the BMP-family of gene products, specifically BMP2 and BMP4, and are the regulation of bone-mass in the adult. Since BMP1 has been shown to cleave and inactivate CHD, and has been isolated with BMP2 and BMP3 from bone (Wozney et al, 1988, Science 242:1528-34; Celeste et al, 1990, Proc. Nat. Acad. Sci. USA 87: 9843-47), CHL2 polypeptides may play a key regulatory role in osteogenesis.
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in diagnosing or treating diseases and conditions affecting bone density.
  • diseases and conditions include, but are not limited to, osteopetrosis and osteoporosis.
  • Other diseases and conditions affecting bone density are encompassed within the scope of this invention.
  • BMP4 or other BMP-family members
  • BMP4 is known to travel only a short distance in vivo (Jones et al, 1996, Curr. Biol. 6:1468-75).
  • CHD the formation of a complex between BMP and CHLH polypeptide may result in the further migration of BMP and formation of a BMP concentration gradient (Jones and Smith, 1998, ev. Biol. 194:12-17).
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in preventing cartilage fibrosis during the early phase of osteoarthritis, regenerating the once-disrupted superficial zone in the later phases of osteoarthritis or rheumatoid arthritis, or generating a proper superficial zone (i.e. surface) in transplanted cartilages.
  • BMP polypeptides have been shown to function in organ formation during late embryogenesis. It has been shown that organ formation in embryonic kidney, lung, and gut are affected by BMP4 expression (Hogan, 1996, Genes Dev. 10:1580-94).
  • a combination of BMP4 and CHL2 polypeptides may be useful for controlling the proliferation and differentiation of progenitor cells, thus permitting the regulation of tissue regeneration or wound healing in vivo.
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in promoting tissue regeneration or wound healing.
  • the CHL2 nucleic acid molecules, polypeptides, agonists and antagonists thereof may also be used in hematopoietic stem cell-genesis and expansion. It may be possible to use BMP/CHL2 polypeptide complexes to regulate primitive hematopoietic stem cells and thereby control adult-marrow repopulating stem cells. Alternatively, it may be possible to control stem cell genesis from a mesodermal stem cell. Thus, the CHL2 polypeptides and nucleic acids of the present invention, along with BMP, may be useful for ex vivo expansion of hematopoietic stem cells and gene therapy performed through such cells. BMP4 is an essential factor for generating hematopoietic progenitor cells from the mouse ES cells.
  • the effective concentration of BMP4 falls into a narrow range (0.5 ng/ml to 5 ng/ml), which is consistent with the idea that the difference in the active concentration of BMP correlates with the difference in the resulting cell-type from the totipotent epiblast.
  • a system for the reproducible in vitro generation of hematopoietic stem cells from ES cells has not yet been disclosed. However, it may be achieved by precise control of the concentration of BMP4.
  • the CHL2 nucleic acid molecules and polypeptides of the present invention may be useful in optimizing the culturing conditions for the in vitro generation of hematopoietic stem cells from ES cells.
  • Primitive hematopoietic stem cells have been recently defined in the mouse yolk sac (Yoder et al, 1997, Proc. Natl. Acad. Sci. USA 94:6776-80). This subclass of hematopoietic stem cells does not exhibit a marrow-repopulating activity in adults. However, when exposed to a newborn liver environment, the primitive stem cells are converted to long-term marrow-repopulating stem cells (i.e., definitive stem cells). This can also be accomplished by culturing the primitive stem cells on certain stroma cell lines. The long-term survival of the definitive stem cells in culture and the long-term maintenance of the primitive stem cells that are spontaneously differentiated into definitive stem cells have not yet been established.
  • CHL2 polypeptides and CHL2 antibodies of the present invention may be useful tools for in vitro long-term maintenance of hematopoietic stem cells and in vitro generation of definitive stem cells from primitive stem cells.
  • BMP4 or putative, novel CHL2-interacting molecules may be useful for controlling these processes.
  • primitive hematopoietic stem cells have yet to be fully characterized. While primitive stem cells may be of a lymphocytic cell type, such cells may also be mesodermal precursors that are able to generate hematopoietic cell types as well as other mesodermal progeny.
  • adult bone marrow has recently been shown to contain endothelial progenitor cells, cells that regenerate liver (Petersen et al, 1999, Science 284:1168-70), and a common stem cell that has a capability of deriving endothelial cells, muscle cells and hematopoietic cells in vivo (Ferrari et al, 1998, Science 279:1528-30).
  • the osteoblast cell lineage which consists of the bone marrow stroma, is known to be derived from a mesenchymal stem cell that is present in bone marrow.
  • the possibility that a common mesodermal stem cell is responsible for the generation of both stroma and hematopoietic cells has also been previously speculated.
  • CHL2 polypeptide expression has been detected in skeletal muscle, CHL2 polypeptides may also play a role in the development and function of skeletal muscle. Accordingly, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may be useful in diagnosing or treating diseases and conditions affecting skeletal muscle. Examples of such diseases and conditions include, but are not limited to, cachexia and muscular dystrophy. Other diseases and conditions associated with skeletal muscle development and function are encompassed within the scope of this invention. Since CHL2 polypeptide expression has been detected in the heart, CHL2 polypeptides may play a role in the development and function of the heart.
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may also be useful in diagnosing or treating diseases and conditions affecting the heart.
  • diseases and conditions include, but are not limited to, arhythmias, angina, hypertension, myocardial infarction, and congestive heart failure.
  • Other diseases and conditions associated with the heart are encompassed within the scope of this invention.
  • CHL2 polypeptides may play a role in the development and function of the stomach. Accordingly, CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may also be useful in diagnosing or treating diseases and conditions involving the stomach. Examples of such diseases and conditions include, but are not limited to, stomach cancer and stomach ulcer. Other diseases and conditions associated with the stomach are encompassed within the scope of this invention. Since CHL2 polypeptide expression has been detected in the liver, CHL2 polypeptides may play a role in the development and function of the liver.
  • CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists thereof may also be useful in diagnosing or treating diseases and conditions involving the liver.
  • diseases and conditions include, but are not limited to, hepatitis and hepatoma.
  • Other diseases and conditions associated with the liver are encompassed within the scope of this invention.
  • Agonists or antagonists of CHL2 polypeptide function may be used (simultaneously or sequentially) in combination with one or more cytokines, growth factors, antibiotics, anti-inflammatories, and/or chemotherapeutic agents as is appropriate for the condition being treated.
  • CHL2 polypeptides Other diseases caused by or mediated by undesirable levels of CHL2 polypeptides are encompassed within the scope of the invention.
  • Undesirable levels include excessive levels of CHL2 polypeptides and sub-normal levels of CHL2 polypeptides.
  • Nucleic acid molecules of the invention may be used to map the locations of the CHL2 gene and related genes on chromosomes. Mapping may be done by techniques known in the art, such as PCR amplification and in situ hybridization.
  • CHL2 nucleic acid molecules may be useful as hybridization probes in diagnostic assays to test, either qualitatively or quantitatively, for the presence of a CHL2 nucleic acid molecule in mammalian tissue or bodily fluid samples.
  • CHL2 polypeptides may also be employed where it is desirable to inhibit the activity of one or more CHL2 polypeptides. Such inhibition may be effected by nucleic acid molecules that are complementary to and hybridize to expression control sequences (triple helix formation) or to CHL2 mRNA.
  • antisense DNA or RNA molecules which have a sequence that is complementary to at least a portion of a CHL2 gene can be introduced into the cell.
  • Anti-sense probes may be designed by available techniques using the sequence of the CHL2 gene disclosed herein. Typically, each such antisense molecule will be complementary to the start site (5' end) of each selected CHL2 gene.
  • Anti-sense inhibitors provide information relating to the decrease or absence of a CHL2 polypeptide in a cell or organism.
  • gene therapy may be employed to create a dominant- negative inhibitor of one or more CHL2 polypeptides.
  • the DNA encoding a mutant polypeptide of each selected CHL2 polypeptide can be prepared and introduced into the cells of a patient using either viral or non-viral methods as described herein. Each such mutant is typically designed to compete with endogenous polypeptide in its biological role.
  • a CHL2 polypeptide may be used as an immunogen, that is, the polypeptide contains at least one epitope to which antibodies may be raised.
  • Selective binding agents that bind to a CHL2 polypeptide may be used for in vivo and in vitro diagnostic purposes, including, but not limited to, use in labeled form to detect the presence of CHL2 polypeptide in a body fluid or cell sample.
  • the antibodies may also be used to prevent, treat, or diagnose a number of diseases and disorders, including those recited herein.
  • the antibodies may bind to a CHL2 polypeptide so as to diminish or block at least one activity characteristic of a CHL2 polypeptide, or may bind to a polypeptide to increase at least one activity characteristic of a CHL2 polypeptide (including by increasing the pharmacokinetics of the CHL2 polypeptide).
  • the CHL2 polypeptides of the present invention can be used to clone CHL2 polypeptide receptors, using an expression cloning strategy.
  • Radiolabeled ( 125 Iodine) CHL2 polypeptide or affinity/activity-tagged CHL2 polypeptide (such as an Fc fusion or an alkaline phosphatase fusion) can be used in binding assays to identify a cell type or cell line or tissue that expresses CHL2 polypeptide receptors.
  • RNA isolated from such cells or tissues can be converted to cDNA, cloned into a mammalian expression vector, and transfected into mammalian cells (such as COS or 293 cells) to create an expression library.
  • a radiolabeled or tagged CHL2 polypeptide can then be used as an affinity ligand to identify and isolate from this library the subset of cells that express the CHL2 polypeptide receptors on their surface.
  • DNA can then be isolated from these cells and transfected into mammalian cells to create a secondary expression library in which the fraction of cells expressing CHL2 polypeptide receptors is many-fold higher than in the original library. This enrichment process can be repeated iteratively until a single recombinant clone containing a CHL2 polypeptide receptor is isolated. Isolation of the CHL2 polypeptide receptors is useful for identifying or developing novel agonists and antagonists of the CHL2 polypeptide signaling pathway.
  • Such agonists and antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide receptor antibodies, small molecules, or antisense oligonucleotides, and they may be used for treating, preventing, or diagnosing one or more of the diseases or disorders described herein.
  • the murine and human CHL2 nucleic acids of the present invention are also useful tools for isolating the corresponding chromosomal CHL2 polypeptide genes.
  • mouse chromosomal DNA containing CHL2 sequences can be used to construct knockout mice, thereby permitting an examination of the in vivo role for CHL2 polypeptide.
  • the human CHL2 genomic DNA can be used to identify heritable tissue-degenerating diseases.
  • Clones containing signal peptide sequences were enriched from the kFGF- based library using kFGF signal trapping technology (U.S. Patent App. No. 09/026,959). Plasmid DNA from this cDNA library was prepared from 10 pools of 100,000 colonies each using standard techniques. This DNA was introduced into NEH 3T3 cells by calcium phosphate transfection and transfected cells were then grown for 14 days in selective media supplemented with 0.5% fetal bovine serum. Only transformants containing plasmids with signal peptide sequences generated colonies.
  • RNA from the colonies was isolated using TRIzon reagent (Gibco-BRL) according to the manufacturer's recommended protocol.
  • Poly-A+ RNA was isolated from the total RNA using an mRNA Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ).
  • first strand cDNA was initially prepared from the poly-A+ RNA isolated above using the SuperscriptTM preamplification system (Gibco- BRL). The first strand cDNA reaction was performed by combining 1 ⁇ g of poly-
  • A+ RNA and 2/zmole of the primer 1605-21 (5'-A-A-T-C-C-G-A-T-G-C-C-C-A- C-G-T-T-G-C-A-G-T-A-3'; SEQ ED NO: 12) in a total volume of 15 ⁇ L and heating the template-primer mixture at 70°C for 10 minutes.
  • the template-primer mixture was transferred to 50°C, and a reaction premixture consisting of 2.5 ⁇ L of 10X buffer, 2.5 ⁇ L of 25 mM MgCl 2 , 1.3 ⁇ L 10 mM dNTPs, and 2.5 ⁇ L 0.1 M dithiothreitol was added.
  • Reverse transcriptase 250 U was then added and the reaction incubated at 50°C for 1 hour.
  • the first strand cDNA reaction was stopped by heating at 70°C for 15 minutes, and the RNA digested by treatment with 2 U of RNAse H for 20 minutes at 37°C.
  • double strand cDNA was generated by PCR amplification in a reaction containing 2 ⁇ L of first strand cDNA, the primers 1239-08 (5'-A-A-A-A-T-C-T-T-A-G-A-C-C-G-A-C-G-A-C- T-G-T-G-T-T-T-3'; SEQ ID NO: 13) and 1605-22 (5'-C-G-T-A-A-A-A-G-A-T- C-C-T-G-C-G-C-T-A-G-A-T-G-C-G-3'; SEQ ED NO: 14) at a final concentration of 0.5 ⁇ M each, 200 ⁇ M of dNTPs, and 2.5 U of Pfu polymerase (PE Biosystems, Norwalk, CT), in a volume of 100 ⁇ L.
  • Pfu polymerase PE Biosystems, Norwalk, CT
  • the PCR reaction was performed at 95°C for 1 minute for one cycle; 95°C for 30 seconds, 66°C for 45 seconds, and 72°C for 2 minutes for 30 cycles; and 72°C for 10 minutes for one cycle.
  • Amplification products were digested with Not I and Sal I and ligated into kFGF7.
  • the ligation reaction was introduced into E. coli using standard transformation techniques to generate a signal enriched cDNA library. Plasmid DNA was prepared from 400 selected clones and analyzed by sequencing.
  • the sequence of one clone (designated ymkz5-00011-cl0) was found to share significant homology with the chordin precursor from Xenopus (GenBank accession no. Q91713). The identified clone was found to contain an insert of 418 bp encoding the N-terminal 115 amino acids of murine CHL2. A full-length cDNA clone was isolated from the cDNA library cloned into pSPORT 1 using the ymkz5-00011-cl0 clone as a probe.
  • FIGS 1A-1C illustrate the nucleotide sequence of the murine CHL2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of murine CHL2 polypeptide (SEQ ID NO: 2).
  • SEQ ID NO: 1 the signal peptide sequence, as predicted by the von Heijne algorithm, is underlined.
  • a human placenta cDNA library was prepared in order to isolate sequences encoding human CHL2 polypeptide.
  • Total RNA was extracted from human placenta and poly-A+ RNA selected using standard extraction and isolation techniques. Oligo d(T)-primed cDNA was then synthesized from poly- A+ RNA using the Superscript Plasmid System for cDNA Synthesis (Gibco- BRL). The resulting cDNA was digested with Not I and fractionated on a 0.8% agarose gel. Elecfrophoresed cDNA of greater than 1.5 kb was isolated and ligated into pSPORT 1. Ligation reactions were introduced into E.
  • Filters were prehybridized in ExpressHyb hybridization solution (Clontech) for 30 minutes at 60°C and then hybridized overnight at 60°C in the same solution containing the murine CHL2 cDNA probe.
  • Hybridized filters were washed twice in 2X SSC and 0.1 % SDS for 10 minutes at room temperature and twice in 0.5X SSC and 0.1% SDS for 30 minutes at 60°C. Following washing, filters were subjected to autoradiography overnight at -80°C in the presence of intensifying screens. Positive cDNA fragments were identified in two of the twelve plasmid pools.
  • FIGS 3A-3C illustrate the nucleotide sequence of the human CHL2 gene (SEQ ED NO: 4) and the deduced amino acid sequence of human CHL2 polypeptide (SEQ ID NO: 5). The amino acid sequence of CHL2 was found to share 26.7%> identity with human chordin.
  • Figure 4 illustrates the amino acid sequence alignment of human CHL2 polypeptide (human CHL2; SEQ ID NO: 5) and human chordin (Af076612; SEQ ID NO: 8).
  • Northern blots Multiple human or murine tissue Northern blots (Clontech) were probed using a 32 P-dCTP-labeled human or murine CHL2 cDNA fragment, respectively.
  • Northern blots were prehybridized in ExpressHyb hybridization solution (Clontech) for 30 minutes at 68°C and then were hybridized in the same solution with the addition of labeled probe overnight at 68°C. Following hybridization, the filters were washed twice in 2X SSC and 0.1% SDS for 10 minutes at room temperature and twice in 0.1X SSC and 0.1% SDS for 30 minutes at 68°C.
  • the blots were subjected to autoradiography for 72 hours at - 80°C in the presence of intensifying screens.
  • Northern blot analysis of human tissue blots revealed predominant CHL2 transcripts of approximately 2 kb in the prostate, testis, uterus (very abundant expression), colon, small intestine, heart, skeletal muscle, and stomach. Weak expression was detected in the trachea, placenta, and bone marrow, and very weak expression was detected in the liver.
  • Northern blot analysis of murine tissue blots revealed predominant CHL2 transcripts of approximately 1.8 kb in liver and kidney. Very weak expression was detected in the heart. Weak expression of an approximately 2 kb transcript was detected in the testis and skeletal muscle.
  • CHD/SOG is expressed at a relatively high level in E7 embryos and at decreased levels in Ell, E15, and E17 embryos (Pappano et al, 1998, Genomics 52:236-39).
  • CHL2 mRNA was similarly detected in E7, El l, El 5, and El 7 embryos, although at lower levels than are CHD/SOG transcripts.
  • CHD/SOG is expressed in spleen, liver, and kidney (Pappano et al, 1998).
  • CHL2 mRNA was similarly detected in liver and kidney.
  • CHL2 mRNA The expression of CHL2 mRNA was localized by in situ hybridization. In situ hybridization to embryonic and adult mouse tissue sections was performed as described (Wilcox, 1993, J. Histochem. Cytochem. 41:1725-33).
  • a murine CHL2 probe was prepared by first removing the 1.2-kb Nco I - Sal I fragment from pSPORTmCHL2 to generate pSPmCHL2COOH, which was then linearized with Eco RI. In addition, the Hind III fragment was removed to obtain pSPmCHL2NH2.
  • Antisense-RNAs were synthesized with SP6 RNA polymerase. The RNAs synthesized from these plasmids contained non-overlapping sequences.
  • CHL2 mRNA expression began in mice at about embryonic day 12.5 in the sternum and persisted in a restricted area of adult articular cartilage. CHL2 mRNA expression was not detected in the sections examined before E10.5. At E13.5, strong CHL2 mRNA expression was detected over the chondrocytes in the limb bones ( Figures 6 and 7) and sternum, as well as in the perichondrial mesenchymal cells adjacent to the developing joints. Strong but highly restricted CHL2 mRNA expression was alos detected in the superficial zone chondrocytes within the developing articular cartilage of vertebra and the epiphyses of the long bones ( Figures 6 - 8).
  • CHL2 mRNA By E18.5, and through to adulthood, the skeletal expression of CHL2 mRNA was restricted to a single layer of superficial zone chondrocytes in articular cartilage.
  • the epiphyseal growth plate did not show CHL2 mRNA expression at any point in mouse development.
  • significant CHL2 mRNA expression were observed in ovary, oviduct, and uterus of female mice, and in testis, epididymus and possibly other accessory glands (e.g., seminal vesicle, coagulating gland, and prostate) of male mice.
  • the strong signal observed in uterine wall was somewhat analogous to the CHLl mRNA expression detected in uterus.
  • CHL2 mRNA A weak expression of CHL2 mRNA was also detected on the colon surface. However, the location of CHL2 expression in the colon clearly differed from that of CHLl, wherein CHL2 mRNA was detected in the fibroblast/corrnective tissue cells dividing the submucosa and muscularis (Nakayama et al, 2001). In contrast with the CHLl gene, CHL2 mRNA expression was not detected in stomach or small intestine. Thus, among soft tissues, the CHL2 gene was expressed specifically in the reproductive organs.
  • Fluorescence in situ hybridization (FISH) analysis was used to determine the chromosomal localization of the murine CHL2 gene (Shi et al, 1997,
  • a FISH probe was prepared from a BAC clone (F1067) isolated from the Mouse ES-129/SvJ II BAC chromosome DNA library (Genome Systems) by PCR using standard techniques and primers corresponding to the 5' untranslated region of the murine CHL2 gene (5'-T-C-C-T-C-T-C-A-T-C-C-T-C- A-C-C-T-T-A-G-3'; SEQ ED NO: 15 and 5'-G-G-A-G-A-A-A-G-T-G-A-G-A-T- A-A-G-G-A-C-A-C-3'; SEQ ID NO: 16).
  • the murine CHL2 gene was localized to chromosome 7 using the chromosome 7 centromere specific PI clone as a co- hybridization probe (Shi et al, 1997). A total of 80 metaphase cells were analyzed, and 10 of the 77 that exhibited specific labeling were used for co- hybridization experiments. As a result, the mCHL2 gene was located at a position
  • the CHL2 gene differs from the CHLl gene in that the CHL2 gene is autosomal.
  • PCR is used to amplify template DNA sequences encoding a CHL2 polypeptide using primers corresponding to the 5' and 3' ends of the sequence.
  • the amplified DNA products may be modified to contain restriction enzyme sites to allow for insertion into expression vectors.
  • PCR products are gel purified and inserted into expression vectors using standard recombinant DNA methodology.
  • An exemplary vector, such as pAMG21 (ATCC no. 98113) containing the lux promoter and a gene encoding kanamycin resistance is digested with Bam HI and Nde I for directional cloning of inserted DNA.
  • the ligated mixture is transformed into an E. coli host strain by electroporation and transformants are selected for kanamycin resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the insert.
  • Transformed host cells are incubated in 2xYT medium containing 30 ⁇ g/mL kanamycin at 30°C prior to induction.
  • Gene expression is induced by the addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a final concentration of 30 ng/mL followed by incubation at either 30°C or 37°C for six hours.
  • the expression of CHL2 polypeptide is evaluated by centrifugation of the culture, resuspension and lysis of the bacterial pellets, and analysis of host cell proteins by SDS-polyacrylamide gel electrophoresis.
  • Inclusion bodies containing CHL2 polypeptide are purified as follows. Bacterial cells are pelleted by centrifugation and resuspended in water. The cell suspension is lysed by sonication and pelleted by centrifugation at 195,000 xg for 5 to 10 minutes. The supernatant is discarded, and the pellet is washed and transferred to a homogenizer. The pellet is homogenized in 5 mL of a Percoll solution (75%) liquid Percoll and 0.15 M NaCl) until uniformly suspended and then diluted and centrifuged at 21,600 xg for 30 minutes. Gradient fractions containing the inclusion bodies are recovered and pooled. The isolated inclusion bodies are analyzed by SDS-PAGE.
  • Murine CHL2 was transiently expressed using the pSR ⁇ mCHL2 vector, which was prepared as follows.
  • the open reading frame for CHL2 polypeptide was first amplified by PCR using standard techniques and the primers 2360-40 (5'-G-C-T-A-T-C-T-A-G-A-G-C-C-A-C-C-A-T-G-G-T-T-C-C-C-G-G-G-G-T-G- A-G-G-A-T-C-A-T-C-3'; SEQ ID NO: 17) and 2360-41 (5'-G-C-T-A-G-T-C-G- A-C-C-T-A-T-A-A-T-G-T-C-T-T-G-G-T-C-A-C-T-T-T-G-T-C-T-G-3 ' ; SEQ ID NO: 18).
  • the amplification product was digested with Xba I and Sal I and then inserted into an SR -based expression plasmid (Takebe et al, 1988, Mol. Cell. Biol. 8:466-72) to yield pSR ⁇ mCHL2.
  • a FLAG-tagged murine CHL2 polypeptide expression construct was prepared as follows.
  • the expression of the mCHL2-FLAG polypeptide was detected by direct western blot analysis using 10 ⁇ g/ml of the anti-FLAG mouse monoclonal antibody, M2 (Sigma), according to the manufacturer's recommendations, with the exception that 5%> (w/v) nonfat dry milk (Trader Joe's, Thousand Oaks CA) was used in place of 3%> (w/v) bovine serum albumin.
  • 293 cells were transfected with linearized pFLAGmCHL2, stable transfectants were selected, and the expression level of the corresponding FLAG-tagged proteins in both the serum-free conditioned media and the cell lysates was compared by western blot analysis (Nakayama et al, 2001, Dev. Biol. in press). Conditioned media was concentrated before western blotting and the proteins were then visualized. Transient transfection-based expression was carried out using 293T cells, and ten-fold concentrated conditioned media was analyzed as described above.
  • Antibodies to CHL2 polypeptides may be obtained by immunization with purified protein or with CHL2 peptides produced by biological or chemical synthesis. Suitable procedures for generating antibodies include those described in Hudson and Bay, Practical Immunology (2nd ed., Blackwell Scientific Publications).
  • mice or rabbits are injected with a CHL2 antigen (such as a CHL2 polypeptide), and those with sufficient serum titer levels as determined by ELISA are selected for hybridoma production.
  • Spleens of immunized animals are collected and prepared as single cell suspensions from which splenocytes are recovered.
  • the splenocytes are fused to mouse myeloma cells (such as Sp2/0-Agl4 cells), are first incubated in DMEM with 200 U/mL penicillin, 200 ⁇ g/mL streptomycin sulfate, and 4 mM glutamine, and are then incubated in HAT selection medium (hypoxanthine, aminopterin, and thymidine). After selection, the tissue culture supernatants are taken from each fusion well and tested for anti-CHL2 antibody production by ELISA.
  • HAT selection medium hyperxanthine, aminopterin, and thymidine
  • anti-CHL2 antibodies may also be employed, such as the immunization of transgenic mice harboring human Ig loci for production of human antibodies, and the screening of synthetic antibody libraries, such as those generated by mutagenesis of an antibody variable domain.
  • Example 7 Biological Activity of Murine CHL2 in Xenopus embryos Chordin is known to dorsalize the gastrulating Xenopus embryo by inhibiting the activity of BMP4.
  • the effects of CHL2 on Xenopus embryo development were examined as follows.
  • the Eco RI - Not I fragment of pSPORTmCHL2 was first cloned into pCS2+ (Rupp et al, 1994, Genes Dev. 8:1311-23), linearized with Not I, and capped mRNAs were synthesized with SP6 polymerase and quantified (Nishinakamura et al, 1999, Dev. Biol. 216:481-90).
  • pSPORTmCHL2 plasmid was also linearized directly with Not I and transcribed with T7 polymerase. Both constructs induced secondary axis formation.
  • EF1 elongation factor 1
  • RNA sample was injected into two ventral blastomeres of a four-cell stage Xenopus embryo. Following injection, the embryos were cultured in 10% Steinberg's solution for 48 hours and were then scored for ectopic axis (Nishinakamura et al, 1999; Figure 9).
  • chordin-CHLl complexes The formation of chordin-CHLl complexes is known to inhibit BMP4 function.
  • the similar axis duplication activity observed for CHL2 polypeptide (Example 7) suggested that CHL2 polypeptide also inhibited BMP4 action directly.
  • CD34 + /CD31 hi lymphohematopoietic progenitor cells, CD34 + /CD31 l0 erythro-myeloid progenitor cells, and CD45 + myelomonocytic cells have been shown to be dependent on the presence of 0.15 to 2 ng/ml of BMP4 during the differentiation of mouse embryonic stem (ES) cells (Nakayama et al, 2000, Blood 95:2275:83).
  • CHL2 polypeptide to inhibit BMP4 action directly was examined as follows. Rosa26 ES cells were transferred to a fibronectin-coated plate, adjusted for two days in serum-free medium, and then subjected to differentiation in serum-free methylcellulose medium in the presence of 0.9 ng/ml BMP4 (Nakayama et al, 2000). A BMP4 concentration of 0.9 ng/ml resulted in a near maximal level. Embryoid bodies (EBs) were collected on day 7 and dissociated with collagenase. The cells were stained with monoclonal antibodies for hematopoietic progenitor cell markers, such as CD31 and CD34, and the stained samples were analyzed on a FACScan (Becton Dickinson, San Jose, CA).
  • FACScan Becton Dickinson, San Jose, CA.
  • BMP2 and BMP4 are known to induce alkaline phospatase expression in C2C12 myoblastic cells.
  • CHL2 polypeptide inhibition of BMP2 and BMP4 action was demonstrated in a C2C12 alkaline phospatase assay.
  • the promyoblast cell line C2C12 (ATCC accession no. CRL-1772) was maintained in DMEM containing 10%> fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 5%> CO . Because receptor sensitivity may decrease in cells that have been highly passaged, cells were discarded after being passaged between 10 and 15 times.
  • C2C12 cells were plated in 96-well microtiter plates in 100 ⁇ l of DMEM containing 2%> calf serum and antibiotics at a density of 3 x 10 4 cells/well. To avoid excessive drying, the peripheral wells of the microtiter plates were filled with 200 ⁇ l of DMEM alone. Cells were then incubated overnight at 37°C in a humidified atmosphere of 5%> CO 2 .
  • C2C12 cells were exposed to serial dilutions (1.47, 2.93, 5.9, 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750, and 1500 ng/ml) of murine CHL2-FLAG in the presence of either 909 ng/ml (35 nM) BMP2 or 566 ng/ml (22 nM) BMP4. Cells were then incubated for three days at 37°C in a humidified atmosphere of 5% CO .
  • the media was removed, the cells were rinsed with 0.1 M Tris, pH 7.4, and 150 ⁇ l of glycine buffer (0.1 M glycine, 1 mM MgCl 2 , pH 10.5) containing 0.1% IGEPAL CA-630 (Sigma), was added to the wells.
  • glycine buffer 0.1 M glycine, 1 mM MgCl 2 , pH 10.5 containing 0.1% IGEPAL CA-630 (Sigma) was added to the wells.
  • the cells were then frozen at -80°C and thawed, 50 ⁇ l of cell supernatant was removed for use in Bradford protein assays, and 100 ⁇ l of p- Nifrophenyl phosphate, disodium (Sigma; diluted to 4 mg/ml in glycine buffer) was added to the remaining cell supernatant.
  • activin A another TGF ⁇ superfamily member - showed no sign of interaction with CHL2 under either set of conditions.
  • TGF ⁇ interaction was never observed.
  • BMP2 and BMP4 form a separate subfamily from BMP5, BMP6, and BMP7 (Celeste et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:9843-47).
  • both CHLl and CHL2 polypeptides may be pan-BMP binding proteins.
  • a construct encoding a CHL2 polypeptide/Fc fusion protein under the control of a liver specific ApoE promoter is prepared.
  • the delivery of this construct is expected to cause pathological changes that are informative as to the function of CHL2 polypeptide.
  • a construct containing the full-length CHL2 polypeptide under the control of the beta actin promoter is prepared.
  • the delivery of this construct is expected to result in ubiquitous expression.
  • PCR is used to amplify template DNA sequences encoding a CHL2 polypeptide using primers that correspond to the 5' and 3 ' ends of the desired sequence and which incorporate restriction enzyme sites to permit insertion of the amplified product into an expression vector.
  • PCR products are gel purified, digested with the appropriate restriction enzymes, and ligated into an expression vector using standard recombinant DNA techniques.
  • amplified CHL2 polypeptide sequences can be cloned into an expression vector under the control of the human ⁇ -actin promoter as described by Graham et al, 1997, Nature Genetics, 17:272-74 and Ray et al, 1991, Genes Dev. 5:2265-73.
  • reaction mixtures are used to transform an E. coli host strain by electroporation and transformants are selected for drug resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of an appropriate insert and absence of mutation.
  • the CHL2 polypeptide expression vector is purified through two rounds of CsCl density gradient centrifugation, cleaved with a suitable restriction enzyme, and the linearized fragment containing the CHL2 polypeptide transgene is purified by gel electrophoresis. The purified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2 mM ⁇ DTA at a concentration of 2 mg/mL.
  • transgenic founder animals and control animals are sacrificed for necropsy and pathological analysis. Portions of spleen are removed and total cellular RNA isolated from the spleens using the Total RNA Extraction Kit (Qiagen) and transgene expression determined by RT-PCR. RNA recovered from spleens is converted to cDNA using the SuperscriptTM Preamplification System (Gibco-BRL) as follows. A suitable primer, located in the expression vector sequence and 3' to the CHL2 polypeptide transgene, is used to prime cDNA synthesis from the transgene transcripts.
  • RNA from transgenic founders and controls is incubated with 1 mM of primer for 10 minutes at 70°C and placed on ice.
  • the reaction is then supplemented with 10 mM Tris- HC1, pH 8.3, 50 mM KC1, 2.5 mM MgCl 2 , 10 mM of each dNTP, 0.1 mM DTT, and 200 U of SuperScri.pt II reverse transcriptase.
  • the reaction is stopped by heating for 15 minutes at 72°C and digested with 2U of RNase H for 20 minutes at 37°C. Samples are then amplified by PCR using primers specific for CHL2 polypeptide.
  • transgenic animals Prior to euthanasia, transgenic animals are weighed, anesthetized by isofluorane and blood drawn by cardiac puncture. The samples are subjected to hematology and serum chemistry analysis. Radiography is performed after terminal exsanguination. Upon gross dissection, major visceral organs are subject to weight analysis.
  • tissues i.e., liver, spleen, pancreas, stomach, the entire gastrointestinal tract, kidney, reproductive organs, skin and mammary glands, bone, brain, heart, lung, thymus, trachea, esophagus, thyroid, adrenals, urinary bladder, lymph nodes and skeletal muscle
  • Zn-Formalin 10% buffered Zn-Formalin for histological examination.
  • the tissues are processed into paraffin blocks, and 3 mm sections are obtained. All sections are stained with hematoxylin and exosin, and are then subjected to histological analysis.
  • the spleen, lymph node, and Peyer's patches of both the transgenic and the control mice are subjected to immunohistology analysis with B cell and T cell specific antibodies as follows.
  • the formalin fixed paraffin embedded sections are deparaffinized and hydrated in deionized water.
  • the sections are quenched with 3%> hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh, PA), and incubated in rat monoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, IN).
  • Antibody binding is detected by biotinylated rabbit anti-rat immunoglobulins and peroxidase conjugated streptavidin (BioGenex, San Ramon, CA) with DAB as a chromagen (BioTek, Santa Barbara, CA).
  • Sections are counterstained with hematoxylin. After necropsy, MLN and sections of spleen and thymus from transgenic animals and control littermates are removed. Single cell suspensions are prepared by gently grinding the tissues with the flat end of a syringe against the bottom of a 100 mm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Cells are washed twice, counted, and approximately 1 x 10 6 cells from each tissue are then incubated for 10 minutes with 0.5 ⁇ g CD16/32(Fc ⁇ III/II) Fc block in a 20 ⁇ L volume.
  • Samples are then stained for 30 minutes at 2-8°C in a 100 ⁇ L volume of PBS (lacking Ca + and Mg + ), 0.1%> bovine serum albumin, and 0.01% sodium azide with 0.5 ⁇ g antibody of FITC or PE-conjugated monoclonal antibodies against CD90.2 (Thy-1.2), CD45R (B220), CDllb(Mac-l), Gr-1, CD4, or CD8 (PharMingen, San Diego, CA). Following antibody binding, the cells are washed and then analyzed by flow cytometry on a FACScan (Becton Dickinson).

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AU4196801A (en) 2001-09-12
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CA2401175A1 (en) 2001-09-07
AU2001241968B2 (en) 2005-09-15
MXPA02008506A (es) 2002-12-13

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