WO2001046418A1 - Polypeptide humain slit, zslit3 - Google Patents

Polypeptide humain slit, zslit3 Download PDF

Info

Publication number
WO2001046418A1
WO2001046418A1 PCT/US2000/034230 US0034230W WO0146418A1 WO 2001046418 A1 WO2001046418 A1 WO 2001046418A1 US 0034230 W US0034230 W US 0034230W WO 0146418 A1 WO0146418 A1 WO 0146418A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid number
polypeptide
zslit3
Prior art date
Application number
PCT/US2000/034230
Other languages
English (en)
Inventor
James L. Holloway
Yasmin A. Chandrasekher
Original Assignee
Zymogenetics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zymogenetics, Inc. filed Critical Zymogenetics, Inc.
Priority to AU22718/01A priority Critical patent/AU2271801A/en
Publication of WO2001046418A1 publication Critical patent/WO2001046418A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Neuronal cell outgrowths grow away from the cell body to form synaptic connections.
  • Long, thin processes that carry information away from the cell body are called axons, and short, thicker processes that carry information to and from the cell body are called dendrites.
  • Axons and dendrites are collectively referred to as neurites.
  • Neurites are extended by means of growth cones, the growing tip of the neurite, which is highly motile and is ultimately responsible for increasing and extending the neuronal network in the body.
  • the growth cones are able to navigate their way to their targets using environmental cues or signals, which encourage or discourage the growth cone from extending the neurite in a particular direction.
  • Such cues and signals include older neurons and orienting glial fibers, chemicals such as nerve growth factor released by astrocytes and other attracting or repelling substances released by target cells.
  • the membrane of the growth cone bears molecules such as nerve cell adhesion molecule (N-CAM) that are attracted or repelled by environmental cues and thus influence the direction and degree of neurite growth.
  • N-CAM nerve cell adhesion molecule
  • the growth cone also engulfs molecules from the environment which are transported to the cell body and influence growth. A number of proteins from vertebrates and invertebrates have been identified as influencing the guidance of neurite growth, either through repulsion or chemoattraction.
  • netrins EPH-related receptor tyrosine kinases and their ligands
  • vitronectin thrombospondin
  • human neuronal attachment factor- 1 NAF-1
  • connectin adhesion molecules such as cell adhesion molecule(s) (CAM(s)) and the semaphorins/collapsins (Neugebauer et al., Neuron 6:345-58, 1991;
  • Semaphorins/collapsins are a family of related transmembrane and secreted molecules. Invertebrate, vertebrate and viral semaphorins are known (Kolodkin et al., Cell 75:1389-99, 1993; Luo et al, Cell 75:217-27, 1993; Ensser and Fleckenstein, J. Gen. Virol. 76:1063-7, 1995; Luo et al, Neuron 14:1131-40, 1995; Adams et al., Mech. Devel. 57:33-45, 1996; Hall et al., Proc. Natl. Acad. Sci.
  • Slit proteins found in Drosophila, humans, mice, and other species are involved in directing neuronal guidance. Slit proteins act primarily in repulsive axon guidance, such as chemorepulsion and collapsing activity, but may have other functions in neuronal growth and organogenesis, e.g., neuronal cell migration. Structurally, slit protein family members are characterized by leucine rich repeats (LRRs) and epidermal growth factor (EGF)-like repeats. However they may also contain Ig-like domains, as well as other structural features.
  • LRRs leucine rich repeats
  • EGF epidermal growth factor
  • slit proteins have been shown to be expressed in neuronal tissues and cells, including forebrain, hippocampus, cerebellar granule cells, glial cells, motor neurons, and spinal cord; however, some data show additional expression in other tissues such as endocrine tissues (e.g. thyroid).
  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusible molecules allow cells to communicate with each other and act in concert to regulate cells and form organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin. Hormones and growth factors influence cellular metabolism by binding to proteins.
  • proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems.
  • Other classes of proteins that hormones and growth factors influence are soluble molecules, such as the transcription factors.
  • Proteins affecting neurite growth cues are of great therapeutic value. Isolating and characterizing novel slit proteins would be of value for example, in modulating neurite growth and development; organ regeneration; treatment of peripheral neuropathies; for use as therapeutics for the regeneration of neurons following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries; diagnosing neurological diseases and in treating neurodegenerative diseases such as- multiple sclerosis, Alzheimer's disease, Parkinson's disease, and the like.
  • slit proteins found in non-neuronal tissues are useful for modulating cellular activation, homing, targeting, adhesion, proliferation and differentiation as well as mediating immunological responses.
  • the present invention addresses these needs and others by providing novel slit proteins and related compositions and methods.
  • the present invention provides novel polynucleotides, polypeptides and related compositions and methods.
  • the present invention provides an isolated polynucleotide that encodes a slit protein polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 53 (Asp), to amino acid number 287 (Phe); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Cys), to amino acid number 673 (De); and (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met), to amino acid number 673 (He).
  • the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 157 to nucleotide 861; (b) a polynucleotide sequence as shown in SEQ JD NO:l from nucleotide 70 to nucleotide 2019; (c) a polynucleotide sequence as shown in SEQ ID NO:l from nucleotide 1 to nucleotide 2019; and (d) a polynucleotide sequence complementary to (a), (b), or (c).
  • the isolated polynucleotide disclosed above comprises nucleotide 1 to nucleotide 2019 of SEQ ID NO: 3.
  • the isolated polynucleotide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ JD NO:2 from amino acid number 53 (Asp), to amino acid number 287 (Phe); (b) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 24 (Cys), to amino acid number 673 (lie); and (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met), to amino acid number 673 (Ee).
  • the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a slit protein polypeptide as shown in SEQ ID NO: 2 from amino acid number 24 (Cys), to amino acid number 673 (He); and a transcription terminator, wherein the promoter is operably linked to the DNA segment, and the DNA segment is operably linked to the transcription terminator.
  • the expression vector disclosed above further comprises a secretory signal sequence operably linked to the DNA segment.
  • the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
  • the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met), to amino acid number 23 (Gly); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Cys), to amino acid number 52 (Pro); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 53 (Asp), to amino acid number 287 (Phe); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 288 (Pro), to amino acid number 408 (Asp); (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 298 (Asn), to amino acid number 350 (Pro); (f) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 4
  • the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as disclosed above; and a transcription terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcription terminator.
  • the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA construct.
  • the present invention provides a method of producing a fusion protein comprising: culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.
  • the present invention provides an isolated slit protein polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 53 (Asp), to amino acid number 287 (Phe); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 24 (Cys), to amino acid number 673 (Ee); and (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 1 (Met), to amino acid number 673 (Ee).
  • the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 53 (Asp), to amino acid number 287 (Phe); (b) the amino acid sequence as shown in SEQ ED NO:2 from amino acid number 24 (Cys), to amino acid number 673 (Ee); and (c) the amino acid sequence as shown in SEQ JD NO:2 from amino acid number 1 (Met), to amino acid number 673 (Ee).
  • the present invention provides a method of producing a slit protein polypeptide comprising: culturing a cell as disclosed above; and isolating the slit protein polypeptide produced by the cell.
  • the present invention provides a method of producing an antibody to a polypeptide comprising: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 30 to 649 amino acids, wherein the polypeptide is identical to a contiguous sequence of amino acids in SEQ JD NO:2 from amino acid number 24 (Cys) to amino acid number 673 (Ee); (b) a polypeptide as disclosed above; (c) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 from amino acid number 24 (Cys), to amino acid number 52 (Pro); (d) a polypeptide consisting of the amino acid sequence of SEQ JD NO:2 from amino acid number 288 (Pro), to amino acid number 408 (Asp); (e) a polypeptide consisting of the amino acid sequence of SEQ JD NO:2 from amino acid number 298 (Asn), to amino acid number 350 (Pro); (f) a polypeptide consisting of:
  • the present invention provides an antibody produced by the method as disclosed above, which binds to a polypeptide.
  • the antibody disclosed above is a monoclonal antibody.
  • the present invention provides an antibody that specifically binds to a polypeptide of SEQ ID NO:2.
  • the present invention provides a method of detecting, in a test sample, the presence of a modulator of ZSL1T3 protein activity, comprising: transfecting a ZSLIT3-responsive cell, with a reporter gene construct that is responsive to a ZSLIT3 -stimulated cellular pathway; and producing a ZSLIT3 polypeptide by the method as disclosed above; and adding the ZSLIT3 polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the ZSLJT3 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the modulator of ZSLIT3 activity in the test sample.
  • the present invention provides a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; producing a first reaction product by incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ JD NO:l or the complement of SEQ ID NO:l, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the first reaction product; and comparing said first reaction product to a control reaction product from a wild type patient, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
  • the present invention provides a method for detecting a cancer in a patient, comprising: obtaining a tissue or biological sample from a patient; incubating the tissue or biological sample with an antibody as disclosed above under conditions wherein the antibody binds to its complementary polypeptide in the tissue or biological sample; visualizing the antibody bound in the tissue or biological sample; and comparing levels of antibody bound in the tissue or biological sample from the patient to a normal control tissue or biological sample, wherein an increase or decrease in the level of antibody bound to the patient tissue or biological sample relative to the normal control tissue or biological sample is indicative of a cancer in the patient.
  • the present invention provides a method for detecting a cancer in a patient, comprising: obtaining a tissue or biological sample from a patient; labeling a polynucleotide comprising at least 14 contiguous nucleotides of SEQ JD NO:l or the complement of SEQ ID NO:l; incubating the tissue or biological sample with under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence; visualizing the labeled polynucleotide in the tissue or biological sample; and comparing the level of labeled polynucleotide hybridization in the tissue or biological sample from the patient to a normal control tissue or biological sample, wherein an increase or decrease in the labeled polynucleotide hybridization to the patient tissue or biological sample relative to the normal control tissue or biological sample is indicative of a cancer in the patient.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate, hi principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • complements of a polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGAGCTT-3' are 5'- AGCTTgagt-3' and 3'-tcgacTACC-5'.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
  • Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
  • an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • a "polynucleotide” is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs”.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".
  • Probes and/or primers can be RNA or DNA.
  • DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements.
  • Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
  • PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis.
  • a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
  • a "protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • a bioactive molecule i.e., a ligand
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
  • PDGF receptor growth hormone receptor
  • IL-3 receptor growth hormone receptor
  • GM-CSF receptor growth hormone receptor
  • G-CSF receptor erythropoietm receptor
  • JL-6 receptor JL-6 receptor
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention is based in part upon the discovery of a novel human member of the LRR-containing protein family, designated "ZSLIT3".
  • the LRR-containing protein family is a diverse family of proteins with differing functions and cellular locations (Kobe, B. and Deisenhofer, J., Trends Biochem Sci. 19:415-21 1994).
  • the ZSLIT3 polypeptide of the present invention shares the closest known structural similarity to Drosophila slit protein (Genbank Accession No. P24014).
  • the human ZSLIT3 nucleotide sequence is represented in SEQ JD NO:l and the deduced amino acid sequence in SEQ JJD NO:2.
  • the novel human ZSLIT3 proteins and polypeptides encoded by polynucleotides of the present invention were initially identified by querying an Expressed Sequence Tag (EST) database for sequences homologous to conserved motifs within the slit protein family.
  • EST Expressed Sequence Tag
  • Sequence analysis of the deduced amino acid sequence as represented in SEQ JD NO:2 indicates a 673 amino acid polypeptide containing a 23 amino acid residue secretory signal sequence (amino acid residues 1 (Met) to 23 (Gly) of SEQ JD NO:2), and a mature polypeptide of 650 amino acids (amino acid residues 24 (Cys) to 673 (Be)).
  • the mature ZSLIT3 polypeptide sequence contains the following domains, and motifs:
  • N-terminal LRR flanking domain comprising amino acid residues 24 (Cys) to 52 (Pro) of SEQ ID NO:2).
  • N-terminal LRR flanking domain Within the N-terminal LRR flanking domain are 4 conserved cysteines comprising amino acid residues 24, 28, 30 and 38 of SEQ ID NO:2.;
  • LRR domain Within the LRR domain there are 10 consecutive LRR motifs, ordered from N-terminus to C-terminus:
  • LRR-1 corresponding to amino acids 53 (Asp) to 76 (Pro) of SEQ BD NO:2
  • LRR-2 corresponding to amino acids 77 (Gly) to 100 (Ala) of SEQ BD
  • LRR domain comprising amino acid residues 228 (Asn) to 350 (Pro) of SEQ BD NO:2.
  • an "EGF domain” comprising amino acid residues 409 (Cys) to 441 (Cys) of SEQ BD NO:2.
  • EGF domain Within the EGF domain are 6 conserved cysteines comprising amino acid residues 409, 414, 420, 430, 432 and 441 of SEQ BD NO:2.
  • the EGF domain is followed by
  • ZSLIT3 has putative N-linked glycosylation sites at Asn residues comprising amino acid residues 101, 117, 273, 500, and 528. Moreover, there are putative prenylation sites Cys residues comprising amino acid residues 304, 420 and
  • genomic structure of ZSLLT3 is readily determined by one of skill in the art by comparing the cDNA sequence of SEQ BD NO:l and the translated amino acid of SEQ BD NO: 2 with the genomic DNA in which the gene is contained (Genbank Accession No. AC012676). For example, such analysis can be readily done using FASTA as described herein. As such, the intron and exon junctions in this region of genomic DNA can be determined for the ZSLJT3 gene.
  • This ZSLIT3 genomic DNA is located on chromosome 16, at 16ql2 (Genbank Accession No. AC007226).
  • the ZSLIT3 genomic DNA appears to have several exons, the entire coding sequence appears to be contained within in a single exon, as there are no putative introns within the genomic DNA.
  • the present invention includes the ZSLJT3 gene as seen in human genomic DNA.
  • domain boundaries are approximations based on sequence alignments, intron positions and splice sites, and may vary slightly; however, such estimates are generally accurate to within ⁇ 4 amino acid residues.
  • the present invention is not limited to the expression of the sequence shown in SEQ ED NO:l.
  • a number of truncated ZSLIT3 polynucleotides and polypeptides are provided by the present invention. These polypeptides can be produced by expressing polynucleotides encoding them in a variety of host cells, hi many cases, the structure of the final polypeptide product will result from processing of the nascent polypeptide chain by the host cell, thus the final sequence of a ZSLIT3 polypeptide produced by a host cell will not always correspond to the full sequence encoded by the expressed polynucleotide.
  • ZSLIT3 sequence in a cultured mammalian cell is expected to result in removal of at least the secretory peptide, while the same polypeptide produced in a prokaryotic host would not be expected to be cleaved.
  • a variety of ZSLJT3 polypeptides can thus be produced. Differential processing of individual chains may result in heterogeneity of expressed polypeptides and the production of heterodimeric ZSLIT3 proteins.
  • ZSLTT3 polypeptides can be produced by other known methods, such as solid phase synthesis, methods for which are well known in the art. See, for example, Merrifield, Am. Chem. Soc.
  • ZSLIT3 polynucleotides are highly expressed in testis and ovary; expressed in brain; moderately expressed in heart, placenta, lung, liver, kidney, prostate and pancreas; and expressed at lower levels in brain, spleen, small intestine and colon and thyroid tissue, and other tissues (See, Example 2).
  • the transcript size agrees with the predicted size of the ZSLJT3 protein as disclosed in SEQ ID NO:2. Additional analysis may reveal a ZSLIT3 transcript in numerous localized brain and neuronal tissues, or cells, and in tumor cell lines. RT-PCR data can also be performed to show where ZSLIT3 mRNA is expressed. Such methods are well known in the art and disclosed herein. Moreover, zslit3 is expressed in caners as evinced by expression in glioblastoma tissue, and intestinal carcinoma, osteogenic sarcoma and breast carcinoma cell lines, but not other tissues and cell lines tested (Example 3).
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the ZSL1T3 polypeptides disclosed herein.
  • polynucleotide molecules including DNA and RNA molecules, that encode the ZSL1T3 polypeptides disclosed herein.
  • a degenerate polynucleotide sequence that encompasses all polynucleotides that encode the ZSLIT3 polypeptide of SEQ ED NO:2 (amino acid residues 1-673) is disclosed in SEQ BD NO:3.
  • ZSLH3 polypeptide-encoding polynucleotides ranging from nucleotide 1-2019 of SEQ BD NO:3 are contemplated by the present invention.
  • fragments as described herein with respect to SEQ BD NO: 1 which are formed from analogous regions of SEQ
  • nucleotides 1-2019 of SEQ BD NO:l correspond to nucleotides 1- 2019 SEQ ED NO: 3.
  • degenerate sequence of SEQ BD NO: 3 also provides all RNA sequences encoding SEQ BD NO:2 by substituting uracil (U) for thymine (T).
  • U uracil
  • T thymine
  • Table 1 sets forth the one- letter nucleotide base codes used within SEQ BD NO: 3 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a nucleotide base code letter.
  • “Complement” indicates the nucleotide base code for the complementary nucleotide(s).
  • the nucleotide base code “Y” denotes either the nucleotide C or T
  • its complement nucleotide base code “R” denotes nucleotides A or G, A being complementary to T, and G being complementary to C.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine WSN
  • the degenerate codon for arginine AGR
  • the degenerate codon for arginine MGN
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ED NO:2. Such variant sequences can be readily tested for functionality as disclosed herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of all of the possible codons encoding each amino acid (See Table 2).
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. The highly conserved amino acids in the LRR and EGF domains of ZSLJT3 can be used as a tool to identify new family members.
  • RT-PCR reverse transcription-polymerase chain reaction
  • RNA obtained from a variety of tissue sources or cell lines can be used to amplify sequences, in particular, those sequences encoding the conserved LRR and EGF domains, especially sequences associated with the conserved cysteine residues, from RNA obtained from a variety of tissue sources or cell lines.
  • highly degenerate primers designed from the ZSLTT3 nucleotide sequences as disclosed in SEQ JD NO:l and SEQ JD NO:3 are useful for this purpose.
  • isolated polynucleotides will hybridize to similar sized regions of SEQ ED NO: 1 , or to sequences complementary thereto, under stringent conditions.
  • isolated polynucleotides will hybridize to similar sized regions of SEQ DD NO:l, other polynucleotide probes, primers, fragments and sequences recited herein or sequences complementary thereto.
  • Polynucleotide hybridization is well known in the art and widely used for many applications, see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-59, 1990. Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.
  • Hybridization will occur between sequences which contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch.
  • the T m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases.
  • Stringent hybridization conditions encompass temperatures of about 5-25°C below the thermal melting point (T m ) of the hybrid and a hybridization buffer having up to 1 M Na + .
  • the above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid.
  • the T m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence.
  • Those conditions which influence the T m include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution.
  • hybridization of longer polynucleotide sequences is done at temperatures of about 20-25°C below the calculated T m .
  • hybridization is typically carried out at the T m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
  • the length of the polynucleotide sequence influences the rate and stability of hybrid formation. Smaller probe sequences, ⁇ 50 bp, come to equilibrium with complementary sequences rapidly, but may form less stable hybrids. Incubation times of anywhere from minutes to hours can be used to achieve hybrid formation. Longer probe sequences come to equilibrium more slowly, but form more stable complexes even at lower temperatures.
  • Incubations are allowed to proceed overnight or longer. Generally, incubations are carried out for a period equal to three times the calculated Cot time.
  • Cot time the time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art.
  • the base pair composition of polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization- buffer.
  • A-T pairs are less stable than G-C pairs in aqueous solutions containing NaCl. Therefore, the higher the G-C content, the more stable the hybrid. Even distribution of G and C residues within the sequence also contribute positively to hybrid stability.
  • Base pair composition can be manipulated to alter the T m of a given sequence, for example, 5-methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the T m . 7-deazo-2'-deoxyguanosine can be substituted for guanosine to reduce dependence on T m . Ionic concentration of the hybridization buffer also effects the stability of the hybrid.
  • Hybridization buffers generally contain blocking agents such as Denhardt's solution (Sigma Chemical Co., St.
  • hybridization buffers contain from between 10 mM-1 M Na + .
  • Premixed hybridization solutions are also available from commercial sources such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, Wl) for use according to manufacturer's instruction.
  • Addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T m of a hybrid.
  • formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures.
  • Formamide also acts to reduce non-specific background when using RNA probes.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for isolating DNA and RNA are well known in the art.
  • RNA is isolated from a tissue or cell that produces large amounts of ZSLIT3 RNA.
  • tissue and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include breast, brain and neuronal tissues, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
  • Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
  • Complementary DNA cDNA is prepared from poly(A) + RNA using known methods.
  • Polynucleotides encoding ZSL1T3 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding ZSLIT3 can be obtained by conventional cloning procedures.
  • Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron.
  • Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
  • Expression libraries can be probed with antibodies to ZSLIT3, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using techniques widely known in the art. See, for example, Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al, Proc. Natl. Acad. Sci.
  • ZSLIT3 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a ZSLIT3 gene.
  • this gene region is expected to provide for testis, ovary, brain, neurological, endrocrinological or tumor- specific expression.
  • Promoter elements from a ZSLIT3 gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of ZSLLT3 proteins by "gene activation" as disclosed in U.S. Patent No.
  • an endogenous ZSLIT3 gene in a cell is altered by introducing into the ZSLIT3 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a ZSLIT3 5' non-coding sequence that permits homologous recombination of the construct with the endogenous ZSLFT3 locus, whereby the sequences within the construct become operably linked with the endogenous ZSLIT 3 coding sequence, hi this way, an endogenous ZSLIT3 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • the present invention further provides counterpart ligands and polynucleotides from other species (orthologs). These species include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are ZSLIT3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human ZSLIT3 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the ligand. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a ligand-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
  • a cDNA can also be cloned by PCR, using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the ligand. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ ED NO:l represents a single allele of the human ZSLJT3 gene and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ED NO:2, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ BD NO:2.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZSLIT3 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • the present invention also provides isolated ZSLIT3 polypeptides that are substantially similar to the polypeptides of SEQ ID NO:2 and their orthologs.
  • the term "substantially similar” is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ JD NO: 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ED NO: 2 or its orthologs). Percent sequence identity is determined by conventional methods. See, for example, Altschul et al offset Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant ZSL1T3.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat. Acad. Sci. USA 85:2444, 1988), and by Pearson, Meth. Enzvmol. 183:63, 1990).
  • the ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allows for amino acid insertions and deletions.
  • ktup l
  • gap opening penalty 10
  • gap extension penalty l
  • substitution matrix BLOSUM62
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other FASTA program parameters set as default.
  • the present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat. Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • the language "conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Variant ZSLIT3 polypeptides or substantially homologous ZSLIT3 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • the present invention thus includes polypeptides of from about 253 amino acid residues to about 700 amino acid residues, that comprise a sequence that is at least 80%, preferably at least 90%, more preferably at least 95% or more identical to the corresponding region of SEQ ED NO:2, and more preferably having conserved cysteine residues corresponding to the amino acid residues 24, 28, 30, and 38; and/or 302, 304, 328, and 349; and/or 409,414, 420, 430, 432, and
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ZSLIT3 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N-methyl- glycine, allo-threonine, methylthreonine, hydroxy- ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3 and 4-methylproline, 3,3-dimethyl- proline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluoro-phenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-3, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the generic code, non-naturally occurring amino acids, and unnatural amino acid residues may be substituted for ZSLIT3 amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991.
  • single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., collapase activity, cellular interaction) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity; in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related LRR and EGF polypeptides.
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight B® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.. Current Opin. Struct. Biol. 6:3-10, 1996).
  • available software e.g., the Insight B® viewer and homology modeling tools; MSI, San Diego, CA
  • secondary structure propensities e.g., binary patterns, complementary packing and buried polar interactions
  • Amino acid sequence changes are made in ZSL ⁇ T3 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • the ZSLIT3 polypeptide comprises one or more helices
  • changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
  • CD circular dichrosism
  • NMR nuclear magnetic resonance
  • digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
  • a Hopp/Woods hydrophilicity profile of the ZSLLT3 protein sequence as shown in SEQ ED NO:2 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824- 3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering LI: 153-169, 1998).
  • the profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored.
  • hydrophilic regions include: (1) amino acid number 123 (Leu) to amino acid number 128 (Arg) of SEQ ED NO:2; (2) amino acid number 156 (Gin) to amino acid number 161 (Arg) of SEQ ED NO:2; (3) amino acid number 225 (Asp) to amino acid number 230 (Arg) of SEQ BD NO:2; (4) amino acid number 322 (Ser) to amino acid number 327 (Arg) of SEQ ED NO:2; and (5) amino acid number 502 (Ser) to amino acid number 507 (Arg) of SEQ ED NO:2.
  • hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a ZSLLT3 polypeptide, so as not to disrupt the overall structural and biological profile.
  • hydrophobic residues selected from the group consisting of Val, Leu and Ee or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
  • residues tolerant of substitution could include those hydrophobic residues as shown in SEQ ED NO: 2.
  • Cysteine residues of SEQ BD NO: 2 will be relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between slit protein family members with ZSLIT3.
  • a variant ZSLIT3 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant ZSLIT3 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, as discussed above.
  • the present invention also includes functional fragments of ZSLIT3 polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" ZSLIT3 or fragment thereof defined herein is characterized by its axon collapsing or slit protein-like activity, proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-ZSLIT3 antibody or ZSL1T3 receptor (either soluble or immobilized).
  • ZSLJT3 is characterized by a LRR domain structure and
  • the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the domains or motifs described above; and (b) functional fragments comprising one or more of these domains or motifs.
  • the other polypeptide portion of the fusion protein may be contributed by another slit protein, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a ZSLIT3 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l or fragments thereof can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vpctors in proper reading frame, and the expressed polypeptides are isolated and tested for ZSLJT3 activity, or for the ability to bind anti-ZSLJT3 antibodies or ZSLH3 receptor.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired ZSLIT3 fragment.
  • particular fragments of a ZSLJT3 polynucleotide can be synthesized using the polymerase chain reaction.
  • variants of the disclosed ZSLJT3 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active ligands or portions thereof e.g., receptor-binding fragments
  • These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • polypeptides that are substantially similar to amino acid residues 24 (Cys) to 673 (Ee), or a fragment thereof, allelic variants thereof and retain the properties of the wild-type ZSLH3 protein.
  • Such polypeptides may include additional amino acids from the N-terminal LRR flanking domain, LRR domain, LRR motifs LRR-1-10, middle region, C-terminal LRR flanking domain, EGF domain, C- terminal region and other domains and motifs as described herein; the secretory signal sequence; affinity tags; and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed herein.
  • the present invention further provides a variety of polypeptide fusions.
  • a ZSLIT3 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584.
  • Preferred dimerizing proteins in this regard include immunoglobulin constant region domains.
  • Immunoglobulin-ZSLIT3 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric ZSLIT3 analogs. Auxiliary domains can be fused to ZSLIT3 polypeptides to target them to specific cells, tissues, or macromolecules.
  • a ZSLIT3 polypeptide or protein could be targeted to a predetermined cell type by fusing a ZSLIT3 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell, hi this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a ZSLIT3 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
  • any ZSLJT3 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the ZSL ⁇ T3 polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor, NY, 1989; and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
  • a DNA sequence encoding a ZSLJT3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the ZSLTT3 polypeptide (amino acid residues 1 (Met) through amino acid residue 23 (Gly) of SEQ JD NO:2), or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is joined to the ZSLIT3 DNA sequence in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid 1 (Met) to amino acid 23 (Gly) of SEQ ED NO: 2 is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • polypeptide fusions, or hybrid ZSLJT3 proteins are constructed using regions or domains of the inventive ZSLJT3 in combination with those of other Slit protein family proteins (e.g. human SLIT-1 and SLtT-2, or murine slit proteins, and the like), or heterologous proteins (Sambrook et al., ibid.; Altschul et al., ibid.; Picard, Cur. Opin.
  • hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, alter activity in neurite or other functional assays, alter immune response, or gene transcription in a cell, alter cytoskeletal organization and cell motility, transformation, or invasiveness, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
  • a polynucleotide encoding various components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a structural or biological function may be swapped between ZSLIT3 of the present invention with the functionally equivalent domain(s) from another family member.
  • Such domains include, but are not limited to the signal peptide, N-terminal LRR flanking domain, LRR domain, LRR motifs LRR 1-10, middle region, C-terminal LRR flanking domain, an EGF domain, C-terminal region and other domains and motifs as described herein.
  • Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known slit protein family proteins (e.g. affecting neurite growth or collapsing activity, and the like) depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the ZSLIT3 polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., a ZSLIT3 active polypeptide or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein.
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising an N-terminal LRR flanking domain, followed by an LRR domain containing at least one LRR motif, followed by a C-terminal LRR flanking domain containing at least one EGF domain; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising an LRR domain followed by an EGF domain ; or a DNA construct would encode from N : terminus to C-terminus a fusion protein comprising a signal peptide, N-terminal LRR flanking domain, LRR domain containing at least one LRR motif, middle region comprising a C-terminal LRR flanking domain, EGF domain, C-terminal region; or for example, any of the above as interchanged with equivalent regions from another slit protein family member.
  • Such fusion proteins can be expressed, isolated, and assayed for activity as described herein. Moreover, such fusion proteins can be used to express and secrete fragments of the ZSLJT3 polypeptide, to be used, for example to inoculate an animal to generate anti-ZSLIT3 antibodies as described herein.
  • a secretory signal sequence can be operably linked to the N-terminal LRR flanking domain, LRR domain containing at least one LRR motif, middle region comprising a C-terminal LRR flanking domain, EGF domain, C-terminal region, or a combination thereof (e.g., operably linked polypeptides comprising operably fused LRR domain and middle region comprising a C-terminal LRR flanking domain, with or without an EGF domain, or other ZSLIT3 polypeptide fragments or combinations described herein), to secrete a fragment of ZSLIT3 polypeptide that can be purified as described herein and serve as an antigen to be inoculated into an animal to produce anti-ZSLtT3 antibodies, as described herein.
  • proteins of the present invention can be joined to other bioactive molecules, particularly other slit proteins, to provide multi-functional molecules.
  • one or more domains from ZSLIT3 can be joined to other slit proteins to enhance their biological properties or efficiency of production.
  • the present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains of ZSLIT3 is fused to another polypeptide. Fusion is preferably done by splicing at the DNA level, as described herein, to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as enhanced or diminished neurite collapsing or repulsing activity, neuronal migration, increased or decreased immune response activity, improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmacodynamics.
  • Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al, Focus 15:80, 1993).
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.
  • CHO-K1 Chinese hamster ovary
  • ATCC No. CCL 61 Chinese hamster ovary
  • DG44 CHO cells Chosin et al., Som. Cell. Molec. Genet. 12:555-66, 1986.
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • ZSLIT3 DNA fragments are subcloned into mammalian expression plasmids, such as pZP9 (ATCC No. 98668) or modifications thereof.
  • mammalian expression plasmids such as pZP9 (ATCC No. 98668) or modifications thereof.
  • Glu-Glu-tagged ZSLIT3 proteins Glu-Glu-tagged for example, such expression plasmids contain the mouse metallothionein- 1 promoter; a TPA leader peptide followed by the sequence encoding a Glu-Glu tag (e.g., SEQ JD NO:4), for expression of N-terminal Glu-Glu ZSLIT3 proteins; the ZSLIT3 polynucleotide sequence without the native signal sequence, and a human growth hormone terminator.
  • the expression cassette can be modified to place the sequence encoding a Glu-Glu tag (e.g., SEQ JJD NO:4) after the ZSLJT3 nucleotide sequence followed by a stop codon and the human growth hormone terminator.
  • a Glu-Glu tag e.g., SEQ JJD NO:4
  • such expression vectors would be transfected and expressed in mammalian cells, such as BHK or CHO cells.
  • Transformed cells can be screened for expression of ZSLIT3 proteins by filter assay.
  • Affinity tagged proteins can be detected using conjugated antibodies to the tag, such as anti-Glu-Glu antibody-HRP conjugate.
  • Colonies expressing ZSL1T3 can be selected and subjected to Western Blot analysis and mycoplasma testing.
  • Preferably individual clones can be expanded and used for large scale production of ZSLIT3 proteins.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 1 L47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King, L.A.
  • the Baculovirus Expression System A Laboratory Guide. London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566- 79, 1993). This system is sold in the Bac-to-BacTM kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the ZSLIT3 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.”
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case ZSLIT3.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
  • the baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
  • Pcor baculovirus basic protein promoter
  • MP promoter baculovirus basic protein promoter
  • transfer vectors can be constructed which replace the native ZSLIT3 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native ZSL ⁇ T3 secretory signal sequence
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ZSLIT3 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T.
  • a transfer vector containing ZSLIT3 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses ZSLIT3 is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and
  • Suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No.5,300,435).
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 BTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 cells to a density of 1-2 x 10 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing fragments or polypeptide fusions.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al, U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose- containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publication WO 9717450.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene.
  • a preferred promoter is that of a P. methanolica alcohol utilization gene (AUG1).
  • P. methanolica contains a second alcohol utilization gene, AUG2, the promoter of which can also be used.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • Preferred such cleavage sites are those that are recognized by restriction endonucleases that cut infrequently within a DNA sequence, such as those that recognize 8-base target sequences (e.g., Not I).
  • a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21).
  • the ADE2 gene when transformed into an ade2 host cell, allows the cell to grow in the absence of adenine.
  • Other nutritional markers that can be used include the P.
  • methanolica ADE1, HIS3, and LEU2 genes which allow for selection in the absence of adenine, histidine, and leucine, respectively.
  • host cells For large- scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and A UG2) are deleted.
  • host cells deficient in vacuolar protease genes PEP4 and PRB1 are preferred.
  • Gene-deficient mutants can be prepared by known methods, such as site-directed mutagenesis.
  • P. methanolica genes can be cloned on the basis of homology with their counterpart Saccharomyces cerevisiae genes.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. See, in general, Neumann et al., EMBO J. 1:841-5, 1982 and Meilhoc et al, Bio/Technology
  • electroporation is most efficient when the cells are exposed to an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kN/cm, preferably about 3.75 kN/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign D ⁇ A sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.).
  • the polypeptide When expressing a ZSLIT3 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added D ⁇ A by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors.
  • a preferred culture medium for P. methanolica is YEPD.
  • Expressed recombinant ZSLIT3 polypeptides can be purified using fractionation and/or conventional purification methods and media.
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
  • polypeptides of the present invention can be isolated by exploitation of their physical or biochemical properties. Methods used to purify mammalian slit proteins, and LRR-containing proteins are exemplary (see, for example, Hu, H., Neuron
  • JJVIAC immobilized metal ion adsorption
  • chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., maltose-binding protein, Glu-Glu tag, or an immunoglobulin domain
  • a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • ZSLIT3 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZSLJT3 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art. Polypeptides containing the receptor-binding region of the ligand can be used for purification of receptor.
  • the ligand polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting media will generally be configured in the form of a column, and fluids containing receptors are passed through the column one or more times to allow receptor to bind to the ligand polypeptide.
  • the receptor is then eluted using changes in salt concentration, chaotropic agents (MnCIj), or pH to disrupt ligand- receptor binding.
  • ZSLIT3 polypeptides or ZSL ⁇ T3 fusion proteins are used, for example, to identify the ZSLIT3 receptor.
  • ZSLJT3 polypeptides Using labeled ZSLJT3 polypeptides, cells expressing the receptor are identified by fluorescence immunocytometry or immunohistochemistry.
  • ZSLIT3 polypeptides are useful in determining the distribution of the receptor on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • An exemplary method to identify a ZSLIT3 receptor in vivo or in vitro, e.g., in cell lines, is to us a ZSLJT3 polypeptide fused to the catalytic domain of Alkaline phosphatase (J P), as described in Feiner, L.
  • J P Alkaline phosphatase
  • Slit proteins have been characterized as chemorepellants in the neurological system, responsible for directing neurite growth and neuronal system organization.
  • Slit protein polypeptides, agonists and antagonists can be used to modulate neurite growth and development and demarcate nervous system structures. Mutations deleting slit proteins result in axon projections in to inappropriate regions of the spinal cord.
  • ZSLJT3 is expressed in various brain tissues and in spinal cord.
  • ZSLIT3 polypeptides and ZSLIT3 antagonists, including anti-ZSLIT3 antibodies, would be useful as in treatment of peripheral neuropathies by increasing spinal cord and sensory neurite outgrowth and patterning by acting as repulsive and attractive guidance cues to the developing sensory or motor neuron.
  • Guidance cues serve to direct or constrain the pattern of neuron growth, channeling axons to their appropriate destination. In the absence of guidance cues neuron growth is random and unstructured.
  • ZSLIT3 polypeptides, agonists, and antagonists, including anti- ZSLIT3 antibodies can be included in the therapeutic treatment for the regeneration and direction of neurite outgrowths following strokes, brain damage caused by head injuries and paralysis caused by spinal injuries.
  • Application may also be made in treating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease and peripheral neuropathies, or demyelinating diseases including multiple sclerosis, by directing neuronal outgrowths.
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's disease Huntington's disease
  • Parkinson's disease and peripheral neuropathies or demyelinating diseases including multiple sclerosis
  • Such an application would be repair of transected axons that are common in lesions of multiple sclerosis (Trapp et al., N. Engl. J. Med. 338:278-85, 1998). Similarly, application may also be made in treating neurodegenerative diseases or conditions as a result of exposure to neurotoxic chemical compounds.
  • ZSLIT3 is expressed in some non-neuronal tissues but likely influences the development and innervation of these tissues.
  • G-Sema I and collapsin are hypothesized to act in vivo as repulsive or inhibitory molecules that prevent neighboring ventral motorneurons from innervating extra thoracic muscle, other situations, G- Sema I and collapsin may also act as an attractive agent to promote innervation (Kolodkin,A.L. et al., Cell 75:1389-99, 1993).
  • ZSLIT3 polypeptides would be useful in directing neuronal development and innervation patterns in various tissues, and in organogenesis, for example in heart, lung, kidney, liver, and pancreas, and the like, by acting as a guidance cue and stimulating the formation of normal synaptic terminal arborizations, for example on a target muscle tissue.
  • ZSLH3 would be useful in directing and defining the growth of developing neuronal and organ tissues, for example as, defining the margins or development of a particular organ or tissue.
  • ZSLIT3 polypeptides would be useful in the defining and directing development of various tissues and organs including those associated with heart, lung, kidney, liver, pancreas, muscle, fibroblasts, reproductive, endocrine and lymphatic tissues.
  • ZSLIT3 molecules of the present invention are useful in lung organogenesis and repair and as permissive or regulatory factors in this process.
  • ZSLIT3 molecules of the present invention can be used to diagnose and treat pulmonary diseases such as those associated with respiration and circulation, cystic fibrosis, asthma, emphysema, lung cancers and the like. Methods for using ZSLH3 as a diagnostic and pharmaceutical composition are described herein.
  • kidney development involves ductile growth and branching, and the formation of the renal epithelium. The latter differentiates onto the nephron. The collecting ducts are formed at this time as well.
  • Secreted polypeptides expressed kidney such as ZSLIT3 polypeptides of the present invention, are useful in kidney organogenesis and repair and in cases of kidney failure, for example, kidney failure associated with diabetes, or in pre-renal, renal, and post-renal complications (e.g., benign prostatic hyperplasia).
  • ZSLIT3 may be a mediator of immunosuppression, in particular the activation and regulation of T lymphocytes.
  • ZSLTT3 polypeptides would be useful additions to therapies for treating immunodeficiencies.
  • ZSLIT3 can be useful in diagnosing and treating conditions where selective elimination of inappropriately activated T cells or other immune cells would be beneficial, such as in autoimmune diseases, in particular insulin dependent diabetes mellitus, rheumatoid arthritis and multiple sclerosis.
  • Such polypeptides could be used to screen serum samples from patients suffering from such conditions in comparison to normal samples.
  • Inappropriately activated T cells would include those specific for self-peptide/self-major histocompatibility complexes and those specific for non-self antigens from transplanted tissues. Use could also be made of these polypeptides in blood screening for removal of inappropriately activated T cells before returning the blood to the donor.
  • ZSLIT3 polypeptides can be used in vivo as an anti-inflammatory, for inhibition of antigen in humoral and cellular immunity and for immunosuppression in graft and organ transplants. Methods of assessing ZSLIT3 pro- or anti-inflammatory effects are well known in the art.
  • ZSLJT3 polynucleotides and/or polypeptides can be used for regulating the proliferation and stimulation of a wide variety of cells, such as T cells, B cells, lymphocytes, peripheral blood mononuclear cells, fibroblasts and hematopoietic cells.
  • ZSLIT3 polypeptides will also find use in mediating metabolic or physiological processes in vivo. Proliferation and differentiation can be measured in vitro using cultured cells. Suitable cell lines are available commercially from such sources as the American Type Culture Collection (Rockville, MD).
  • Bioassays and ELISAs are available to measure cellular response to ZSLIT3, in particular are those which measure changes in cytokine production as a measure of cellular response (see for example, Current Protocols in Immunology ed. John Coligan et al., NJH, 1996).
  • apoptosis assays such as the DNA fragmentation assay described by Wiley et al. (Immunity, 3:673-82, 1995, and the cell death assay described by Pan et al., Science, 276:111-13, 1997).
  • Assays to measure other cellular responses including antibody isotype, monocyte activation, NK cell formation and antigen presenting cell function are also known.
  • the ZSLIT3 polypeptides may also be used to stimulate lymphocyte development, such as during bone marrow transplantation and as therapy for some cancers.
  • ZSLIT3 polypeptides can also be measured by administering polypeptides of the claimed invention to the appropriate animal model.
  • Well established animal models are available to test in vivo efficacy of ZSLIT3 polypeptides for certain disease states.
  • ZSLIT3 polypeptides can be tested in vivo in a number of animal models of autoimmune disease, such as the NOD mice, a spontaneous model system for insulin-dependent diabetes mellitus (JDDM), to study induction of non-responsiveness in the animal model.
  • Administration of ZSLIT3 polypeptides prior to or after onset of disease can be monitored by assay of urine glucose levels in the NOD mouse.
  • induced models of autoimmune disease such as experimental allergic encephalitis (EAE)
  • EAE experimental allergic encephalitis
  • ZSLTT3 polypeptides can be tested in vivo in animal models for cancer, where suppression or apoptosis of introduced tumor cells can be monitored following administration of ZSLIT3.
  • the activity of ZSLIT3 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer (Molecular Devices, Sunnyvale, CA).
  • CytosensorTM Microphysiometer Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al, Science 257:1906-12, 1992; Pitchford et al., Meth. Enzvmol. 228:84-108, 1997; Arimilli et al., J. Immunol. Meth.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including ZSLJT3 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a ZSLIT3-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to ZSLIT3 polypeptide.
  • ZSLTT3- responsive eukaryotic cells comprise cells into which a receptor for ZSLIT3 has been transfected creating a cell that is responsive to ZSLIT3; or cells naturally responsive to ZSLJT3 such as cells derived from neurological, endrocrinological or tumor tissue. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to ZSLIT3 polypeptide, relative to a control not exposed to ZSLJT3, are a direct measurement of ZSLJT3- modulated cellular responses. Moreover, such ZSLIT3-modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of ZSLIT3 polypeptide comprising providing cells responsive to a ZSLTT3 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • culturing a third portion of the cells in the presence of ZSLIT3 polypeptide and the absence of a test compound can be used as a positive control for the ZSLJT3-responsive cells, and as a control to compare the agonist activity of a test compound with that of the ZSLIT3 polypeptide.
  • a method of identifying antagonists of ZSL1T3 polypeptide comprising providing cells responsive to a ZSLJT3 polypeptide, culturing a first portion of the cells in the presence of ZSLIT3 and the absence of a test compound, culturing a second portion of the cells in the presence of ZSLIT3 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • Antagonists and agonists, for ZSLIT3 polypeptide can be rapidly identified using this method.
  • ZSLIT3 can be used to identify cells, tissues, or cell lines which respond to a ZSLIT3 -stimulated pathway.
  • the microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to ZSLIT3 of the present invention.
  • Cells can be cultured in the presence or absence of ZSLIT3 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of ZSLIT3 are responsive to ZSLIT3.
  • Such cell lines can be used to identify antagonists and agonists of ZSLTT3 polypeptide as described above.
  • ZSLLT3 polypeptides can also be used to identify inhibitors (antagonists) of its activity.
  • ZSLIT3 antagonists include anti-ZSLJT3 antibodies and soluble ZSLIT3 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
  • Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of ZSLJT3.
  • samples can be tested for inhibition of ZSLIT3 activity within a variety of assays designed to measure receptor binding or the stimulation inhibition of ZSLIT3-dependent cellular responses.
  • ZSLIT3-responsive cell lines can be transfected with a reporter gene construct that is responsive to a ZDMF-7-stimulated cellular pathway.
  • Reporter gene constructs of this type are known in the art, and will generally comprise a ZSLIT3-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem.
  • compounds or other samples can be tested for direct blocking of ZSLrT3 binding to receptor using ZSLTT3 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FJTC, and the like).
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FJTC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • ZSLTT3 antagonists would find use to modulate or down regulate one or more detrimental biological processes in cells, tissues and/or biological fluids, such as over-responsiveness, unregulated or inappropriate growth, and inflammation or allergic reaction.
  • ZSLIT3 antagonists would have beneficial therapeutic effect in diseases where the inhibition of activation of certain B lymphocytes and/or T cells would be effective.
  • diseases would include autoimmune diseases, such as multiple sclerosis, insulin-dependent diabetes and systemic lupus erythematosus.
  • benefit would be derived from using ZSLIT3 antagonists for chronic inflammatory and infective diseases.
  • Antagonists could be used to dampen or inactivate ZSLJT3 during activated immune response.
  • the activity of slit protein polypeptides, agonists, antagonists and antibodies of the present invention can be measured, and compounds screened to identify agonists and antagonists, using a variety of assays, such as assays that measure axon guidance and growth and neuronal migration. Such assays are suitable to assess the axon guidance and growth and neuronal migration activity of the ZSLIT3 polypeptides of the present invention. Of particular interest are assays that indicate changes in neuron growth patterns, see for example, Hastings, WIPO Patent Application No:97/29189 and Walter et al., Development 101:685-96, 1987. Assays to measure the effects of proteins on neuron growth are well known in the art.
  • the C assay see for example, Raper and Kapfhammer, Neuron 4:21-9, 1990 and Luo et al., Cell 75:217-27, 1993
  • other chemorepulsion and collapsing assays see, e.g., Ba-Charvet, K.T.N, et al., Neuron 22:463-473, 1999
  • slit protein chemorepulsion of neuronal migration can be measured (see, Hu, H., Neuron 23:703- 711, 1999; Wu, W. Nature 400:331-336, 1999).
  • Other methods which assess protein induced inhibition of neurite extension or divert such extension are also known, see
  • Conditioned media from cells expressing a slit protein can by placed in a gel matrix near suitable neural cells, such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been co- cultured with nerve growth factor.
  • a slit protein such as ZSLIT3, a slit protein agonist or slit protein antagonist, or aggregates of such cells.
  • suitable neural cells such as dorsal root ganglia (DRG) or sympathetic ganglia explants, which have been co- cultured with nerve growth factor.
  • ZSLIT3 protein-induced changes in neuron growth can be measured (see, for example, Messersmith et al., Neuron 14:949-59, 1995; Puschel et al., Neuron 14:941-8, 1995); or in a motor neuron repulsion assay (Brose, K. et al, Cell 96:795-806, 1999).
  • neurite outgrowth can be measured using neuronal cell suspensions grown in the presence of molecules of the present invention. See for example, O'Shea et al., Neuron 7:231-7, 1991 and
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair.
  • complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 ⁇ M ⁇ l. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art.
  • Such systems include Scatchard analysis for determination of binding affinity (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253:545-8, 1991; Cunningham et al., Science 245:821-5, 1991).
  • viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997).
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high- titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein.
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy 9:671- 679, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, A. et al, J. Virol. 72:926-933, 1998).
  • adenovirus system can also be used for protein production in vitro.
  • adenovirus-infected non-293 cells By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made.
  • Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products, and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population.
  • Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al, J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages.
  • the novel polypeptides of the present invention may be useful for studies to isolate mesenchymal stem cells and myocyte or other progenitor cells, both in vivo and ex vivo.
  • the present invention includes stimulating or inhibiting the proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes, neuronal and endothelial cells.
  • Molecules of the present invention may while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of the affect on their common precursor/stem cells.
  • molecules of the present invention may have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
  • Assays measuring differentiation include, for example, measuring cell markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference).
  • ZSLIT3 polypeptide itself can serve as an additional cell-surface or secreted marker associated with stage-specific expression of a tissue.
  • direct measurement of ZSLIT3 polypeptide, or its loss of expression in a tissue as it differentiates can serve as a marker for differentiation of tissues.
  • direct measurement of ZSLIT3 polypeptide, or its loss of expression in a tissue can be determined in a tissue or cells as they undergo tumor progression.
  • Increases in invasiveness and motility of cells, or the gain or loss of expression of ZSLLT3 in a pre-cancerous or cancerous condition, in comparison to normal tissue can serve as a diagnostic for transformation, invasion and metastasis in tumor progression.
  • knowledge of a tumor's stage of progression or metastasis will aid the physician in choosing the most proper therapy, or aggressiveness of treatment, for a given individual cancer patient.
  • Methods of measuring gain and loss of expression are well known in the art and described herein and can be applied to ZSLJT3 expression.
  • polypeptides that regulate cell motility can be used to aid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999).
  • ZSLIT3 gain or loss of expression may serve as a diagnostic for prostate and other cancers.
  • ZSLIT3 is expressed in cancerous glioblastoma tissue, and osteogenic sarcoma, breast, and intestinal cancer cells.
  • polynucleotides, polypeptides, and anti-ZSLIT3 antibodies and binding partners can be used to detect these cancers in biopsies, tissue samples, histologic sections, and in vivo.
  • PSA prostate specific antigen
  • increased levels of ZSLIT3 polypeptides, or anti-ZSLIT3 antibodies in a patient, relative to a normal control can be indicative of brain, testis and ovarian diseases, such as brain, testis and ovarian cancer, and possibly lung, live, kidney, prostate and pancreatic cancers (See, e.g., Mulders, TMT, et al.. Eur. J. Surgical Oncol. 16:37-41. 1990).
  • ZSLH3 expression appears to be restricted to specific human tissues
  • lack of ZSLIT3 expression in those tissues or strong ZSLIT3 expression in tissues where ZSLIT3 is not normally expressed would serve as a diagnostic of an abnormality in the cell or tissue type, of invasion or metastasis of cancerous testicular tissues into non-testicular tissue, and could aid a physician in directing further testing or investigation, or aid in directing therapy.
  • polynucleotide probes, anti-ZSLJT3 antibodies, and detection the presence of ZSLH3 polypeptides in tissue can be used to assess whether these tissues are present, for example, after surgery involving the excision of a diseased or cancerous testis or ovarian tissue.
  • the polynucleotides, polypeptides, and antibodies of the present invention can be used as an aid to determine whether all such tissue is excised after surgery, for example, after surgery for cancer. In such instances, it is especially important to remove all potentially diseased tissue to maximize recovery from the cancer, and to minimize recurrence.
  • Preferred embodiments include fluorescent, radiolabeled, or calorimetrically labeled anti-ZSLIT3 antibodies and ZSLH3 polypeptide binding partners, that can be used histologically or in situ.
  • the activity and effect of ZSLIT3 on tumor progression and metastasis can be measured in vivo.
  • Several syngeneic mouse models have been developed to study the influence of polypeptides, compounds or other treatments on tumor progression.
  • tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor. The cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
  • Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others. These are both commonly used tumor lines, syngeneic to the C57BL6 mouse, that are readily cultured and manipulated in vitro.
  • Tumors resulting from implantation of either of these cell lines are capable of metastasis to the lung in C57BL6 mice.
  • the Lewis lung carcinoma model has recently been used in mice to identify an inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79: 315-328,1994).
  • C57BL6/J mice are treated with an experimental agent either through daily injection of recombinant protein, agonist or antagonist or a one time injection of recombinant adenovirus. Three days following this treatment, 10 5 to IO 6 cells are implanted under the dorsal skin.
  • the cells themselves may be infected with recombinant adenovirus, such as one expressing ZSLIT3, before implantation so that the protein is synthesized at the tumor site or intracellularly, rather than systemically.
  • adenovirus such as one expressing ZSLIT3
  • the mice normally develop visible tumors within 5 days.
  • the tumors are allowed to grow for a period of up to 3 weeks, during which time they may reach a size of 1500 - 1800 mm 3 in the control treated group. Tumor size and body weight are carefully monitored throughout the experiment.
  • the tumor is removed and weighed along with the lungs and the liver.
  • the lung weight has been shown to correlate well with metastatic tumor burden. As an additional measure, lung surface metastases are counted.
  • the resected tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry, and in situ hybridization, using methods known in the art and described herein.
  • the influence of the expressed polypeptide in question, e.g., ZSLIT3, on the ability of the tumor to recruit vasculature and undergo metastasis can thus be assessed.
  • the implanted cells can be transiently transfected with ZSLIT3.
  • Use of stable ZSLJT3 transfectants as well as use of induceable promoters to activate ZSLIT3 expression in vivo are known in the art and can be used in this system to assess ZSL1T3 induction of metastasis.
  • ZSLJ 3 or ZSLIT3 conditioned media can be directly injected in to this mouse model, and hence be used in this system.
  • ZSLJT3 is expressed in heart, it could be useful as modulator blood pressure, muscle tension or and osmotic balance.
  • blood pressure modification is important in situations such as heart attack, stroke, traumatic shock, surgery, and any number of bleeding complications.
  • ZSLIT3 may modulate contractility in the organ systems and tissues that it effects.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure cell contractility and discussed below. Such assays are well known in the art.
  • ZSLIT 3 is expressed in tissues that contract, thus it can be used to modulate such contractile tissues.
  • contractile tissues where ZSLIT3 is expressed include, tissues in testis, e.g., vas deferens; prostate tissues; gastrointestinal tissues, e.g., colon and small intestine; and heart, lung, and ovary, and may include uterus.
  • the effects of ZSLIT3 polypeptide, its antagonists and agonists, on tissue contractility can be measured in vitro using a tensiometer with or without electrical field stimulation.
  • Such assays are known in the art and can be applied to tissue samples, such as aortic rings, vas deferens, ilium, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and' ' can be used to determine whether ZSLIT3 polypeptide, its agonists or antagonists, enhance or depress contractility.
  • Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo.
  • molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.
  • the effect of the ZSLIT 3 polypeptides, antagonists and agonists of the present invention on contractility of tissues including uterus, prostate, testis, gastrointestinal tissues, and heart can be measured in a tensiometer that measures contractility and relaxation in tissues.
  • a tensiometer that measures contractility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:767, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, M.B., Endocrinol. 114:364-368, 1984; and Downing, SJ. and Sherwood, O.D, Endocrinol. 116:1206-1214, 1985.
  • measuring vasodilatation of aortic rings is well known in the art. Briefly, aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6
  • the tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing.
  • the integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour. To test a sample for vasodilatation, or relaxation of the aortic ring tissue, the rings are contracted to two grams tension and allowed to stabilize for fifteen minutes. A ZSLIT3 polypeptide sample is then added to 1, 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of contractility by ZSLIT3 polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as gastrointestinal , lung, prostate, and testis.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes.
  • assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes.
  • changes in contractility of smooth muscle cells For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1:150-159, 1989, incorporated herein by reference).
  • An exemplary in vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longitudinally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60:S384-390, 1995).
  • Gastric motility is generally measured in the clinical setting as the time required for gastric emptying and subsequent transit time through the gastrointestinal tract.
  • Gastric emptying scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently.
  • a test food or liquid is radiolabeled with an isotope (e.g. 99m Tc), and after ingestion or administration, transit time through the gastrointestinal tract and gastric emptying are measured by visualization using gamma cameras (Meyer et aladmi Am. J. Dig. Dis. 21:296. 1976; Collins et al., Gut 24:1117, 1983; Maughan et al, Diabet. Med.
  • polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention can also be used in treatment of disorders associated with gastrointestinal cell contractility, secretion of digestive enzymes and acids, gastrointestinal motility, recruitment of digestive enzymes; inflammation, particularly as it affects the gastrointestinal system; reflux disease and regulation of nutrient absorption.
  • Specific conditions that will benefit from treatment with molecules of the present invention include, but are not limited to, diabetic gastroparesis, post-surgical gastroparesis, vagotomy, chronic idiopathic intestinal pseudo-obstruction and gastroesophageal reflux disease. Additional uses include, gastric emptying for radiological studies, stimulating gallbladder contraction and antrectomy.
  • the motor and neurological affects of molecules of the present invention make it useful for treatment of obesity and other metabolic disorders where neurological feedback modulates nutritional absorption.
  • the molecules of the present invention are useful for regulating satiety, glucose absorption and metabolism, and neuropathy-associated gastrointestinal disorders.
  • Molecules of the present invention are also useful as additives to anti-hypoglycemic preparations containing glucose and as adsorption enhancers for oral drugs which require fast nutrient action. Additionally, molecules of the present invention can be used to stimulate glucose-induced insulin release.
  • tissues in which the polypeptides of the present invention are expressed are comprised in part of epithelial cells where ZSLIT3 polypeptides, agonists or antagonists thereof may be therapeutically useful for promoting wound healing.
  • ZSLIT3 polypeptides, agonists or antagonists of the present invention such ZSLFI 3 polypeptides, agonists or antagonists are evaluated with respect to their ability to facilitate wound healing according to procedures known in the art.
  • ZSLH3 polypeptide performance in this regard can be compared to growth factors, such as EGF, NGF, TGF- ⁇ , TGF- ⁇ , insulin, IGF-I, IGF-B, fibroblast growth factor (FGF) and the like.
  • ZSLIT3 polypeptides, agonists or antagonists thereof can be evaluated with respect to their ability to enhance wound contractility involved in wound healing.
  • ZSLLT3 polypeptides or agonists or antagonists thereof may be evaluated in combination with one or more growth factors to identify synergistic effects.
  • the molecules of the present invention are useful as components of defined cell culture media, as described herein, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation, and/or maturation of ovarian cells in culture, and may also prove useful in the study of the ovarian cycle, reproductive function, ovarian and testicular cell-cell interactions, sperm capacitation and fertilization.
  • the present invention also provides methods for studying steroidogenesis and steroid hormone secretion.
  • Such methods generally comprise incubating ovarian cells in culture medium comprising ZSLIT3 polypeptides, monoclonal antibodies, agonists or antagonists thereof with and without gonadotropins and/or steroid hormones, and subsequently observing protein and steroid secretion.
  • gonadotropin hormones include luteinizing hormone and follicle stimulating hormone (Rouillier et al., Mol. Reprod. Dev. 50:170-7, 1998).
  • Exemplary steroid hormones include estradiol, androstenedione, and progesterone.
  • Effects of ZSLJT3 on steroidogenesis or steroid secretion can be determined by methods known in the art, such as radioimmunoassay (to detect levels of estradiol, androstenedione, progesterone, and the like), and immunoradiometric assay (JPJVIA).
  • radioimmunoassay to detect levels of estradiol, androstenedione, progesterone, and the like
  • JPJVIA immunoradiometric assay
  • Molecules expressed in the ovary, testis and prostate such as ZSLJT3 polypeptide which is specifically highly expressed in testis and ovary, and which may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade or are involved in oocyte or ovarian development, spermatogenesis, or the like, would be useful as markers for cancer of reproductive organs and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells.
  • Human reproductive system cancers such as ovarian, uterine, cervical, testicular and prostate cancers are common.
  • Diagnostic methods of the present invention involve the detection of ZSLIT3 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive cancers, e.g., uterine, testicular or prostate cancer.
  • Such polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing ZSLJT3 polypeptide epitopes.
  • the present invention contemplates methods for detecting ZSLIT3 polypeptides comprising: exposing a test sample potentially containing ZSLIT3 polypeptides to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a ZSLIT 3 polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a ZSLJT3 polypeptide, wherein the second antibody is associated with a detectable label; and detecting the detectable label.
  • Altered levels of ZSLJT3 polypeptides in a test sample can be monitored as an indication of reproductive function or of reproductive cancer or disease, when compared against a normal control.
  • detecting altered levels of ZSLIT3 polypeptides in a test sample can also be used to detect cancers in other organs or tissues as well.
  • detecting altered levels of ZSLH3 mRNA expression in a test sample can be used as a diagnostic.
  • ZSLIT3 is expressed in several cancer cell lines (See, Example 3).
  • a PCR-based method such as that shown in Example 3 can be used as a cancer or disease diagnostic, as described above.
  • probes or primers derived, for example, from the nucleotide sequences disclosed herein can also be used to detect ZSLIT3 expression in a patient sample, such as a blood, saliva, sweat, biopsy, tissue sample, or the like.
  • probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization.
  • ZSLIT3 sequences can also be detected by PCR amplification using cDNA generated by reverse translation of sample mRNA as a template (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995).
  • both increases or decreases of ZSL ⁇ T3 expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease.
  • the polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome, uterine cancer, endometriosis, libido, mylagia and neuralgia associated with reproductive phenomena, male sexual dysfunction, impotency, prostate cancer, testicular cancer, colon and stomach cancer, gastrointestinal mobility and dysfunction.
  • the molecules of the present invention may be used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as neuronal tissues, heart and ovary.
  • certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention.
  • natural functions, such as embryo implantation or spermatogenesis may be suppressed or controlled for use in birth control by molecules of the present invention.
  • ZSLIT3 polypeptide is expressed in the ovary and may have additional biological activity independent of prostate or testis function, as described herein.
  • Oogenesis is the process by which a diploid stem cell proceeds through multiple stages of differentiation, culminating in the formation of a terminally differentiated cell with a unique function, an oocyte. Unlike spermatogenesis, which begins at puberty and continues on through the life of a male, oogenesis begins during fetal development and by birth, a female's entire supply of primary oocytes are stored in the ovaries in primordial follicles and await maturation and release. hi the adult ovary, folliculogenesis starts when the follicles enter the growth phase.
  • ZSLFT3 As ZSLFT3 is expressed in the ovary, it may serve a role in modulating ovarian function by regulating folliculogenesis and dominant follicle selection, by affecting proliferation or differentiation of follicular cells, affecting cell- cell interactions, modulating hormones involved in the process, and the like.
  • the ovarian cycle in mammals includes the growth and maturation of follicles, followed by ovulation and transformation of follicles into corpea lutea.
  • the physiological events in the ovarian cycle are dependent on interactions between hormones and cells within the hypothalamic-pituitary-ovarian axis, including gonadotropin releasing hormone (GnRH), LH, and FSH.
  • estradiol synthesized in the follicle, primes the hypothalamic-pituitary axis and is required for the mid-cycle surge of gonadotropin that stimulates the resumption of oocyte meiosis and leads to ovulation and subsequent extrusion of an oocyte from the follicle.
  • This gonadotropin surge also promotes the differentiation of the follicular cells from secreting estradiol to secreting progesterone.
  • Progesterone secreted by the corpus luteum, is needed for uterine development required for the implantation of fertilized oocytes.
  • hypothalamic-pituitary-gonadal hormones in the ovarian cycle and reproductive cascade, and the role of sex steroids on target tissues and organs, e.g., uterus, breast, adipose, bones and liver, has made modulators of their activity desirable for therapeutic applications.
  • Such applications include treatments for precocious puberty, endometriosis, uterine leiomyomata, hirsutism, infertility, pre menstrual syndrome (PMS), amenorrhea, and as contraceptive agents.
  • ZSLIT3 polypeptides, agonists and antagonists which modulate the actions of such hormones can be of therapeutic value.
  • Such molecules can also be useful for modulating steroidogenesis, both in vivo and in vitro, and modulating aspects of the ovarian cycle such as oocyte maturation, ovarian cell-cell interactions, follicular development and rupture, luteal function, menstruation, and promoting uterine implantation of fertilized oocytes.
  • Molecules which modulate hormone action can be beneficial therapeutics for use prior to or at onset of puberty, or in adult women.
  • puberty in females is marked by an establishment of feed-back loops to control hormone levels and hormone production. Abnormalities resulting from hormone imbalances during puberty have been observed and include precocious puberty, where pubertal changes occur in females prior to the age of 8.
  • Hormone- modulating molecules can be used, in this case, to suppress hormone secretion and delay onset of puberty.
  • the level and ratio of gonadotropin and steroid hormones can be used to assess the existence of hormonal imbalances associated with diseases, as well as determine whether normal hormonal balance has been restored after administration of a therapeutic agent. Determination of estradiol, progesterone, LH, and FSH, for example, from serum is known by one of skill in the art. Such assays can be used to monitor the hormone levels after administration of ZSLH3 in vivo, or in a transgenic mouse model where the ZSLIT3 gene is expressed or the murine ortholog is deleted.
  • ZSLIT3 polypeptides can have therapeutic application for treating, for example, breakthrough menopausal bleeding, as part of a therapeutic regime for pregnancy support, or for treating symptoms associated with polycystic ovarian syndrome (PCOS), endometriosis, PMS and menopause.
  • PCOS polycystic ovarian syndrome
  • other in vivo rodent models are known in the art to assay effects of ZSLIT3 polypeptide on, for example, polycystic ovarian syndrome (PCOS).
  • Proteins of the present invention may also be used in applications for enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer, and gamete intrafallopian transfer. Such methods are useful for assisting those who may have physiological or metabolic disorders that prevent or impede natural conception.
  • animal breeding programs e.g., for livestock, racehorses, domestic and wild animals, and could be used as methods for the creation of transgenic animals.
  • ZSLIT3 polypeptides could be used in the induction of ovulation, either independently or in conjunction with a regimen of gonadotropins or agents such as clomiphene citrate or bromocriptine (Speroff et al., Induction of ovulation, Clinical Gynecologic Endocrinology and Infertility, 5 th ed., Baltimore, Williams & Wilkins, 1994).
  • proteins of the present invention can be administered to the recipient prior to fertilization or combined with the sperm, an egg or an egg-sperm mixture prior to in vitro or in vivo fertilization.
  • Such proteins can also be mixed with oocytes prior to cryopreservation to enhance viability of the preserved oocytes for use in assisted reproduction.
  • the ZSLIT3 polypeptides, agonists and antagonists of the present invention may be directly used as or incorporated into therapies for treating reproductive disorders. Disorders such as luteal phase deficiency would benefit from such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation" in, Infertility: Evaluation and Treatment, Keye et al., eds., Philadelphia, WB Saunders, 1995). Moreover, administration of gonadotropin-releasing hormone is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. 11:481-5, 1983; Kadar et al., Physiol. Behav. 51:601-5, 1992 and Silver et al., J. Neruoendocrin.
  • polypeptides of the present invention can be used to inhibit normal reproduction in the form of birth control, for example, by decreasing spermatogenesis or preventing uterine implantation of a fertilized egg.
  • the ZSLH3 polypeptides of the present invention can be used to study ovarian cell proliferation, maturation, and differentiation, i.e., by acting as a luteinizing agent that converts granulosa cells from estradiol to progesterone-producing cells.
  • Such methods of the present invention generally comprise incubating granulosa cells, theca cells, oocytes or a combination thereof, in the presence and absence of ZSLIT3 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in cell proliferation, maturation and differentiation. See for example, Basini et al.,(L Rep. Immunol. 37:139-53, 1998); Duleba et al.,(Fert. Ster.
  • Molecules expressed in the ovary, testis and prostate such as ZSLIT3 polypeptide which is specifically highly expressed in testis and ovary, and which may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade or are involved in oocyte or ovarian development, spermatogenesis, or the like, would be useful as markers for cancer of reproductive organs and as therapeutic agents for hormone-dependent cancers, by inhibiting hormone-dependent growth and/or development of tumor cells.
  • Human reproductive system cancers such as ovarian, uterine, cervical, testicular and prostate cancers are common.
  • ZSLIT3 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible reproductive tissue cancers e.g., ovarian, uterine, testicular, osteogenic sarcoma, intestinal carcinoma, breast carcinoma, glioblastoma or prostate cancer.
  • reproductive tissue cancers e.g., ovarian, uterine, testicular, osteogenic sarcoma, intestinal carcinoma, breast carcinoma, glioblastoma or prostate cancer.
  • Such polypeptides can be detected using immunoassay techniques and antibodies, described herein that are capable of recognizing ZSLIT3 polypeptide epitopes.
  • both increases or decreases of ZSLIT3 expression in a patient sample, relative to that of a control can be monitored and used as an indicator or diagnostic for disease.
  • the present invention contemplates methods for detecting ZSLIT3 polypeptides and mRNA, described above, and can be applied to the detection and monitoring of reproductive disease, for example, in the testis and ovary.
  • the ZSLJT3 polypeptides, antagonists of agonists, of the present invention can also modulate sperm capacitation. Before reaching the oocyte or egg and initiating an egg-sperm interaction, the sperm must be activated. The sperm undergo a gradual capacitation, lasting up to 3 or 4 hours in vitro, during which the plasma membrane of the sperm head and the outer acrosomal membrane fuse to form vesicles that facilitate the release of acrosomal enzymes.
  • the acrosomal membrane surrounds the acrosome or acrosomal cap which is located at the anterior end of the nucleus in the sperm head.
  • the sperm In order for the sperm to fertilize egg the sperm must penetrate the oocyte. To enable this process the sperm must undergo acrosomal exocytosis, also known as the acrosomal reaction, and release the acrosomal enzymes in the vicinity of the oocyte. These enzymes enable the sperm to penetrate the various oocyte layers, (the cumulus oophorus, the corona radiata and the zona pellucida).
  • the released acrosomal enzymes include hyaluronidase and proacrosin, in addition to other enzymes such as proteases.
  • proacrosin is converted to acrosin, the active form of the enzyme, which is required for and must occur before binding and penetration of the zona pellucida is possible.
  • a combination of the acrosomal lytic enzymes and sperm tail movements allow the sperm to penetrate the oocyte layers. Numerous sperm must reach the egg and release acrosomal enzymes before the egg can finally be fertilized. Only one sperm will successfully bind to, penetrate and fertilize the egg, after which the zona hardens so that no other sperm can penetrate the egg (Zaneveld, in Male Infertility
  • Peptide hormones such as insulin homologs are associated with sperm activation and egg-sperm interaction. For instance, capacitated sperm incubated with relaxin show an increased percentage of progressively motile sperm, increased zona penetration rates, and increased percentage of viable acrosome-reacted sperm (Carrell et al., Endocr. Res. 21:697-707, 1995). Similarity of the ZSLIT3 polypeptide structure with peptide hormones and localization of ZSLTT3 to the testis, prostate and uterus suggests that the ZSLIT3 polypeptides described herein play a role in these and other reproductive processes.
  • proteins of the present invention can have applications in enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo transfer and gamete intrafallopian transfer. Such methods are useful for assisting men and women who have physiological or metabolic disorders preventing natural conception or can be used to enhance in vitro fertilization.
  • Such methods are also used in animal breeding programs, such as for livestock breeding and could be used as methods for the creation of transgenic animals.
  • Proteins of the present invention can be combined with sperm, an egg or an egg-sperm mixture prior to fertilization of the egg. In some species, sperm capacitate spontaneously during in vitro fertilization procedures, but normally sperm capacitate over an extended period of time both in vivo and in vitro.
  • sperm activation during such procedures to enhance the likelihood of successful fertilization.
  • the washed sperm or sperm removed from the seminal plasma used in such assisted reproduction methods has been shown to have altered reproductive functions, in particular, reduced motility and zona interaction.
  • sperm is capacitated using exogenously added compounds.
  • Polypeptides of the present invention can used in such methods to enhance viability of cryopreserved sperm, enhance sperm motility and enhance fertilization, particularly in association with methods of assisted reproduction.
  • ZSLIT3 polypeptide or polypeptide fragments may function as germ-cell-specific antigens for use as components in "immunocontraceptive" or "anti-fertility” vaccines to induce formation of antibodies and/or cell mediated immunity to selectively inhibit a process, or processes, critical to successful reproduction in humans and animals.
  • the use of sperm and testis antigens in the development of immunocontraceptives have been described (O'Hern et al., Biol Reprod.
  • HCG human chorionic gonadotrophin
  • Such methods of immunocontraception using vaccines would include a ZSLIT3 testes-specific protein or fragment thereof.
  • the ZSLIT3 protein or fragments can be conjugated to a carrier protein or peptide, such as tetanus or diphtheria toxoid.
  • An adjuvant, as described above, can be included and the protein or fragment can be noncovalently associated with other molecules to enhance intrinsic immunoreactivity.
  • Methods for administration and methods for determining the number of administrations are known in the art. Such a method might include a number of primary injections over several weeks followed by booster injections as needed to maintain a suitable antibody titer.
  • Regulation of reproductive function in males and females is controlled in part by feedback inhibition of the hypothalamus and anterior pituitary by blood-borne hormones.
  • Testis proteins such as activins and inhibins, have been shown to regulate secretion of active molecules including follicle stimulating hormone (FSH) from the pituitary (Ying, Endodcr. Rev. 9:267-93, 1988; Plant et al., Hum. Reprod. 8:41- 44,1993).
  • FSH follicle stimulating hormone
  • Inhibins also expressed in the ovaries, have been shown to regulate ovarian functions (Woodruff et al., Endocr. 132:2332-42,1993; Russell et al., J. Reprod. Fertil. 100:115-22, 1994).
  • Relaxin has been shown to be a systemic and local acting hormone regulating follicular and uterine growth (Bagnell et al., J. Reprod. Fertil. 48:127-38, 1993). As such, the polypeptides of the present invention may also have effects on female gametes and reproductive tract. These functions may also be associated with
  • ZSLJT3 polypeptides may be used to regulate testicular or ovarian functions.
  • the activity of molecules of the present invention may be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i.e., proliferation) of cardiac cells based on the potential effects of activity of ZSLIT3.
  • Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic factor; and factor for expanding mesenchymal stem cell and precursor populations.
  • Proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the present invention to the appropriate animal model. Generally, proliferative effects are seen as an increase in cell number, and may include inhibition of apoptosis as well as stimulation of mitogenesis.
  • Cultured cells for use in these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, and human umbilical vein endothelial cells from primary cultures. Suitable established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No.
  • assays measuring cell proliferation are well known in the art.
  • assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al.,
  • In vivo assays for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention.
  • the animals' cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function.
  • Post-mortem methods for assessing cardiac decline or improvement include: increased or decreased cardiac weight, nuclei/cytoplasmic volume, and staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci.
  • PCNA proliferating cell nuclear antigen
  • the present invention also provides methods of studying mammalian cellular metabolism. Such methods of the present invention comprise incubating cells to be studied, for example, human vascular endothelial cells, + ZSLIT3 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, or the like.
  • a pharmaceutical composition comprising a purified ZSLTT3 polypeptide in combination with a pharmaceutically acceptable vehicle. This pharmaceutical composition may be used to modulate energy balance in mammals or to protect endothelial cells from injury.
  • ZSL1T3 polypeptides may be used to modulate cellular metabolic reactions. Such metabolic reactions include adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, lipid metabolism, detoxification and excretion, glucose uptake, protein synthesis, thermogenesis, oxygen utilization and the like.
  • the expression pattern of ZSLJT3 polypeptide indicates expression in major metabolic organs, e.g., liver, pancreas and thyroid, and may have intra- and extra-hepatic and -thyroidal effects on endothelial cell tissues. Such effects involve protection, regeneration, growth and development of liver, pancreas, thyroid or other tissues.
  • ZSLIT3 polypeptides may be used in organ preservation, for cryopreservation, for surgical pretreatment to prevent injury due to ischemia and/or inflammation or in like procedures.
  • organ preservation for cryopreservation, for surgical pretreatment to prevent injury due to ischemia and/or inflammation or in like procedures.
  • ZSL1T3 polypeptides may find utility in modulating nutrient uptake, as demonstrated, for example, by 2-deoxy-glucose uptake in the brain or the like.
  • the ZSL1T3 polypeptides may modulate mammalian energy balance.
  • the thyroid expression pattern of ZSLTT3 suggests that ZSLH3 may exhibit effects on glucose uptake, e.g. through GLUT-1, and thermogenesis (thermoregulation).
  • mammalian energy balance may be evaluated by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, oxygen utilization or the like. These metabolic functions are monitored by techniques (assays or animal models) known to one of ordinary skill in the art, as is more fully set forth below.
  • the glucoregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue, sulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result in, for example, the inhibition of glucose production and the stimulation of glucose utilization.
  • insulin stimulates glucose uptake and inhibits gluconeogenesis and glycogenolysis.
  • skeletal muscle and adipose tissue insulin acts to stimulate the uptake, storage and utilization of glucose.
  • ZSL1T3 polypeptide is expressed in thyroid but may exhibit extrathyroidal activity in organs that affect metabolic functions.
  • pharmaceutical compositions of the present invention may be useful in prevention or treatment of pancreatic disorders.
  • ZSLIT3 may be associated with pathological regulation of the expansion of neurocrine and exocrine cells in the pancreas, as evident in JDDM, pancreatic cancer or the like.
  • Pharmaceutical compositions of the present invention may also be involved in prevention or treatment of pancreatic conditions characterized by dysfunction associated with pathological regulation of blood glucose levels, insulin resistance or digestive function.
  • Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of mammalian energy balance, which may be evaluated by known techniques using, for example, ob/ob mice or db/db mice.
  • the ob/ob mice are inbred mice that are homozygous for an inactivating mutation at the ob (obese) locus. Such ob/ob mice are hyperphagic and hypometabolic, and are believed to be deficient in production of circulating OB protein.
  • the db/db mice are inbred mice that are homozygous for an inactivating mutation at the db (diabetes) locus.
  • db/db mice display a phenotype similar to that of ob/ob mice, except db/db mice display a more severe diabetic phenotype.
  • Such db/db mice are believed to be resistant to the effects of circulating OB protein.
  • various in vitro methods of assessing these parameters are known in the art.
  • Insulin-stimulated lipogenesis may be monitored by measuring the incorporation of l ⁇ C-acetate into triglyceride (Mackall et al. J. Biol. Chem. 251:6462-6464, 1976) or triglyceride accumulation (Kletzien et al., Mol. Pharmacol. 41:393-398. 1992).
  • Glucose uptake may be evaluated, for example, in an assay for insulin- stimulated glucose transport.
  • Non-transfected, differentiated L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g/1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mM glutamine.
  • the medium is replaced with fresh, glucose-free DMEM containing 0.5 or 1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine.
  • Appropriate concentrations of insulin or IGF-1, or a dilution series of the test substance, are added, and the cells are incubated for 20-30 minutes, - H or 14c_ ⁇ a b e ⁇ ec ⁇ deoxyglucose is added to -50 1 M final concentration, and the cells are incubated for approximately 10-30 minutes.
  • the cells are then quickly rinsed with cold buffer (e.g. PBS), then lysed with a suitable lysing agent (e.g.
  • Protein synthesis may be evaluated, for example, by comparing precipitation of 35 S-methionine-labeled proteins following incubation of the test cells with 35 S-methionine and 35 S-methionine and a putative modulator of protein synthesis.
  • Thermogenesis may be evaluated as described by B. Stanley in The Biology of Neuropeptide Y and Related Peptides, W. Colmers and C. Wahlestedt
  • metabolic rate which may be measured by a variety of techniques, is an indirect measurement of thermogenesis.
  • Oxygen utilization may be evaluated as described by Heller et al., Pflugers Arch 369(1): 55-9, 1977. This method also involved an analysis of hypothalmic temperature and metabolic heat production. Oxygen utilization and thermoregulation have also been evaluated in humans as described by Haskell et al., 1 Appl. Physiol. 51(4): 948-54. 1981.
  • the ZSLJT3 polypeptides of the present invention may act in the neuroendocrine/exocrine cell fate decision pathway and is therefore capable of regulating the expansion of neuroendocrine and exocrine cells in the pancreas.
  • One such regulatory use is that of islet cell regeneration.
  • ZSL1T3 polypeptide is a developmental gene involved in cell partitioning.
  • Assays and animal models are known in the art for monitoring the exocrine/neuroendocrine cell lineage decision, for observing pancreatic cell balance and for evaluating ZSLIT3 polypeptide, fragment, fusion protein, antibody, agonist or antagonist in the prevention or treatment of the conditions set forth above.
  • the cardiac activity of molecules of the present invention may be measured using a Langendorff assay. This preferred assay measures ex vivo cardiac function for an experimental animal, and is well known in the art. Experimental animals are, for example but not limited to, rats, rabbits and guinea pigs. Chronic effects on heart tissue can be measured after treating a test animal with ZSLIT3 polypeptide for 1 to 7 days, or longer. Control animals will have only received buffer. After treatment, the heart is removed and perfused retrograde through the aorta. During perfusion, several physiologic parameters are measured: coronary blood flow per time, left ventricular (LV) pressures, and heart rate. These perameters directly reflect cardiac function.
  • LV left ventricular
  • the molecules of the present invention may be useful for proliferation of cardiac tissue cells, such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • cardiac tissue cells such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • molecules of the present invention are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture.
  • Molecules of the present invention are particularly useful in specifically promoting the growth and/or development of myocytes in culture, and may also prove useful in the study of cardiac myocyte hyperplasia and regeneration.
  • polypeptides, nucleic acids and/or antibodies of the present invention may be used in treatment of disorders associated with myocardial infarction, congestive heart failure, hypertrophic cardiomyopathy and dilated cardiomyopathy.
  • Molecules of the present invention may also be useful for limiting infarct size following a heart attack, aiding in recovery after heart transplantation, promoting angiogenesis and wound healing following angioplasty or endarterectomy, to develop coronary collateral circulation, for revascularization in the eye, for complications related to poor circulation such as diabetic foot ulcers, for stroke, following coronary reperfusion using pharmacologic methods, and other indications where angiogenesis is of benefit.
  • Molecules of the present invention may be useful for improving cardiac function, either by inducing cardiac myocyte neogenesis and/or hyperplasia, by inducing coronary collateral development, or by inducing remodeling of necrotic myocardial area.
  • Other therapeutic uses for the present invention include induction of skeletal muscle neogenesis and/or hyperplasia, kidney regeneration and/or for treatment of systemic and pulmonary hypertension.
  • ZSLIT3 induced coronary collateral development is measured in rabbits, dogs or pigs using models of chronic coronary occlusion (Landau et al., Amer. Heart J. 29:924-931, 1995; Sellke et al., Surgery 120(2):182-188. 1996; and Lazarous et al., 1996, ibid.)
  • ZSLIT3 efficacy for treating stroke is tested in vivo, in rats, utilizing bilateral carotid artery occlusion and measuring histological changes, as well as maze performance (Gage et al., Neurobiol. Aging 9:645-655, 1988).
  • ZSLJT3 efficacy in hypertension is tested in vivo utilizing spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl. S114- 116, 1995).
  • SHR spontaneously hypertensive rats
  • ZSLIT3 polypeptides can also be used to prepare antibodies that bind to ZSLIT3 epitopes, peptides or polypeptides.
  • the ZSLIT3 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a ZSLIT3 polypeptide (e.g., SEQ ID NO:2).
  • Polypeptides comprising a larger portion of a ZSLIT3 polypeptide, i.e., from 30 to 10 residues up to the entire length of the amino acid sequence are included.
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the ZSLLT3 polypeptide encoded by SEQ JD NO:2 from amino acid number 24 (Cys) to amino acid number 673 (Ee) or a contiguous 9 to 649 amino acid fragment thereof.
  • antigens include the N-terminal LRR flanking domain, LRR domain, LRR motifs LRR- 1-10, middle region, C-terminal LRR flanking domain, EGF domain, C-terminal region and other domains and motifs as described herein.
  • Preferred peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot.
  • ZSLTT3 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 123 (Leu) to amino acid number 128 (Arg) of SEQ ID NO:2; (2) amino acid number 156 (Gin) to amino acid number 161 (Arg) of SEQ ED NO:2; (3) amino acid number 225 (Asp) to amino acid number 230 (Arg) of SEQ JD NO:2; (4) amino acid number 322 (Ser) to amino acid number 327 (Arg) of SEQ BD NO:2; and (5) amino acid number 502 (Ser) to amino acid number 507 (Arg) of SEQ BD NO:2.
  • ZSLIT3 antigenic epitopes as predicted by a Jameson- Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wl) serve as suitable antigens.
  • Antibodies from an immune response generated by inoculation of an animal with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a ZSLIT3 polypeptide or a fragment thereof.
  • the immunogenicity of a ZSLTT3 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZSLIT3 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in W1PO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
  • Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules.
  • a threshold level of binding is determined if anti- ZSLIT3 antibodies herein bind to a ZSLIT3 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-ZSLIT3) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M " or greater, preferably 10 M or greater, more preferably 10 M ⁇ or greater, and most preferably 10 9 M -1 or greater.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
  • anti-ZSLIT3 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting ZSLIT3 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family, Screening can also be done using non-human ZSLJT3, and ZSL ⁇ T3 mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the ZSLIT3 polypeptides.
  • antibodies raised to ZSLIT3 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZSLJT3 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol.
  • Specifically binding anti-ZSLIT3 antibodies can be detected by a number of methods in the art, and disclosed below.
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to ZSLIT3 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ZSLIT3 protein or polypeptide.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to ZSLH3 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZSLH3 protein or peptide).
  • Genes encoding polypeptides having potential ZSLH3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the ZSLIT3 sequences disclosed herein to identify proteins which bind to ZSL1T3. These "binding polypeptides" which interact with ZSLIT3 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • the binding polypeptides can also be used for diagnostic assays for determining circulating levels of ZSLLT3 polypeptides; for detecting or quantitating soluble ZSLIT3 polypeptides as marker of underlying pathology or disease.
  • These binding polypeptides can also act as ZSL1T3 "antagonists" to block ZSLIT3 binding and signal transduction in vitro and in vivo. These anti-ZSLLT3 binding polypeptides would be useful for inhibiting ZSLFT3 activity or protein-binding.
  • Antibodies to ZSLIT3 may be used for tagging cells that express ZSLIT3; for isolating ZSL1T3 by affinity purification; for diagnostic assays for determining circulating levels of ZSLLT3 polypeptides; for detecting or quantitating soluble ZSLIT3 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZSLIT3 activity in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to ZSLIT3 or fragments thereof may be used in vitro to detect denatured ZSLJT3 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • the ZSLH3 polypeptide itself can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • These ZSLIT3 polypeptide fusions would be useful for identifying, monitoring or activating ZSLIT3 activity or used to specifically kill cells in which SLIT3 receptor is over-expressed, for example in ZSL ⁇ T3 receptor-expressing cancers.
  • Genes encoding polypeptides having potential ZSLJT3 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the binding polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the ZSLIT3 sequences disclosed herein to identify proteins which bind to ZSL1T3.
  • binding polypeptides which interact with ZSLIT3 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as ZSLH3 "antagonists" to block ZSLIT3 binding and signal transduction in vitro and in vivo. These anti-ZSLIT3 binding polypeptides would be useful for inhibiting ZSLIT3 binding.
  • ZSLIT3 polypeptides and polynucleotides may be used within diagnostic systems.
  • Antibodies or other agents that specifically bind to ZSLJT3 may be used to detect the presence of circulating ligand or receptor polypeptides.
  • detection methods include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • Immunohistochemically labeled ZSLH3 antibodies can be used to detect ZSLIT3 receptor and/or ligands in tissue samples and identify ZSL1T3 receptors.
  • ZSLFT3 levels can also be monitored by such methods as RT-PCR, where ZSLIT3 mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of ZSLTT3 polypeptides in various diseases and biological processes, and as such would serve as diagnostic tools for diseases for which altered levels of ZSLJT3 are significant.
  • Nucleic acid molecules disclosed herein can be used to detect the expression of a ZSLIT3 gene in a biological sample.
  • probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequences of SEQ ED NO:l or SEQ ED NO:3, or fragments thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequences of SEQ ED NO: 1 or SEQ
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • suitable probes include nucleic acid molecules that bind with a portion of a ZSLIT3 domain or motif disclosed herein, such as the ZSLIT3 slit protein domain.
  • Other probes include those to the N-terminal LRR flanking domain, LRR domain, LRR motifs LRR- 1-10, middle region, C-terminal LRR flanking domain, EGF domain, C-terminal region and other domains and motifs as described herein.
  • RNA detection In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target ZSLH3 RNA species. After separating unbound probe from hybridized molecules, the level and length of the hybrid is detected.
  • Well-established hybridization methods of RNA detection include northern analysis and dot/slot blot hybridization, see, for example, Ausubel ibid, and Wu et al. (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997), and methods described herein.
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • ZSLJT3 RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, h e, 1993).
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Elustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • ZSLJT3 oligonucleotide probes are also useful for in vivo diagnosis.
  • 18 F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4:467, 1998).
  • numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods.
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, h e.
  • PCR primers can be designed to amplify a sequence encoding a full-length or partial ZSLJT3 polynucleotide, or a particular ZSLIT 3 domain or motif, such as the ZSLIT3 LRR or EGF domains as disclosed herein.
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZSL1T3 primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, CRC Press, Inc., pages 15-28, 1997).
  • ZSL1T3 primers see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in Methods in Gene Biotechnology, CRC Press, Inc., pages 15-28, 1997.
  • PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the guanidinium-isothiocyanate cell lysis procedure described herein.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or ZSL1T3 anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • ZSLIT3 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers.
  • PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled ZSLIT3 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • Another approach is to use real time quantitative PCR (Perkin-Elmer Cetus, Norwalk, Ct.).
  • a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • ZSLIT 3 probes and primers can also be used to detect and to localize ZSLIT3 gene expression in tissue samples.
  • Methods for such in situ hybridization are well-known to those of skill in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols, Humana Press, Inc., 1994; Wu et al. (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization (RISH),” in Methods in Gene Biotechnology. CRC Press, Inc., pages 259-278, 1997 and Wu et al.
  • ZSLIT3 polynucleotides and/or polypeptides disclosed herein can be useful as therapeutics, wherein ZSLIT3 agonists and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids.
  • ZSLIT3 antagonists provided by the invention bind to ZSLIT3 polypeptides or, alternatively, to a receptor to which ZSLIT3 polypeptides bind, thereby inhibiting or eliminating the function of ZSLIT3.
  • ZSLH3 antagonists would include antibodies; oligonucleotides which bind either to the ZSLLT3 polypeptide or to its ligand; natural or synthetic analogs of
  • ZSLIT3 ligands which retain the ability to bind the receptor but do not result in either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZSLIT3 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZSLTT3 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZSLJT3 receptor or ligand would be beneficial.
  • the invention also provides nucleic acid-based therapeutic treatment. If a mammal lacks or has a mutated ZSLJT3 gene, the ZSLIT3 gene can be introduced into the cells of the mammal. Using such methods, cells altered to express the nerve growth factor neurotrophin-3 (NT-3) were grafted to a rat model for spinal injury and stimulated axon regrowth at the lesion site and the rats thus treated recovered some ability to walk (Grill et al., J. Neuroscience 17:5560-72, 1997). In one embodiment, a gene encoding a ZSLJT3 polypeptide is introduced in vivo in a viral vector.
  • NT-3 nerve growth factor neurotrophin-3
  • Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno- associated virus (AAV), and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector (Kaplitt et al, Molec. Cell. Neurosci.
  • an attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-30, 1992), and a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., Cell 33:153, 1983; Ternin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; Dougherty et al., WIPO Publication WO 95/07358; and Kuo et al, Blood 82:845-52, 1993.
  • a retroviral vector e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., Cell 33:153, 1983; Ternin et al., U.S. Patent No. 4,
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; and Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988).
  • Another aspect of the present invention involves antisense polynucleotide compositions that are complementary to a segment of the polynucleotide set forth in SEQ ED NO:l.
  • Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZSLIT3 polypeptides and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides are used to inhibit expression of ZSLH3 polypeptide- encoding genes in cell culture or in a subject.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the ZSLIT3 gene a probe comprising ZSLIT3 DNA or RNA or a subsequence thereof can be used to determine if the ZSLJT3 gene is present on a human chromosome, such as chromosome 16, or if a mutation has occurred. Based on annotation of a fragment of human genomic DNA containing the
  • ZSLIT3 genomic DNA (Genbank Accession No. AC006208), ZSLIT3 is located at the 16ql2 region of chromosome 16.
  • Detectable chromosomal aberrations at the ZSLIT3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, translocations, insertions, deletions, loss of heterogeneity, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et.
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • the precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
  • the ZSLIT3 gene is located on chromosome 16.
  • genes of known function map to chromosome 16.
  • IL-4 interleukin 4
  • cytokine receptor alpha-subunit a member of the hematopoietin receptor family
  • ZSLIT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with defects in EL-4 receptor, such as those that are implicated in some allergic inflammatory disorders and asthma (Deichman, K.A. et al., Exp. Allergy 28:151-155; 1998; Mitsuyasu, H. et al., Nature Genet. 19:119-120, 1998).
  • ZSL1T3 polynucleotide probes can be used to detect abnormalities or genotypes associated with inflammatory bowel disease, where a susceptibility marker maps to 16pl2-ql3 (Cho, J.H. et al, Proc. Nat. Acad. Sci. 95:7502-7507, 1998).
  • ZSLIT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with hemoglobin loci located at 16pter-pl3.3, and particularly hemoglobin-alpha defects associated with alpha-thalassemia syndromes, such as hydrops fetalis (for review, see Chui, M.P., and Waye, J.S. Blood 91:2213-2222, 1998).
  • chromosome 16 trisomy results in an increase of mutant APRT expression (16q24.3) associated with disease; and a chromosomal breakpoint exists at 16ql2-22.
  • ZSLIT3 probes could be used specifically to detect such abnormalities in addition to detection of mutations within the ZSLIT gene itself.
  • a diagnostic could assist physicians in determining the type of genetic disease and appropriate associated therapy, or assistance in genetic counseling.
  • inventive anti-ZSLIT3 antibodies, polynucleotides, and polypeptides can be used for the detection of ZSL1T3 polypeptide, mRNA or anti-ZSLlT3 antibodies, thus serving as markers and be directly used for detecting or diagnosing diseases or cancers, as described herein, using methods known in the art and described herein.
  • ZSLIT 3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 16 deletions, translocations and aneuploidy associated with human diseases, such as those associated with the fragile site at 16q22 which are expected to be involved in chromosome rearrangements in cancers and other disease states.
  • ZSLIT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 16 trisomy and chromosome loss associated with human diseases such as Wilms Tumor (above; 16q loss), and polycystic kidney disease (above), Tuberous sclerosis 2 (above).
  • ZSLIT3 gene itself may result in a heritable human disease state. Such defects may result in abnormal neuronal development, neurologic disease, retardation, organ failure or other disease states.
  • Molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a ZSLIT3 genetic defect.
  • ZSLJT3 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the ZSLJT3 chromosomal locus.
  • the ZSLIT3 sequences can be used not only as diagnostics for disease, but as as diagnostics in forensic DNA profiling.
  • ZSLIT3 is located near genes involved in human disease, and one of skill in the art would appreciate that defects in 16ql2 are known to cause disease states in humans.
  • defects in ZSL1T3 can cause a disease state or susceptibility to disease.
  • ZSLIT3 is a slit protein in a chromosomal hot spot for translocations and LOH involved in genetic diseases and is shown to be expressed in intestinal, brain, breast and osteogenic sarcoma cancer cells, the molecules of the present invention could also be directly involved in cancer formation or metastasis.
  • polynucleotide probes can be used to detect chromosome 16ql2 loss, trisomy, duplication or translocation associated with human diseases, such as brain tumors, breast, intestinal and osteogenic tumors and diseased brain, ovary or testis cancers, or diseases.
  • molecules of the present invention such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention would aid in the detection, diagnosis prevention, and treatment associated with a ZSLIT3 genetic defect.
  • a diagnostic could assist physicians in determining the type of disease and appropriate associated therapy, or assistance in genetic counseling.
  • inventive anti-ZSLtT3 antibodies, polynucleotides, and polypeptides can be used for the detection of ZSL1T3 polypeptide, mRNA or anti-ZSLJT3 antibodies, thus serving as markers and be directly used for detecting or genetic diseases or cancers, as described herein, using methods known in the art and described herein.
  • ZSLTT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 16ql2 deletions and translocations associated with human diseases, other translocations involved with malignant progression of tumors or other 16ql2 mutations, which are expected to be involved in chromosome rearrangements in malignancy; or in other cancers, or in spontaneous abortion.
  • ZSLIT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with chromosome 16ql2 trisomy and chromosome loss associated with human diseases.
  • ZSLIT3 polynucleotide probes can be used to detect abnormalities or genotypes associated with these defects.
  • ZSLIT3 polynucleotide probes can be used to detect allelic differences between diseased or non-diseased individuals at the ZSLIT3 chromosomal locus. As such, the ZSLIT3 sequences can be used as diagnostics in forensic DNA profiling.
  • Analytical probes will be generally at least 20 nt in length, although somewhat shorter probes can be used (e.g., 14-17 nt).
  • PCR primers are at least 5 nt in length, preferably 15 or more, more preferably 20-30 nt.
  • a ZSLTT3 polynucleotide probe may comprise an entire exon or more. Exons are readily determined by one of skill in the art by comparing ZSLIT3 sequences (SEQ ED NO: 1) with the human genomic DNA for ZSLJT3 (Genbank Accession No. AC007226).
  • diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient, are known in the art.
  • Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a ZSLIT3 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a ZSLIT3 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing
  • a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre-implantation embryo.
  • a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the ZSL1T3 genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
  • Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ JD NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof.
  • Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • Aberrations associated with the ZSLIT3 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-as
  • mice engineered to express the ZSLLT3 gene referred to as “transgenic mice,” and mice that exhibit a complete absence of ZSLIT3 gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al., Science 257:1083, 1992;
  • transgenic mice that over-express ZSLIT3, either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild-type ZSLIT3 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which ZSLIT3 expression is functionally relevant and may indicate a therapeutic target for the ZSLIT3, its agonists or antagonists.
  • a preferred transgenic mouse to engineer is one that over- expresses the mature human ZSLIT3 polypeptide (residue 24 (Cys) to residue 673 (Ee) of SEQ ED NO: 2).
  • such over-expression may result in a phenotype that shows similarity with human diseases.
  • knockout ZSLJT3 mice can be used to determine where ZSL1T3 is absolutely required in vivo.
  • the phenotype of knockout mice is predictive of the in vivo effects of that a ZSLIT3 antagonist, such as those described herein, may have.
  • the murine ZSL1T3 mRNA, and cDNA can be isolated and used to isolate mouse ZSLIT3 genomic DNA, which are subsequently used to generate knockout mice.
  • These transgenic and knockout mice may be employed to study the ZSLIT3 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human or animal diseases (such as those in commercially viable animal populations).
  • the mouse models of the present invention are particularly relevant as tumor models for the study of cancer biology and progression. Such models are useful in the development and efficacy of therapeutic molecules used in human cancers. Because increases in ZSLIT3 expression, as well as decreases in ZSLIT3 expression are associated with specific human cancers, both transgenic mice and knockout mice would serve as useful animal models for cancer. Moreover, in a preferred embodiment, ZSL1T3 transgenic mouse can serve as an animal model for specific tumors, particularly glioblastoma, intestinal and breast cancers and osteogenic sarcomas. Moreover, transgenic mice expression of ZSLIT3 antisense polynucleotides or ribozymes directed against ZSLTT3, described herein, can be used analogously to transgenic mice described above.
  • the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
  • Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
  • pharmaceutical formulations will include a ZSLIT3 polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
  • Example 1 Identification and Cloning of ZSLJT3 Scanning of a translated human cDNA database resulted in identification of the expressed tag (EST) sequence which was used to identify a human full length cDNA sequence.
  • the cDNA sequence has homology to the slit family of proteins and been designated ZSLFT3.
  • the entire insert of the plasmid containing the EST sequence was sequenced and found to have the initiation Met and contain most of the 5' sequence.
  • a human genomic sequence was identified corresponding to the zslit3 gene which completed the 3' end of the cDNA thus providing a full length coding sequence for ZSLIT3.
  • the full length cDNA sequence is shown in SEQ BD NO:l and its corresponding polypeptide sequence is shown in SEQ BD NO:2.
  • Northern blot analysis was performed using Human Multiple Tissue NorthernTM Blots (MTN I, MTN B, and MTN BI) (Clontech).
  • a SAOS2 marathon library (a human primary osteogenic sarcoma cell line ) was used in a PCR reaction using oligos ZC23,641 (SEQ BD NO:5), and ZC23,642 (SEQ ED NO:6) as primers.
  • PCR conditions were as follows: 94°C for 1 minute; then 30 cycles of 94°C, 20 seconds; 61°C, 30 seconds; 72°C, 30 seconds; then ended with a final extension at 72°C for 5 minutes.
  • a sample of the PCR reaction product was run on a 4% agarose gel.
  • the 116 bp PCR fragment was gel purified using a commercially available kit (QiaQuick Gel Extraction Kit; Qiagen) and re-amplified according to conditions above except for 25 cycles instead of 30 for additional fragment and gel purified as above.
  • This fragment was then radioactively labeled with 32 P-dCTP using Rediprime ETM (Amersham), a random prime labeling system, according to the manufacturer's specifications.
  • the probe was then purified using a Nuc-TrapTM column (Stratagene) according to the manufacturer's instructions. ExpressHybTM (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots.
  • Hybridization took place overnight at 65°C using 1.05 x IO 6 cpm/ml of labeled probe. The blots were then washed 4 times for 15 minutes in 2X SSC/1% SDS at 25°C, followed by two 30 minute washes in 0.1X SSC/0.1% SDS at 55°C. Transcripts of approximately 3 kb were detected in heart, brain, placenta, lung , liver, kidney, pancreas, spleen, prostate, testis, ovary, small intestine, colon, stomach, thyroid, spinal cord, lymph node, trachea and adrenal gland.
  • ZSLIT3 is expected to show expression in human fetal brain blots as well. Dot Blots were also performed using Human RNA Master BlotsTM
  • Dot Blot had strongest signals in aorta, liver, kidney, small intestine, lung, placenta, fetal kidney and fetal spleen.
  • the dot blot fetal tissue signals were strongest in heart, kidney, liver, spleen, and lung, and weaker in brain and thymus.
  • a PCR cDNA panel screen was performed on ZSLIT3 using Marathon cDNAs generated in house using the Marathon cDNA Amplification Kit (Clontech).
  • the panel included the following tissues and cell lines: Daudi, HuH7, adrenal gland, bladder, bone marrow, brain, CaCO2, CD4+, CD8+, cervix, fetal brain, fetal heart, fetal kidney, fetal lung, fetal muscle, fetal skin, glioblastoma, heart, liver, HOS, K562, kidney, lung, lymph node, melanoma, MLR, MG63, pancreas, pituitary gland, placenta, prostate, rectum, SAOS2, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, T47D, testis , thymus, thyroid, trachea, U2OS and uterus.
  • the PCR reactions were performed with primers ZC23,641 (SEQ ED NO:5) and ZC23,642 (SEQ ED NO: 6) using the same conditions as described in Example 2.
  • CaCO2 intestinal carcinoma
  • SAOS2 primary osteogenic sarcoma
  • MG63 osteogenic sarcoma
  • T47D breast carcinoma
  • Example 4 Chromosomal Assignment and Placement of ZSL1T3 ZSLLT3 is mapped to a human chromosome, such as chromosome 16, using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL).
  • the GeneBridge 4 Radiation Hybrid Panel contains DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient).
  • a publicly available WWW server http://www- genome.wi.mit.edu/cgi-bin/contig/ rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR” radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.
  • Each of the 95 PCR reactions consist of 2 ⁇ l 10X KlenTaq PCR reaction buffer (Clontech), 1.6 ⁇ l dNTPs mix (2.5 mM each, PERKTN-ELMER, Foster City, CA), 1 ⁇ l sense primer, 1 ⁇ l antisense primer, 2 ⁇ l Red ⁇ Load (Research Genetics, Inc.), 0.4 ⁇ l 50X Advantage KlenTaq Polymerase Mix (Clontech), 25 ng of DNA from an individual hybrid clone or control and ddH 2 O for a total volume of 20 ⁇ l.
  • the reactions are overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions are, for example, as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 66°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions are separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction, as describe above, to specifically detect this site in the presence of all other genomic sequences.
  • STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences, or on the WICGR radiation hybrid map. Proximal and distal framework markers can be determined as well. The use of surrounding markers will position ZSLJT3 in a defined region on the integrated LDB chromosome map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar. genetics.soton.ac.uk/public_html/).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des polypeptides de la protéine slit, des polynucléotides codant pour ces polypetides et des compositions et des procédés relatifs. Les polypeptides sont exprimés dans les tissus cérébraux et organiques humains. Ces polypeptides peuvent être utilisés dans des procédés de détection de récepteurs qui induisent une excroissance du neurite et modulent l'organogenèse, la prolifération et/ou la différenciation cellulaire et la réaction immunitaire.
PCT/US2000/034230 1999-12-21 2000-12-14 Polypeptide humain slit, zslit3 WO2001046418A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU22718/01A AU2271801A (en) 1999-12-21 2000-12-14 Human slit polypeptide zslit3

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46984799A 1999-12-21 1999-12-21
US09/469,847 1999-12-21

Publications (1)

Publication Number Publication Date
WO2001046418A1 true WO2001046418A1 (fr) 2001-06-28

Family

ID=23865268

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/034230 WO2001046418A1 (fr) 1999-12-21 2000-12-14 Polypeptide humain slit, zslit3

Country Status (2)

Country Link
AU (1) AU2271801A (fr)
WO (1) WO2001046418A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002012346A2 (fr) * 2000-08-10 2002-02-14 Pharmacia Corporation Nouvelle proteine du type slit obtenue d'une banque d'adn-c a partir d'une ligne de cellules stromales hs-5
WO2003018805A1 (fr) * 2001-08-23 2003-03-06 Japan Science And Technology Agency Regulateurs de la proliferation de cellules souches hematopoietiques et polynucleotides codant ces regulateurs
EP1364965A1 (fr) * 2002-05-21 2003-11-26 metaGen Pharmaceuticals GmbH Isoformes de Slit1 et MEGF4 et leurs utilisations
EP1556516A2 (fr) * 2002-11-01 2005-07-27 Decode Genetics EHF. Gene slit-3 du diabete de type ii humain situe sur le chromosome 5q35
WO2010138709A1 (fr) * 2009-05-28 2010-12-02 Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Marqueurs de diagnostic de l'apoptose induite par des anti-tnf (atia) et thérapies afférentes
CN110494151A (zh) * 2017-02-14 2019-11-22 耶路撒冷希伯来大学伊森姆研究发展有限公司 Naf-1衍生肽及其用途
CN112638402A (zh) * 2018-07-03 2021-04-09 Sotio有限责任公司 与增强葡萄糖输入的反式代谢分子组合的嵌合受体及其治疗用途
US11555172B2 (en) 2014-12-02 2023-01-17 Ocugen, Inc. Cell and tissue culture container

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1087699A (ja) * 1996-07-16 1998-04-07 Asahi Chem Ind Co Ltd スリット様ポリペプチド
WO1999023219A1 (fr) * 1997-10-31 1999-05-14 Osiris Therapeutics, Inc. Polypeptide humain slit et polynucleotides le codant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1087699A (ja) * 1996-07-16 1998-04-07 Asahi Chem Ind Co Ltd スリット様ポリペプチド
WO1999023219A1 (fr) * 1997-10-31 1999-05-14 Osiris Therapeutics, Inc. Polypeptide humain slit et polynucleotides le codant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL SEQUENCES 2 July 1998 (1998-07-02), RICKE D.O.: "Homo sapiens chromosome 16, cosmid clone RT163 (LANL)", XP002162951 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002012346A2 (fr) * 2000-08-10 2002-02-14 Pharmacia Corporation Nouvelle proteine du type slit obtenue d'une banque d'adn-c a partir d'une ligne de cellules stromales hs-5
WO2002012346A3 (fr) * 2000-08-10 2002-09-12 Pharmacia Corp Nouvelle proteine du type slit obtenue d'une banque d'adn-c a partir d'une ligne de cellules stromales hs-5
WO2003018805A1 (fr) * 2001-08-23 2003-03-06 Japan Science And Technology Agency Regulateurs de la proliferation de cellules souches hematopoietiques et polynucleotides codant ces regulateurs
US7262026B2 (en) 2001-08-23 2007-08-28 Japan Science And Technology Agency Hematopoietic stem cell proliferation regulators and polynucleotides encoding the same
EP1364965A1 (fr) * 2002-05-21 2003-11-26 metaGen Pharmaceuticals GmbH Isoformes de Slit1 et MEGF4 et leurs utilisations
EP1556516A2 (fr) * 2002-11-01 2005-07-27 Decode Genetics EHF. Gene slit-3 du diabete de type ii humain situe sur le chromosome 5q35
EP1556516A4 (fr) * 2002-11-01 2007-11-28 Decode Genetics Ehf Gene slit-3 du diabete de type ii humain situe sur le chromosome 5q35
WO2010138709A1 (fr) * 2009-05-28 2010-12-02 Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Marqueurs de diagnostic de l'apoptose induite par des anti-tnf (atia) et thérapies afférentes
US9989533B2 (en) 2009-05-28 2018-06-05 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anti-TNF induced apoptosis (ATIA) diagnostic markers and therapies
US11555172B2 (en) 2014-12-02 2023-01-17 Ocugen, Inc. Cell and tissue culture container
CN110494151A (zh) * 2017-02-14 2019-11-22 耶路撒冷希伯来大学伊森姆研究发展有限公司 Naf-1衍生肽及其用途
CN112638402A (zh) * 2018-07-03 2021-04-09 Sotio有限责任公司 与增强葡萄糖输入的反式代谢分子组合的嵌合受体及其治疗用途

Also Published As

Publication number Publication date
AU2271801A (en) 2001-07-03

Similar Documents

Publication Publication Date Title
WO2001046418A1 (fr) Polypeptide humain slit, zslit3
CA2402614A1 (fr) Polypeptide zins4 homologue de l'insuline
US20010044134A1 (en) Novel secreted polypeptide zsig87
US20020012967A1 (en) Insulin homolog polypeptide zins4
US6372889B1 (en) Soluble protein ZTMPO-1
WO2001025444A2 (fr) Nouvelle phosphodiesterase humaine: zcytor13
US20050250184A1 (en) Peptide hormones zalpha48 and zsig97
US6514741B1 (en) Tryptase-like polypeptide ztryp1
AU2182900A (en) Pancreatic polypeptide zsig66
US6573069B1 (en) Crib protein ZMSE1
US20020045210A1 (en) Neuropeptide-like polypeptide zpep17
US20010049432A1 (en) Human semaphorin ZSMF-16
US20020042094A1 (en) RING finger protein zapop2
WO2000023591A2 (fr) Proteine secretee zsig49
US6440697B1 (en) Ring finger protein zapop3
WO2001042292A2 (fr) Nouveau polypeptide secrete zsig87
US20030119035A1 (en) Tryptase-like polypeptide ztryp1
US20030083478A1 (en) Prostate, testis and uterine polypeptide zpep14
WO2001012788A2 (fr) Polypeptide ztryp1 de type tryptase
WO2001057080A2 (fr) Polypeptide zpep17 similaire au neuropeptide
US20020182677A1 (en) Pancreatic and ovarian polypeptide, zsig58
WO2002012330A2 (fr) Zzp1, proteine humaine secretee
WO2001040278A2 (fr) Semaphorine humaine zsmf-16
JP2002512033A (ja) 可溶性タンパク質ｚｔｍｐｏ−１
EP1151096A2 (fr) Polypeptide zpep14 de la prostate, des testicules et de l'uterus

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP