WO2003040345A2 - Type 2 cytokine receptor and nucleic acids encoding same - Google Patents

Type 2 cytokine receptor and nucleic acids encoding same Download PDF

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Publication number
WO2003040345A2
WO2003040345A2 PCT/US2002/036316 US0236316W WO03040345A2 WO 2003040345 A2 WO2003040345 A2 WO 2003040345A2 US 0236316 W US0236316 W US 0236316W WO 03040345 A2 WO03040345 A2 WO 03040345A2
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polypeptide
crf2
nucleic acid
seq
polynucleotide
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PCT/US2002/036316
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French (fr)
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WO2003040345A3 (en
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Wei Liu
Lynette Fouser
Vikki Spaulding
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Wyeth
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Priority to AU2002343671A priority Critical patent/AU2002343671B2/en
Priority to CA002464765A priority patent/CA2464765A1/en
Priority to JP2003542592A priority patent/JP2005508640A/en
Priority to EP02780631A priority patent/EP1451307A4/en
Publication of WO2003040345A2 publication Critical patent/WO2003040345A2/en
Publication of WO2003040345A3 publication Critical patent/WO2003040345A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates generally to nucleic acids and polypeptides and more specifically to nucleic acids and polypeptides encoding type II cytokine receptors, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides.
  • Cytokines such as interferons are soluble proteins that influence the growth and differentiation of many cell types. Cytokines exert their effects through cytokine receptors, which are located on the surface of cells responsive to the effects of cytokines. Cytokine receptors are composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the receptor subunits.
  • Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains.
  • the receptor chains responsible for binding and/or transducing the effect of interferons cytokine are members of the type II cytokine receptor family (CRF2), based upon the presence of a characteristic 200-250 residue extracellular domain.
  • CRF2 family Members of the CRF2 family have been reported to act as receptors for a variety of cytokines, including interferon alpha, interferon beta, interferon gamma, IL-10, IL-20, and BL-22. Recently identified members of the CRF2 family are candidate ligands for the IL-10- like molecules IL-19, AK155 and mda-7. The demonstrated in vivo activities of these interferons illustrate the clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
  • the invention is based, in part, upon the discovery of polynucleotide sequences encoding CRF2-13, novel member of the CRF2 family.
  • the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO: 1 , or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can include, e.g., a nucleic acid sequence encoding a polypeptide at least 70%, e.g., 80%, 85%, 90%, 95%, 98%, or even 99% or more identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:2.
  • the nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
  • nucleic acid that encodes a polypeptide that includes amino acid sequences 21-520 of SEQ ID NO:2, e.g., a nucleic acids 61-1560 of SEQ ID NO:l.
  • nucleic acid molecules are that encode polypeptides with the amino acid sequences of SEQ ID NO:2.
  • Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
  • the invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
  • the invention includes a pharmaceutical composition that includes an CRF2-13 nucleic acid and a pharmaceutically acceptable carrier or diluent.
  • the invention includes a substantially purified CRF2-13 polypeptide, e.g., any ofthe CRF2-13 polypeptides encoded by an CRF2-13 nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition that includes an CRF2-13 polypeptide and a pharmaceutically acceptable carrier or diluent.
  • the invention provides an antibody that binds specifically to an CRF2-13 polypeptide.
  • the antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition including CRF2-13 antibody and a pharmaceutically acceptable carrier or diluent.
  • the invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
  • the invention also includes kits comprising any ofthe pharmaceutical compositions described above.
  • the invention further provides a method for producing an CRF2-13 polypeptide by providing a cell containing an CRF2-13 nucleic acid, e.g., a vector that includes an CRF2-13 nucleic acid, and culturing the cell under conditions sufficient to express the CRF2-13 polypeptide encoded by the nucleic acid.
  • the expressed CRF2-13 polypeptide is then recovered from the cell.
  • the cell produces little or no endogenous CRF2-13 polypeptide.
  • the cell can be, e.g., a prokaryotic cell or eukaryotic cell.
  • the invention is also directed to methods of identifying an CRF2-13 polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
  • the invention further provides methods of identifying a compound that modulates the activity of an CRF2-13 polypeptide by contacting an CRF2-13 polypeptide with a compound and determining whether the CRF2-13 polypeptide activity is modified.
  • the invention is also directed to compounds that modulate CRF2-13 polypeptide activity identified by contacting an CRF2-13 polypeptide with the compound and determining whether the compound modifies activity ofthe CRF2-13 polypeptide, binds to the CRF2-13 polypeptide, or binds to a nucleic acid molecule encoding an CRF2-13 polypeptide.
  • the invention provides a method of determining the presence of or predisposition of an CRF2-13 -associated disorder in a subject.
  • the method includes providing a sample from the subject and measuring the amount of CRF2-13 polypeptide in the subject sample.
  • the amount of CRF2-13 polypeptide in the subject sample is then compared to the amount of CRF2-13 polypeptide in a control sample.
  • An alteration in the amount of CRF2-13 polypeptide in the subject protein sample relative to the amount of CRF2-13 polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition.
  • a control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation-associated condition.
  • the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder.
  • the CRF2-13 is detected using an CRF2-13 antibody.
  • the invention provides a method of determining the presence of or predisposition of an CRF2-13 -associated disorder in a subject. The method includes providing a nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount ofthe CRF2-13 nucleic acid in the subject nucleic acid sample.
  • the amount of CRF2-13 nucleic acid sample in the subject nucleic acid is then compared to the amount of an CRF2-13 nucleic acid in a control sample.
  • An alteration in the amount of CRF2-13 nucleic acid in the sample relative to the amount of CRF2-13 in the control sample indicates the subject has a tissue proliferation-associated disorder.
  • the invention provides a method of treating or preventing or delaying an CRF2-13 -associated disorder.
  • the method includes administering to a subject in which such treatment or prevention or delay is desired an CRF2-13 nucleic acid, an CRF2-13 polypeptide, or an CRF2-13 antibody in an amount sufficient to treat, prevent, or delay a tissue proliferation-associated disorder in the subject.
  • disorders include rheumatoid arthritis and multiple sclerosis.
  • FIG. 1 is a phylogram showing polypeptide sequences related to a CRF2-13 polypeptide according to the invention.
  • the invention is based in part on the discovery of novel nucleic acid sequences encoding a polypeptide showing homology to CRF2 polypeptides. Included in the invention is a 1563 nucleotide sequence (SEQ ID NO:l) shown in Table 1. Nucleotides 1-1560 of SEQ ID NO:l encode a 520 amino acid CRF2-like polypeptide. The amino acid sequences ofthe encoded polypeptide is shown in Table 2 (SEQ ID NO:2). A nucleic acid having a portion of the 5' untranslated region and a portion of the coding sequence shown in Table 1 was identified in a human placental cDNA library.
  • the nucleic acid of Table 1 encodes the 520 amino acid sequence (SEQ ID NO:2) shown in Table 2.
  • SEQ ID NO:2 The nucleic acid of Table 1 encodes the 520 amino acid sequence (SEQ ID NO:2) shown in Table 2.
  • Signal P and Psort results predict that CRF2-13 protein contains a signal peptide, and is likely to be localized to the plasma membrane with a certainty of 0.460.
  • the most likely cleavage site for a CRF2-13 polypeptide is between amino acids 246 and 247, at:AGG-VI.
  • the CRF2-13 amino acid sequence is related to other previously described interleukin- binding proteins. The relationship is schematically represented in FIG. 1.
  • the CRF2-13 amino acid sequence of SEQ ID NO:2 has 40 of 111 amino acid residues (36%) identical to, and 56 of 111 (50%) amino acid residues similar to, the 231 amino acid residue human interleukin 22-binding protein CRF2-10 (gi
  • the CRF2-13 amino acid sequence has 32 of 86 amino acid residues (37%) identical to, and 43 of 86 (49%) amino acid residues similar to, the 130 amino acid residue human interleukin 22-binding protein CRF2-10S (gi
  • the CRF2-13 amino acid sequence has 41 of 142 amino acid residues (28%) identical to, and 58 of 142 (39%) amino acid residues similar to, the 130 amino acid residue human interleukin 22-binding protein CRF2-10L (gi
  • CRF2-13 polypeptide also shows homology to the amino acid sequences shown in the BLASTP data listed in Table 3A. Homologies are calculated according to the method of Altschul and coworkers (Nucleic Acids Res. 25:3389-3402, 1997).
  • the "E- value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject ("Sbjct") retrieved from the IIT BLAST analysis, matched the Query IIT sequence purely by chance is the E value.
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • Blasting is performed against public nucleotide databases such as GenBank databases and the GeneSeq patent database. For example, BLASTX searching is performed against public protein databases, which include GenBank databases, SwissProt, PDB and PIR.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • the Expect value is also used instead of the P value (probability) to report the significance of matches.
  • P value probability
  • an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/.
  • Cytokines e.g., lymphokines; interleukin and interferon
  • cytokine receptor class 2 family includes interleukin- 10 receptor; interferon-gamma receptor; interferon- alpha/beta receptor; and tissue factor (Konigsberg et al, Nature 380:41-46, 1996).
  • Table 5 presents a correlation between the genomic sequence shown in Table 4 and the location ofthe corresponding regions ofthe cDNA sequence shown in Table 1.
  • a CRF2-encoding nucleic acid is also present in the genomic nucleic acid sequence shown in Table 7:
  • Table 8 presents a correlation between the genomic sequence shown in Table 7 and the locations of the corresponding regions of the cDNA sequence shown in Table 1.
  • CRF2-like nucleic acids and polypeptides ofthe invention are referred to herein as "CRF2-13 " nucleic acids and polypeptides.
  • a CRF2-13 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • sequence comparison reveals that the disclosed CRF2-13 nucleic acid (Table 1) encodes a Type II cytokine receptor.
  • One or more secreted receptor chains may be associated with, and/or modulate the activity of, another membrane bound member of CRF2, or a membrane bound receptor of another family.
  • the receptor chains disclosed herein may act alone or in combination with another soluble receptor. In effect, the receptor can also be a ligand.
  • a soluble form ofthe CRF2-13 polypeptide ofthe invention may additionally be used as a soluble receptor antagonist.
  • Soluble receptor antagonists that block the activity of specific cytokines, e.g., TNF, are known in the art.
  • a soluble CRF2-13 polypeptide of the invention can similarly block the activity of a cytokine that acts through a CRF2 member. Examples of such polypeptides include IL-10, IL-19, IL-20, IL-22, AK155, mda-7 or an interferon, such as interferon alpha, interferon beta, or interferon gamma.
  • a soluble CRF2-13 polypeptide of the invention is used to antagonize the function of IL-22.
  • IL-22 is distantly related in sequence to IL-10 and is produced by activated T cells.
  • IL-22 signaling into a cell is mediated by its receptor chains, IL-22R and CRF2-4, both members of the
  • the CRF2-4 receptor was originally reported to serve as a second component in IL-10 signaling.
  • IL-22 has been reported to inhibit IL-4 production from human Th2 T cells and to induce acute phase proteins in the liver of mice.
  • CRF2-13 nucleic acids and polypeptides according to the invention may additionally be used to identify cell types that make the invention or bind to the invention in a population of cells.
  • the CRF2-13 nucleic acids and polypeptides can also be used for immunomodulation, inflammation, immunosuppression, allergy, asthma, autoimmunity (including rheumatoid arthritis and multiple sclerosis), repair of vascular smooth muscle cell after vascular injury or disease, transplantation and cancer based on the ligand that associates with this soluble receptor, alone or in conjunction with another receptor, and the impact that this ligand has on the above mechanisms and/or pathologies.
  • a CRF2-13 polypeptide and/or soluble form of a CRF2-13 polypeptide ofthe invention may exhibit one or more of the following activities: (1) modulation, e.g., it may antagonize a signal transduction pathway mediated by a cytokine (such as EL- 10 or IL- 22); (2) modulation of cytokine production and/or secretion (e.g., production and/or secretion of a proinflammatory cytokine); (3) modulation of lymphokine production and/or secretion; (4) modulation of expression or activity of nuclear transcription factors (5) competition with cytokine receptors for cytokine ligands; (6) modulation of cell proliferation, development or differentiation, e.g., cytokine-stimulated (such as IL-10 or IL-22) production, development, or differentiation; (7) modulation of cellular immune responses; modulation of cytokine- meditated proinflammatory actions; and/or promotion and/or potentiation of immune reactions.
  • a CRF2-13 polypeptide of the invention may directly, by association with a membrane bound receptor, or indirectly, by its association with a soluble ligand affect or effect one or more ofthe following cell types: circulating or tissue-associated cells: T cells, B cells, NK cells, NK T cells, dendritic cells, macrophages, monocytes, neutrophils, mast cells, basophils, eosinophils, as well as cells in the respiratory tract, pancreas, kidney, liver, small and large intestine.
  • a CRF2-13 polypeptide ofthe invention may additionally modulate upregulation of humoral immune responses and cell-mediated immune reactions; modulate the synthesis of proinflammatory cytokines and chemokines; and modulate inflammatory responses associated with injury, sepsis, gastrointestinal and cardiovascular disease, or inflammation following surgery.
  • the CRF2-13 sequences may be placed under the control of expression control sequences optimized for expression in a desired host.
  • the sequences may include optimized transcriptional and/or translational regulatory sequences (such as altered Kozak sequences).
  • the mature amino terminus of a CRF2-13 protein may be operably linked to a non-CRF2-13 signal sequence based on a hypothetical or empirically determined of the mature amino terminal end of the protein.
  • a CRF2-13 fusion protein can be used to identify and determine binding partners using assays known in the art. These assays include, e.g., either histological, immunochemical, BIACORE or cell biology based assays.
  • Assays can also be performed in order to determine whether a CRF2-13 protein ofthe invention associates with cell types that already express other members ofthe CRF2 family.
  • a CRF2-13 of the invention can also be examined for its ability to modulate the activity of known or novel cytokines (e.g., by inhibiting or otherwise antagonizing the functions of a cytokine).
  • IL-22 is one of these molecules. It has been reported that this molecule blocks the production of EL-4 by Th2 cells (human) and initiates an acute phase response (mice).
  • a finding that CRF2-13 binds to and inhibits IL-22 (or other IL-10 like molecules) indicates a CRF2-13 invention can be used to treat or prevent diseases associated with high levels of the IL-22 polypeptide.
  • a CRF2-13 polypeptide of the invention associates with other receptors and/or their associated cytokines within the CRF2 family.
  • a CRF2-13 ofthe invention may associate with either chain of the IL-22R and affect the function of the receptor or the IL-22 ligand.
  • the nucleic acids of the invention include those that encode a CRF2-13 polypeptide or protein.
  • the terms polypeptide and protein are interchangeable.
  • a CRF2-13 nucleic acid encodes a mature CRF2-13 polypeptide.
  • a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein.
  • An example of a CRF2-13 nucleic acid encoding a mature form of a CRF2-13 polypeptide is a nucleotide sequence encoding amino acids 21-520 of SEQ ID NO:2 (e.g., nucleotides 61- 1560 of SEQ ID NO: 1).
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal of the N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
  • Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • CRF2-13 nucleic acids ofthe invenation are the nucleic acid whose sequence is provided in nucleotides 1-1560 of SEQ ID NO: 1, SEQ ID NO: 1 itself, or a fragment of one of these sequences.
  • the invention includes mutant or variant nucleic acids of SEQ ID NO: 1 , or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO:l, while still encoding a protein that maintains at least one of its CRF2-13 -like activities and physiological functions (i.e., modulating angiogenesis, neuronal development).
  • the invention further includes the complement of the nucleic acid sequence of SEQ ID NO: 1 , including fragments, derivatives, analogs and homologs thereof.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • One aspect of the invention pertains to isolated nucleic acid molecules that encode CRF2-13 proteins or biologically active portions thereof.
  • nucleic acid fragments sufficient for use as hybridization probes to identify CRF2-13 -encoding nucleic acids (e.g., CRF2-13 mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of CRF2-13 nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated CRF2-13 nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • CRF2- 13 nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et ⁇ l., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • oligonucleotides corresponding to CRF2-13 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO:l, or a complement thereof.
  • Oligonucleotides may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1 , or a portion of this nucleotide sequence.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:l is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of CRF2-13 .
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of a CRF2-13 polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a CRF2-13 polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human CRF2-13 protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:2, as well as a polypeptide having CRF2- 13 activity. Biological activities of the CRF2- 13 proteins are described below.
  • a homologous amino acid sequence does not encode the amino acid sequence of a human CRF2-13 polypeptide.
  • the nucleotide sequence determined from the cloning of the human CRF2-13 gene allows for the generation of probes and primers designed for use in identifying and/or cloning CRF2-13 homologues in other cell types, e.g., from other tissues, as well as CRF2-13 homologues from other mammals.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1 ; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1 ; or of a naturally occurring mutant of SEQ ID NO: 1.
  • Probes based on the human CRF2-13 nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. , the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CRF2-13 protein, such as by measuring a level of a CRF2-13-encoding nucleic acid in a sample of cells from a subject e.g., detecting CRF2-13 mRNA levels or determining whether a genomic CRF2- 13 gene has been mutated or deleted.
  • a "polypeptide having a biologically active portion of CRF2-13” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of CRF2-13 " can be prepared by isolating a portion of SEQ ID NO:l that encodes a polypeptide having a CRF2-13 biological activity (biological activities of the CRF2-13 proteins are described below), expressing the encoded portion of CRF2-13 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of CRF2-13 .
  • a nucleic acid fragment encoding a biologically active portion of CRF2-13 can optionally include a cytokine-binding domain.
  • a nucleic acid fragment encoding a biologically active portion of CRF2-13 includes one or more regions.
  • polymorphisms in CRF2-13 associated sequences The invention also provides polymorphic forms of CRF2-13 nucleic acid sequences as well as methods of detecting polymorphic sequences in CRF2-13 sequences
  • the polymorphic forms include genomic sequences corresponding to exons and/or introns associated with CRF2-13.
  • the polymorphisms can be provided on various isolated CRF2-13 nucleic acids.
  • the polymorphism can be provided on an isolated polynucleotide comprising at least 10 contiguous nucleotides of SEQ ID NO:3 that include the polymorphic sequences shown in Table 6.
  • the polymorphism can be provided on an isolated polynucleotide comprising at least 10 nucleotides of SEQ ID NO:2 that include alternative forms of the polymorphic sequences shown in Table 9.
  • an isolated CRF2-13 polymorphic sequence can include from nucleotide 30957 to nucleotide 30967 of SEQ ID NO:3, provided that position 30962 is "A or "G”.
  • the isolated CRF2-13 polymorphic sequence can include at least 10 contiguous nucleotides from nucleotide 30650 to nucleotide 30660 of SEQ ID NO:3, provided that position 30655 is "A" or "G”.
  • the isolated CRF2-13 nucleic acid sequence includes at least 10 contiguous nucleotides from nucleotide 28739 to nucleotide 28749 of SEQ ID NO:3, wherein position 28744 is "A” or “G”; at least 10 contiguous nucleotides from nucleotide 28442 to 28452 of SEQ ID NO:3, wherein position 28448 is "C” or "T”; additional examples include an isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 9421 to 9431 of SEQ ID NO:3, wherein position 9426 of the polynucleotide is "A" or "G", or an isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 8806 to 8816 of SEQ ID NO:3, wherein position 8811 of the polynucleotide is "C or "T”.
  • an isolated CRF2-13 polymorphic sequence can include from nucleotide 32954 to nucleotide 32964 of SEQ ID NO:22, provided that position 30962 is “C” or “A”.
  • the polymorphic sequence can include from nucleotide 31262 to 31272 of SEQ ID NO:22, provided that position 31266 is “C” or “T”; or nucleotides 30955 to 20965 of SEQ ID NO:22, provided that nucleotide 30960 is “T” or “C”; or nucleotides 29043 to 29053 of SEQ ID NO:22, provided that nucleotide 29048 is “C” or “T”; or nucleotides 28748 to 28758 of SEQ ID NO:22, provided that nucleotide 28753 is “G” or “A”; or nucleotides 23825 to 23835 of SEQ ID NO:22, provided that nucleotide 23830 is "G”
  • the polymorphic nucleic acid includes at least 15, 20, 25, 50, 75, 100, 150, 250, 500, 750, or 1000 or more contiguous nucleotides from SEQ ID NO:3.
  • the polymorphic nucleotide sequence is 10-1000 nucleotides in length.
  • the polymorphic nucleotide sequence can be 20-750 nucleotides, 50-625 nucleotides, 75-500 nucleotides, 100-250 nucleotides in length.
  • polymorphic alleles of the invention may be detected at either the DNA, the RNA, or the protein level using a variety of techniques that are well known in the art. Strategies for identification and detection are described in e.g., EP 730,663, EP 717,113, and PCT US97/02102.
  • the present methods usually employ pre-characterized polymorphisms. That is, the genotyping location and nature of polymorphic forms present at a site have already been determined. The availability of this information allows sets of probes to be designed for specific identification of the known polymorphic forms.
  • PCR. (1989), B. for detecting polymorphisms See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Apphcations 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202.
  • the genomic DNA used for the diagnosis may be obtained from any nucleated cells of the body, such as those present in peripheral blood, urine, saliva, buccal samples, surgical specimen, and autopsy specimens.
  • the DNA may be used directly or may be amplified enzymatically in vitro through use of PCR (Saiki et al. Science 239:487-491 (1988)) or other in vitro amplification methods such as the ligase chain reaction (LCR) (Wu and Wallace Genomics 4:560-569 (1989)), strand displacement amplification (SDA) (Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992)), self-sustained sequence replication (3SR) (Fahy et al. PCR Methods P&J& 1:25-33 (1992)), prior to mutation analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • 3SR self-sustained sequence replication
  • the detection of polymorphisms in specific DNA sequences can be accomplished by a variety of methods including, but not limited to, restriction-fragment-length-polymo hism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy Lancet ii:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl. Acids Res. 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al. Proc. Natl. Acad. SCI.
  • gap-LCR Alpha-LCR
  • CRF2-13 Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO:l due to the degeneracy ofthe genetic code. These nucleic acids thus encode the same CRF2-13 protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, e.g., the polypeptide of SEQ ID NO:2.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of CRF2-13 may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the CRF2-13 gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a CRF2- 13 protein, preferably a mammalian CRF2- 13 protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the CRF2-13 gene.
  • nucleotide variations and resulting amino acid polymorphisms in CRF2-13 that are the result of natural allelic variation and that do not alter the functional activity of CRF2-13 are intended to be within the scope of the invention.
  • nucleic acid molecules encoding CRF2-13 proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of SEQ JO NO: 1 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe CRF2-13 cDNAs ofthe invention can be isolated based on their homology to the human CRF2-13 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human CRF2-13 cDNA can be isolated based on its homology to human membrane-bound CRF2-13 .
  • a membrane-bound human CRF2-13 cDNA can be isolated based on its homology to soluble human CRF2-13 .
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding CRF2-13 proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm,
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al.
  • a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, or fragments, analogs or derivatives thereof, under conditions of low stringency is provided.
  • a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM
  • Tris-HCl pH 7.5
  • 5 mM EDTA 0.02% PVP
  • 0.02% Ficoll 0.2% BSA
  • 100 mg/ml denatured salmon sperm DNA 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al.
  • allelic variants of the CRF2-13 sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, thereby leading to changes in the amino acid sequence ofthe encoded CRF2-13 protein, without altering the functional ability ofthe CRF2-13 protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CRF2-13 without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the CRF2-13 proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2.
  • the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO:2, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2.
  • An isolated nucleic acid molecule encoding a CRF2-13 protein homologous to the protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into the nucleotide sequence of SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in CRF2-13 is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a CRF2-13 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CRF2-13 biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
  • a mutant CRF2-13 protein can be assayed for (1) the ability to form protei protein interactions with other CRF2-13 proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant CRF2-13 protein and a CRF2- 13 receptor; (3) the ability of a mutant CRF2- 13 protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind CRF2-13 protein; or (5) the ability to specifically bind an anti-CRF2-13 protein antibody.
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire CRF2-13 coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a CRF2-13 protein of SEQ ID NO:2, or antisense nucleic acids complementary to a CRF2-13 nucleic acid sequence of SEQ ID NO:l are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding CRF2-13 .
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human CRF2-13 corresponds to SEQ ID NO:2).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding CRF2-13 .
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding CRF2-13 disclosed herein (e.g., SEQ ID NO: 1
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of CRF2-13 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of CRF2-13 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CRF2-13 mRNA:
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CRF2-13 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CRF2-13 mRNA transcripts to thereby inhibit translation of CRF2- 13 mRNA.
  • a ribozyme having specificity for a CRF2-13 -encoding nucleic acid can be designed based upon the nucleotide sequence of a CRF2- 13 DNA disclosed herein (i.e., SEQ ID NO:l).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CRF2-13 -encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
  • CRF2-13 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g. , Bartel et al, (1993) Science 261:1411-1418.
  • CRF2-13 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CRF2-13 (e.g., the CRF2-13 promoter and/or enhancers) to form triple helical structures that prevent transcription of the CRF2-13 gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
  • the nucleic acids of CRF2-13 can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • the terms "peptide nucleic acids” or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
  • PNAs of CRF2-13 can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of CRF2-13 can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of CRF2-13 can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of CRF2-13 can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
  • PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Pro
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • a CRF2-13 polypeptide ofthe invention includes the CRF2-13 -like protein whose sequence is provided in SEQ ID NO:2.
  • a CRF2-13 polypeptide includes amino acid sequences 21-520, amino acids 21-230 of SEQ ID NO:2, amino acids v 21-246 of SEQ ID NO:2, amino acids 231-520 of SEQ ID NO:2, amino acids 247-520 of SEQ ID NO:2.
  • the invention also includes a mutant or variant form of the disclosed CRF2- 13 polypeptide, or of any of the fragments of the herein disclosed CRF2-13 polypeptide sequences.
  • a CRF2-13 polypeptide includes one in which any residues may be changed from the corresponding residue shown in SEQ ID NO:2 while still encoding a protein that maintains its CRF2-13 -like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more of the residues may be so changed in the mutant or variant protein. In some embodiments, the CRF2-13 polypeptide according to the invention is a mature polypeptide.
  • a CRF2-13 -like variant that preserves CRF2-13 -like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • CRF2-13 proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • polypeptide fragments suitable for use as immunogens to raise anti-CRF2-13 antibodies are provided.
  • native CRF2-13 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • CRF2-13 proteins are produced by recombinant DNA techniques.
  • a CRF2-13 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • a “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the CRF2-13 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of CRF2-13 protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of CRF2-13 protein having less than about 30% (by dry weight) of non-CRF2-13 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-CRF2-13 protein, still more preferably less than about 10% of non-CRF2-13 protein, and most preferably less than about 5 % non-CRF2- 13 protein.
  • non-CRF2-13 protein also referred to herein as a "contaminating protein”
  • the CRF2-13 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of CRF2-13 protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of CRF2-13 protein having less than about 30% (by dry weight) of chemical precursors or non-CRF2-13 chemicals, more preferably less than about 20% chemical precursors or non-CRF2-13 chemicals, still more preferably less than about 10% chemical precursors or non-CRF2-13 chemicals, and most preferably less than about 5% chemical precursors or non-CRF2-13 chemicals.
  • Biologically active portions of a CRF2-13 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the CRF2-13 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 that include fewer amino acids than the full length CRF2-13 proteins, and exhibit at least one activity of a CRF2-13 protein.
  • biologically active portions comprise a domain or motif with at least one activity of the CRF2-13 protein.
  • a biologically active portion of a CRF2-13 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a CRF2-13 protein of the present invention may contain at least one ofthe above-identified domains conserved between the CRF2-13 proteins.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native CRF2-13 protein.
  • the CRF2-13 protein has an amino acid sequence shown in SEQ ID NO:2.
  • the CRF2-13 protein is substantially homologous to SEQ ID NO:2 and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
  • the CRF2- 13 protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the CRF2-13 proteins of SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either ofthe sequences being compared for optimal alignment between the sequences).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 910 J Mol Biol 48: 443-453.
  • GAP software provided in the GCG program package. See, Needleman and Wunsch 910 J Mol Biol 48: 443-453.
  • GAP creation penalty of 5.0 and GAP extension penalty of 0.3 the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • percentage of positive residues is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
  • the invention also provides CRF2-13 chimeric or fusion proteins.
  • a CRF2-13 "chimeric protein” or “fusion protein” comprises a CRF2-13 polypeptide operatively linked to a non-CRF2- 13 polypeptide.
  • An "CRF2-13 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to CRF2-13
  • a “non-CRF2-13 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the CRF2-13 protein, e.g., a protein that is different from the CRF2-13 protein and that is derived from the same or a different organism.
  • a CRF2- 13 fusion protein can correspond to all or a portion of a CRF2- 13 protein.
  • An example of a CRF2- 13 fusion polypeptide is one that includes amino acids 21-230 of SEQ ID NO:2 (e.g., a polypeptide that includes amino acids 1-246 or amino acids 21-246 of SEQ ID NO:2).
  • a CRF2-13 fusion protein comprises at least one biologically active portion of a CRF2-13 protein.
  • a CRF2-13 fusion protein comprises at least two biologically active portions of a CRF2-13 protein.
  • operatively linked is intended to indicate that the CRF2-13 polypeptide and the non-CRF2- 13 polypeptide are fused in-frame to each other.
  • the non-CRF2-13 polypeptide can be fused to the N-terminus or C-terminus ofthe CRF2-13 polypeptide.
  • a CRF2-13 fusion protein comprises a CRF2-13 polypeptide operably linked to either an extracellular domain of a second protein, i.e., non- CRF2-13 protein, or to the transmembrane and intracellular domain of a second protein, ie., non-CRF2-13 protein.
  • fusion proteins can be further utilized in screening assays for compounds that modulate CRF2-13 activity (such assays are described in detail below).
  • the fusion protein is a GST-CRF2-13 fusion protein in which the CRF2-13 sequences are fused to the C-terminus ofthe GST (i.e., glutathione
  • Such fusion proteins can facilitate the purification of recombinant CRF2-13 .
  • the fusion protein is a CRF2-13 -immunoglobulin fusion protein in which the CRF2-13 sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.
  • the CFR2-13 fusion proteins (e.g., CRF2-13 -immunoglobulin fusion proteins) ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit or augment an interaction between a cell surface receptor and its ligand. This could occur either by 1) binding to and removing available ligand for the receptor (Fc mediated scavenging ofthe ligand affecting bioavailability); 2) binding to the ligand and blocking its ability to bind to the cell receptor (antagonizing or neutralizing); 3) associating with another CRF member and thereby modulating (e.g., inhibiting) a downstream signal transduction cascade; 4) associating with either a ligand or another CRF and facilitating the activity ofthe ligand.
  • Fc mediated scavenging ofthe ligand affecting bioavailability binding to the ligand and blocking its ability to bind to the cell receptor (antagonizing or neutralizing)
  • a CRF2-13 protein may be used to modulate the interaction between a CRF2 receptor and its cognate ligand (e.g., an interaction between IL-10 and an IL-10 receptor and interaction between JL-22 and an IL-22 receptor).
  • Inhibition of the CRF2-13 ligand/CRF2-13 interaction can be used therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer, modulating (e.g., promoting or inhibiting) cell survival as well as immunomodulatory disorders, autoimmunity, transplantation, and inflammation by alteration of cyotokine and chemokine cascade mechanisms.
  • the CRF2-13 -immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-CRF2-13 antibodies in a subject, to purify CRF2-13 ligands, and in screening assays to identify molecules that inhibit the interaction of CRF2- 13 with a CRF2-13 ligand.
  • a CRF2-13 chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g
  • a CRF2-13 -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CRF2-13 protein.
  • libraries of fragments of the CRF2-13 protein coding sequence can be used to generate a variegated population of CRF2-13 fragments for screening and subsequent selection of variants of a CRF2-13 protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CRF2-13 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the CRF2- 13 protein.
  • REM Recursive ensemble mutagenesis
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Ig immunoglobulin
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a b, F ab 1 and F( a b-)2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG 2 , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated CRF2-13 -related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecificaUy bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO:2, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of CRF2-13 -related protein that is located on the surface of the protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human CRF2-13 -related protein sequence will indicate which regions of a CRF2-13 -related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.
  • a protein of the invention may be utilized as an immunogen in the generation of antibodies that immunospecificaUy bind these protein components.
  • an appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59- 103).
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368. 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non- human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F a b expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F a b fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F( a b')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (ab' )2 fragment; (iii) an F a fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature. 305:537-539 (1983)).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted fromE. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioco ⁇ jugated antibodies. Examples include 212 Bi, 131 1, 131 In, 90 Y, and 186 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis--
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • vectors preferably expression vectors, containing a nucleic acid encoding a CRF2-13 protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous rephcation in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CRF2-13 proteins, mutant forms of CRF2-13 proteins, fusion proteins, etc.).
  • the recombinant expression vectors ofthe invention can be designed for expression of CRF2-13 proteins in prokaryotic or eukaryotic cells.
  • CRF2- 13 proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
  • nucleic acid sequence of the nucleic acid is altered by e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118).
  • Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
  • the CRF2-13 expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baldari, et al, 1987. EMBO I. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invirrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif.).
  • CRF2-13 can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO I. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to CRF2-13 mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • CRF2- 13 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding CRF2-13 or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) CRF2-13 protein.
  • the invention further provides methods for producing CRF2-13 protein using the host cells ofthe invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding CRF2-13 protein has been introduced) in a suitable medium such that CRF2-13 protein is produced.
  • the method further comprises isolating CRF2-13 protein from the medium or the host cell.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which CRF2-13 protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous CRF2-13 sequences have been introduced into their genome or homologous recombinant animals in which endogenous CRF2-13 sequences have been altered. Such animals are useful for studying the function and/or activity of CRF2-13 protein and for identifying and/or evaluating modulators of CRF2-13 protein activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CRF2-13 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development of the animal.
  • a transgenic animal ofthe invention can be created by introducing CRF2-13 -encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retro viral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Sequences including SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue of the human CRF2-13 gene such as a mouse CRF2-13 gene, can be isolated based on hybridization to the human CRF2-13 cDNA (described further supra) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably-linked to the CRF2-13 transgene to direct expression of CRF2-13 protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence ofthe CRF2-13 transgene in its genome and/or expression of CRF2-13 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding CRF2-13 protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a CRF2-13 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the CRF2-13 gene.
  • the CRF2-13 gene can be a human gene (e.g., the DNA of SEQ ID NO: 1), but more preferably, is a non-human homologue of a human CRF2-13 gene.
  • a mouse homologue of human CRF2-13 gene of SEQ ID NO: 1 can be used to construct a homologous recombination vector suitable for altering an endogenous CRF2-13 gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous CRF2-13 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous CRF2-13 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous CRF2-13 protein).
  • the altered portion ofthe CRF2-13 gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the CRF2-13 gene to allow for homologous recombination to occur between the exogenous CRF2-13 gene carried by the vector and an endogenous CRF2- 13 gene in an embryonic stem cell.
  • flanking CRF2-13 nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5'- and 3'-termini
  • the vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced CRF2-13 gene has homologously-recombined with the endogenous CRF2-13 gene are selected. See, e.g., Li, etal, 1992. Cell 69: 915.
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously- recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2:
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236.
  • Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments ofthe antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst, 81(19): 1484 (1989).
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
  • Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drag Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M.
  • antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are prefe ⁇ ed.
  • liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and or produced by recombinant DNA technology. See, e.g., Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893.
  • the formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drag delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate non-degradable ethylene- vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the isolated nucleic acid molecules ofthe invention can be used to express CRF2-13 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect CRF2-13 mRNA (e.g., in a biological sample) or a genetic lesion in a CRF2-13 gene, and to modulate CRF2-13 activity, as described further, below.
  • the CRF2- 13 proteins can be used to screen drugs or compounds that modulate the CRF2-13 protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of CRF2-13 protein or production of CRF2-13 protein forms that have decreased or abe ⁇ ant activity compared to CRF2-13 wild-type protein .
  • the anti-CRF2-13 antibodies of the invention can be used to detect and isolate CRF2-13 proteins and modulate CRF2-13 activity.
  • CRF2- 13 activity includes T-cell or NK cell growth and differentiation, antibody production, and tumor growth.
  • the invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to CRF2-13 proteins or have a stimulatory or inhibitory effect on, e.g., CRF2-13 protein expression or CRF2-13 protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to CRF2-13 proteins or have a stimulatory or inhibitory effect on, e.g., CRF2-13 protein expression or CRF2-13 protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to CRF2-13 proteins or have a stimulatory or inhibitory effect on, e.g., C
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a CRF2- 13 protein or polypeptide or biologically-active portion thereof.
  • the test compounds ofthe invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 ' . Anticancer Drug Design 12: 145.
  • a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to a CRF2-13 protein determined.
  • the cell for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the CRF2-13 protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the CRF2-13 protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds CRF2-13 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CRF2-13 protein, wherein determining the ability ofthe test compound to interact with a CRF2-13 protein comprises determining the ability of the test compound to preferentially bind to CRF2-13 protein or a biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface with a test compound and determimng the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe CRF2-13 protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of CRF2-13 or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the CRF2-13 protein to bind to or interact with a CRF2-13 target molecule.
  • a "target molecule” is a molecule with which a CRF2-13 protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a CRF2-13 interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a CRF2-13 target molecule can be a non-CRF2-13 molecule or a CRF2-13 protein or polypeptide of the invention
  • a CRF2- 13 target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with CRF2-13 .
  • Determining the ability of the CRF2-13 protein to bind to or interact with a CRF2-13 target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability ofthe CRF2-13 protein to bind to or interact with a CRF2-13 target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e.
  • a reporter gene comprising a CRF2-13 -responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, cell survival, cellular differentiation, or cell proliferation.
  • an assay of the invention is a cell-free assay comprising contacting a CRF2-13 protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the CRF2-13 protein or biologically-active portion thereof. Binding of the test compound to the CRF2-13 protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the CRF2-13 protein or biologically-active portion thereof with a known compound which binds CRF2-13 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a CRF2-13 protein, wherein determining the ability ofthe test compound to interact with a CRF2-13 protein comprises determining the ability of the test compound to preferentially bind to CRF2-13 or biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting CRF2-13 protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the CRF2-13 protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of CRF2-13 can be accomplished, for example, by determining the ability ofthe CRF2-13 protein to bind to a CRF2-13 target molecule by one of the methods described above for determining direct binding.
  • determining the ability of the test compound to modulate the activity of CRF2-13 protein can be accomplished by determimng the ability of the CRF2-13 protein further modulate a CRF2- 13 target molecule.
  • the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described above.
  • the cell-free assay comprises contacting the CRF2-13 protein or biologically-active portion thereof with a known compound which binds CRF2-13 protein to form an assay mixture, contacting the assay mixture with a test compound, and dete ⁇ mning the ability of the test compound to interact with a CRF2-13 protein, wherein determining the ability of the test compound to interact with a CRF2-13 protein comprises determining the ability of the CRF2-13 protein to preferentially bind to or modulate the activity of a CRF2-13 target molecule.
  • the cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of CRF2-13 protein.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-114, Thesit ® , Isotridecypoly(ethylene glycol ether) n , N-dodecyl ⁇ N,N-dimethyl-3-ammonio-l-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholarmdopropyl)dimemylanuniniol-2-hydroxy-l-propane sulfonate (CHAPSO).
  • non-ionic detergents such as n-oct
  • CRF2-13 protein or its target molecule it may be desirable to immobilize either CRF2-13 protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation ofthe assay.
  • Binding of a test compound to CRF2-13 protein, or interaction of CRF2-13 protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST-CRF2-13 fusion proteins or GST- target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or CRF2-13 protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtiter plates that are then combined with the test compound or the test compound and either the non-adsorbed target protein or CRF2-13 protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions
  • the complexes can be dissociated from the matrix, and the level of CRF2-13 protein binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • either the CRF2-13 protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated CRF2-13 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with CRF2-13 protein or target molecules can be derivatized to the wells ofthe plate, and unbound target or CRF2-13 protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the CRF2-13 protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the CRF2-13 protein or target molecule.
  • modulators of CRF2-13 protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of CRF2- 13 mRNA or protein in the cell is determined.
  • the level of expression of CRF2-13 mRNA or protein in the presence of the candidate compound is compared to the level of expression of CRF2-13 mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of CRF2-13 mRNA or protein expression based upon this comparison. For example, when expression of CRF2-13 mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of CRF2-13 mRNA or protein expression.
  • the candidate compound when expression of CRF2-13 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CRF2-13 mRNA or protein expression.
  • the level of CRF2-13 mRNA or protein expression in the cells can be determined by methods described herein for detecting CRF2-13 mRNA or protein.
  • the CRF2-13 proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, etal, 1993. Cell 72: 223-232; Madura, etal, 1993. /. Biol Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, etal, 1993.
  • CRF2-13 -binding proteins or "CRF2-13 -bp"
  • CRF2-13 -binding proteins are also likely to be involved in the propagation of signals by the CRF2-13 proteins as, for example, upstream or downstream elements ofthe CRF2-13 pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for CRF2-13 is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with CRF2-13 .
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with CRF2-13 .
  • the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
  • Example 1 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:4.
  • the variant amino acid sequence is shown in bold-font.
  • a valine at position 30 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an alanine in SEQ ID NO:4.
  • Example 2 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:5.
  • the variant amino acid sequence is shown in bold-font.
  • a leucine at position 39 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an isoleucine in SEQ ID NO:5.
  • Example 3 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:6.
  • the variant amino acid sequence is shown in bold-font.
  • An asparagine at position 49 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a threonine in SEQ ID NO:6.
  • Example 4 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:7.
  • the variant amino acid sequence is shown in bold-font.
  • An arginine at position 65 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO:7.
  • Example 5 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 8.
  • the variant amino acid sequence is shown in bold-font.
  • a lysine at position 78 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an arginine in SEQ ID NO:8.
  • Example 6 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:9.
  • the variant amino acid sequence is shown in bold-font.
  • a Q ⁇ glutamine? ⁇ at position 90 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an asparagine in SEQ ID NO:9.
  • Example 7 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 10.
  • the variant amino acid sequence is shown in bold-font.
  • a arginine at position 99 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an lysine in SEQ ID NO: 10.
  • Example 8 A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence ofSEQ ID NO:2 is shown in SEQ ID NO:11.
  • the variant amino acid sequence is shown in bold-font.
  • a valine atposition 112 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an leucine in SEQ ID NO:.11.
  • Example 9 A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 12.
  • the variant amino acid sequence is shown in bold-font.
  • a tyrosine at position 119 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a phenylalanine in SEQ ID NO: 12.
  • Example 10 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 13.
  • the variant amino acid sequence is shown in bold-font.
  • a valine at position 129 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an isoleucine in SEQ ID NO: 13.
  • Example 11 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 14.
  • the variant amino acid sequence is shown in bold-font.
  • a threonine at position 144 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an asparagine in SEQ ED NO: 14.
  • Example 12 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 15.
  • the variant amino acid sequence is shown in bold-font.
  • a leucine at position 154 in the polypeptide sequence shown in SEQ ED NO:2 is replaced with an alanine in SEQ ID NO: 15.
  • Example 13 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2) A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 16. The variant amino acid sequence is shown in bold-font. A lysine at position 170 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an arginine in SEQ ID NO: 16.
  • Example 14 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 17.
  • the variant amino acid sequence is shown in bold-font.
  • a valine at position 175 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a leucine in SEQ ID NO: 17.
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 18.
  • the variant amino acid sequence is shown in bold-font.
  • An alanine at position 189 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a valine in SEQ ID NO: 18.
  • Example 16 A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 19
  • the variant amino acid sequence is shown in bold-font.
  • An arginine at position 199 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO:.19
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:20.
  • the variant amino acid sequence is shown in bold-font.
  • a phenylalanine at position 212 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an a tryptophan in SEQ ID NO:20.
  • Example 18 A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
  • a polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:21.
  • the variant amino acid sequence is shown in bold-font.
  • An arginine at position 230 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO21:.
  • a 310 nucleotide fragment corresponding to nucleotides XX to XX [41-352 of SEQ ID No.l] in Table 1 was identified in a human placental cDNA library (BD Biosciences Clontech, Palo Alto, CA, USA) by PCR using an Advantage II PCR kit (BD Biosciences Clontech, Palo Alto, CA, USA) and primers specific for the 5' region of the human CRF2-13.
  • the primers included Ax5-1 (GCTGCAGGCCGCTCCAGGGAGGCCCCG; SEQ ID:23) and Ax3-1 (CCAGGTATTCGGACTCCACCCAGGGGGAC; SEQ ID NO:24). The primers were used for thirty eight thermal cycles of PCR.
  • the CRF2-13 nucleic acid product was gel purified and sequenced. The sequence corresponds to the corresponding sequences in the CRF2-13 sequence disclosed in Table 1.
  • cDNAs ofthe Human Placenta Sub-Plate 2H human placental library were directionally-cloned into the CMV expression vector pCMV6-XL4, a vector-derived 5' PCR primer was used in conjunction with a gene-specific 3' reverse primer to identify the CRF2- 13 clone.
  • the cDNA library was screened by a PCR-based procedure using the Advantage II PCR kit (BD Biosciences Clontech, Palo Alto, CA, USA) and Ax5-1 (SEQ ID:25 and Ax3-2 (TTGGTTCCCGCACACTCTTCCACTTCG; SEQ ID NO:26) as PCR primers.
  • PCR analysis was carried out in a 96-well arrayed at 50 clones per well.
  • the PCR positive well (E2) was identified and the E. coli cells from that well were subsequently diluted, plated out and analyzed to yield the clone full-length CRF2-13 clone. The identity of the CRF2-13 clone was then verified by sequence analysis.

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Abstract

The present invention provides novel isolated CRF2-13 polynucleotides and polypeptides encoded by the CRF2-13 polynucleotides. Also provided are the antibodies that immunospecifically bind to a CRF2-13 polypeptide or any derivative (including fusion derivative), variant, mutant or fragment of the CRF2-13 polypeptide, polynucleotide or antibody. The invention additionally provides methods in which the CRF2-13 polypeptide, polynucleotide and antibody are utilized in the detection and treatment of a broad range of pathological states, as well as to other uses.

Description

TYPE 2 CYTOKINE RECEPTOR AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTION The invention relates generally to nucleic acids and polypeptides and more specifically to nucleic acids and polypeptides encoding type II cytokine receptors, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION Cytokines such as interferons are soluble proteins that influence the growth and differentiation of many cell types. Cytokines exert their effects through cytokine receptors, which are located on the surface of cells responsive to the effects of cytokines. Cytokine receptors are composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the receptor subunits.
Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains. For example, the receptor chains responsible for binding and/or transducing the effect of interferons cytokine are members of the type II cytokine receptor family (CRF2), based upon the presence of a characteristic 200-250 residue extracellular domain.
Members of the CRF2 family have been reported to act as receptors for a variety of cytokines, including interferon alpha, interferon beta, interferon gamma, IL-10, IL-20, and BL-22. Recently identified members of the CRF2 family are candidate ligands for the IL-10- like molecules IL-19, AK155 and mda-7. The demonstrated in vivo activities of these interferons illustrate the clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists. SUMMARY OF THE INVENTION
The invention is based, in part, upon the discovery of polynucleotide sequences encoding CRF2-13, novel member of the CRF2 family.
Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO: 1 , or a fragment, homolog, analog or derivative thereof. The nucleic acid can include, e.g., a nucleic acid sequence encoding a polypeptide at least 70%, e.g., 80%, 85%, 90%, 95%, 98%, or even 99% or more identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:2. The nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
Also within the invention is a nucleic acid that encodes a polypeptide that includes amino acid sequences 21-520 of SEQ ID NO:2, e.g., a nucleic acids 61-1560 of SEQ ID NO:l. Examples of such nucleic acid molecules are that encode polypeptides with the amino acid sequences of SEQ ID NO:2.
Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
The invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
In another aspect, the invention includes a pharmaceutical composition that includes an CRF2-13 nucleic acid and a pharmaceutically acceptable carrier or diluent. In a further aspect, the invention includes a substantially purified CRF2-13 polypeptide, e.g., any ofthe CRF2-13 polypeptides encoded by an CRF2-13 nucleic acid, and fragments, homologs, analogs, and derivatives thereof. The invention also includes a pharmaceutical composition that includes an CRF2-13 polypeptide and a pharmaceutically acceptable carrier or diluent. In still a further aspect, the invention provides an antibody that binds specifically to an CRF2-13 polypeptide. The antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof. The invention also includes a pharmaceutical composition including CRF2-13 antibody and a pharmaceutically acceptable carrier or diluent. The invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
The invention also includes kits comprising any ofthe pharmaceutical compositions described above. The invention further provides a method for producing an CRF2-13 polypeptide by providing a cell containing an CRF2-13 nucleic acid, e.g., a vector that includes an CRF2-13 nucleic acid, and culturing the cell under conditions sufficient to express the CRF2-13 polypeptide encoded by the nucleic acid. The expressed CRF2-13 polypeptide is then recovered from the cell. Preferably, the cell produces little or no endogenous CRF2-13 polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.
The invention is also directed to methods of identifying an CRF2-13 polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
The invention further provides methods of identifying a compound that modulates the activity of an CRF2-13 polypeptide by contacting an CRF2-13 polypeptide with a compound and determining whether the CRF2-13 polypeptide activity is modified.
The invention is also directed to compounds that modulate CRF2-13 polypeptide activity identified by contacting an CRF2-13 polypeptide with the compound and determining whether the compound modifies activity ofthe CRF2-13 polypeptide, binds to the CRF2-13 polypeptide, or binds to a nucleic acid molecule encoding an CRF2-13 polypeptide.
In another aspect, the invention provides a method of determining the presence of or predisposition of an CRF2-13 -associated disorder in a subject. The method includes providing a sample from the subject and measuring the amount of CRF2-13 polypeptide in the subject sample. The amount of CRF2-13 polypeptide in the subject sample is then compared to the amount of CRF2-13 polypeptide in a control sample. An alteration in the amount of CRF2-13 polypeptide in the subject protein sample relative to the amount of CRF2-13 polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition. A control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation-associated condition. Alternatively, the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder. In some embodiments, the CRF2-13 is detected using an CRF2-13 antibody. In a further aspect, the invention provides a method of determining the presence of or predisposition of an CRF2-13 -associated disorder in a subject. The method includes providing a nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount ofthe CRF2-13 nucleic acid in the subject nucleic acid sample. The amount of CRF2-13 nucleic acid sample in the subject nucleic acid is then compared to the amount of an CRF2-13 nucleic acid in a control sample. An alteration in the amount of CRF2-13 nucleic acid in the sample relative to the amount of CRF2-13 in the control sample indicates the subject has a tissue proliferation-associated disorder.
In a still further aspect, the invention provides a method of treating or preventing or delaying an CRF2-13 -associated disorder. The method includes administering to a subject in which such treatment or prevention or delay is desired an CRF2-13 nucleic acid, an CRF2-13 polypeptide, or an CRF2-13 antibody in an amount sufficient to treat, prevent, or delay a tissue proliferation-associated disorder in the subject. Examples of such disorders include rheumatoid arthritis and multiple sclerosis.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a phylogram showing polypeptide sequences related to a CRF2-13 polypeptide according to the invention.
DETAILED DESCRIPTION OF THE INVENTION The invention is based in part on the discovery of novel nucleic acid sequences encoding a polypeptide showing homology to CRF2 polypeptides. Included in the invention is a 1563 nucleotide sequence (SEQ ID NO:l) shown in Table 1. Nucleotides 1-1560 of SEQ ID NO:l encode a 520 amino acid CRF2-like polypeptide. The amino acid sequences ofthe encoded polypeptide is shown in Table 2 (SEQ ID NO:2). A nucleic acid having a portion of the 5' untranslated region and a portion of the coding sequence shown in Table 1 was identified in a human placental cDNA library.
Table 1
ATGGCGGGGCCCGAGCGCTGGGGCCCCCTGCTCCTGTGCCTGCTGCAGGCCGCTCCAGGGAGGCCCCGTCTGGCC CCTCCCCAGAATGTGACGCTGCTCTCCCAGAACTTCAGCGTGTACCTGACATGGCTCCCAGGGCTTGGCAACCCC CAGGATGTGACCTATTTTGTGGCCTATCAGAGCTCTCCCACCCGTAGACGGTGGCGCGAAGTGGAAGAGTβTGCG GGAACCAAGGAGCTGCTATGTTCTATGATGTGCCTGAAGAAACAGGACCTGTACAACAAGTTCAAGGGACGCGTG CGGACGGTTTCTCCCAGCTCCAAGTCCCCCTGGGTGGAGTCCGAATACCTGGATTACCTTTTTGAAGTGGAGCCG GCCCCACCTGTCCTGGTGCTCACCCAGACGGAGGAGATCCTGAGTGCCAATGCCACGTACCAGCTGCCCCCCTGC ATGCCCCCACTGGATCTGAAGTATGAGGTGGCATTCTGGAAGGAGGGGGCCGGAAACAAGACCCTATTTCCAGTC ACTCCCCATGGCCAGCCAGTCCAGATCACTCTCCAGCCAGCTGCCAGCGAACACCACTGCCTCAGTGCCAGAACC ATCTACACGTTCAGTGTCCCGAAATACAGCAAGTTCTCTAAGCCCACCTGCTTCTTGCTGGAGGTCCCAGAAGCC AACTGGGCTTTCCTGGTGCTGCCATCGCTTCTGATACTGCTGTTAGTAATTGCCGCAGGGGGTGTGATCTGGAAG ACCCTCATGGGGAACCCCTGGTTTCAGCGGGCAAAGATGCCACGGGCCCTGGACTTTTCTGGACACACACACCCT GTGGCAACCTTTCAGCCCAGCAGACCAGAGTCCGTGAATGACTTGTTCCTCTGTCCCCAAAAGGAACTGACCAGA GGGGTCAGGCCGACGCCTCGAGTCAGGGCCCCAGCCACCCAACAGACAAGATGGAAGAAGGACCTTGCAGAGGAC GAAGAGGAGGAGGATGAGGAGGACACAGAAGATGGCGTCAGCTTCCAGCCCTACATTGAACCACCTTCTTTCCTG GGGCAAGAGCACCAGGCTCCAGGGCACTCGGAGGCTGGTGGGGTGGACTCAGGGAGGCCCAGGGCTCCTCTGGTC CCAAGCGAAGGCTCCTCTGCTTGGGATTCTTCAGACAGAAGCTGGGCCAGCACTGTGGACTCCTCCTGGGACAGG GCTGGGTCCTCTGGCTATTTGGCTGAGAAGGGGCCAGGCCAAGGGCCGGGTGGGGATGGGCACCAAGAATCTCTC CCACCACCTGAATTCTCCAAGGACTCGGGTTTCCTGGAAGAGCTCCCAGAAGATAACCTCTCCTCCTGGGCCACC TGGGGCACCTTACCACCGGAGCCGAATCTGGTCCCTGGGGGACCCCCAGTTTCTCTTCAGACACTGACCTTCTGC TGGGAAAGCAGCCCTGAGGAGGAAGAGGAGGCGAGGGAATCAGAAATTGAGGACAGCGATGCGGGCAGCTGGGGG GCTGAGAGCACCCAGAGGACCGAGGACAGGGGCCGGACATTGGGGCATTACATGGCCAGGTGA (SEQ ID Nθ:l)
Table 2
MAGPERWGP LLCLLQAAPGRPR APPQ VTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEY DYLFEVEPAPPVVLTQTEEILSANATYQ PPC MPPLDLKYEVAF KEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVI T MGNPWFQRAKMPRA DFSGHTHPVATFQPSRPESVNDLF CPQKELTR GVRPTPRVRAPATQQTRWKKD AEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAP V PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGT PPEPNLVPGGPPVSLQTLTFCWESSPEEEEΞARESΞIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 2)
The nucleic acid of Table 1 encodes the 520 amino acid sequence (SEQ ID NO:2) shown in Table 2. Signal P and Psort results predict that CRF2-13 protein contains a signal peptide, and is likely to be localized to the plasma membrane with a certainty of 0.460. The most likely cleavage site for a CRF2-13 polypeptide is between amino acids 246 and 247, at:AGG-VI. The CRF2-13 amino acid sequence is related to other previously described interleukin- binding proteins. The relationship is schematically represented in FIG. 1. The CRF2-13 amino acid sequence of SEQ ID NO:2 has 40 of 111 amino acid residues (36%) identical to, and 56 of 111 (50%) amino acid residues similar to, the 231 amino acid residue human interleukin 22-binding protein CRF2-10 (gi|15212826|). Similarly, the CRF2-13 amino acid sequence has 32 of 86 amino acid residues (37%) identical to, and 43 of 86 (49%) amino acid residues similar to, the 130 amino acid residue human interleukin 22-binding protein CRF2-10S (gi|15212830|). Moreover, the CRF2-13 amino acid sequence has 41 of 142 amino acid residues (28%) identical to, and 58 of 142 (39%) amino acid residues similar to, the 130 amino acid residue human interleukin 22-binding protein CRF2-10L (gi|15212828|).
CRF2-13 polypeptide also shows homology to the amino acid sequences shown in the BLASTP data listed in Table 3A. Homologies are calculated according to the method of Altschul and coworkers (Nucleic Acids Res. 25:3389-3402, 1997).
In all BLAST alignments herein, the "E- value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the IIT BLAST analysis, matched the Query IIT sequence purely by chance is the E value. The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. Blasting is performed against public nucleotide databases such as GenBank databases and the GeneSeq patent database. For example, BLASTX searching is performed against public protein databases, which include GenBank databases, SwissProt, PDB and PIR.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/.
Table 3A. BLAST results for NOV10
Gene Index/ Protein/ Length Identity Positives Expect Identifier Organism (aa) (%) (%) gi 115212826 gb interleukin 231 40/111 56/111 2e-08 |AAK85714.1 22-binding (36%) (50%) (AY040566) protein CRF2- 10 [Homo sapiens] gi|15212830 gb interleukin 130 32/86 43/86 2e-05 |AAK85716.1 22-binding (37%) (49%) (AY040568) protein CRF2- 10S [Homo sapiens] gi 115212828 gb interleukin 263 41/142 58/142 3e-05 |AAK85715.1 22-binding (28%) (39%) (AY040567) protein CRF2- 10L [Homo sapiens] gi|432 |eιrib|CAA interferon 560 40/170 75/170 0.001 48484.1| receptor type (23%) (43%) (X68443) 1 [Bos taurus] gi|163188 gb A alpha- 560 0/170 75/170 0.001 AA02571.1 interferon (23%) (43%) (L06320) receptor [Bos taurus] The homology of these sequences are graphically depicted in the ClustalW analysis of Table 3B.
Table 3B. ClustalW Analysis of CRF2-13 Protein
1) CFR2-13-EX ( SEQ ID NO : 2 )
2) gi 115212826 I interleukin 22-binding protein CRF2-10 [Homo sapiens] (SEQ ID NO:XX)
3) gi j 15212830 j interleukin 22-binding protein CRF2-10S [Homo sapiens] (SEQ ID NO:XX)
4) gi|15212828|interleukin 22-binding protein CRF2-10L [Homo sapiens] (SEQ ID NO:XX)
5) gi 1 321 interferon receptor type 1 [Bos taurus] (SEQ ID NO:XX)
6) gi 1163188 I alp a-interferon receptor [Bos taurus] (SEQ ID NO:XX)
1 10 20 30 40 50
CRF2-13-EX MAGPERWGPLLLCLLQAAPGRPRLAPgQNVT|3LSQNFSVYJ33WLP@ gi 1 1521282 MTyiHJKHCFfflRFIil S -ij-aHMGVAlBT gi 1 1521283 MMBKHCFJIGFLI S -I331ΠGVA51T gi 1 1521282 MMEKHCFHIGFLI S -133-CilGVABiτ gi I 432 I MLALLGATTLMIiVAflRWVLPAASGEAN KBEMVEIHI IDDNJ33IKWNS S S gi 1163188 1 MLALLGATTLMLVAGR VLPAASGEAN KØE VEIHIIDDNSSJKWNSSS
60 70 80 90 100
CRF2-13-EX GNPQDVTYFVAYQSSPTRRRWREVEECAGTSE LCSMMCLKKQD YNKFK gi 1 1521282 Q§TH ESLJjgPQRVQ Q|R FH|J]lLQWQP gi 1 1521283 Q@TH ESLi3PQRVQ[3Q@RNFH|J]lLQWQP gi 1 1521282 C TH ESL|2PQRVQJ3Q@RNFH|ϊ]lLQWQP gi I 432 I E§VKNVTFSADYQILGTDN-WKK SGCQHITSTKCNJ sgVELEJϊlVFEKIE gi 1 163188 | E§VKOTTFSADYQILGTDN-WKKLSGCQHITSTKC J3S§VELEJJlVFEKIE
110 120 130 140 150
CRF2-13-EX @RVJ3τVSPSgKSP VESEY DJ LFEVEPAPPVLV TQTEEI SAN- gi 1 1521282 @— j^L' S JjSgVQYKø gi 1 1521283 @— jggi is iB33voγκfl gi 1 1521282 |— |0L' |S BΠUVOYKHM FSCSMKSSHQKPSGCW g-i I 432 I RIJ23E STWYEVEPFSPilLEAQBGPPDVHLEAEDKAI ILS I SPPG gi 1 163188 I RI^E TSTWYEVEPFJSPELEAQØGPPDVHLEAEDKAI ILS I SPPG
160 170 180 190 200
CRF2 -13 -EX ATYOLPPCMPPLDLKHEVAFWEGAG NKTLFPVTPHG- gi 1 1521282 HGORCUEffl K^DCWGTQELS- gi 1 1521283 HGOROiBigSl κJ|DC GTQELS- gi 1 1521282 OHI SπNFPGCRTLAKBlGORθlCTl K0DCWGTQELS gi I 432 I TKDSI AMDRSSFR0SWlESiSSSLEERT[ τVYPEDKIYKLSPEITYC gi 1 163188 | TKDSIMWAMDRSSFRHSWlRB3SISSSLEERTigτ-yYPEDKIYKT.,SPEITYC
210 220 230 240 250
CRF2-13-EX QPVQITLQPAASEHHgLSARTIYTFSVøKYSKFSKPTCFLLEVPE gi 11521282 gDL0S rSDIQEJJ3 gi 11521283 gDL0S@TSDIQEg gi 11521282 gDLøsg SDIQEg gi|432| LKVKAELRLQSRVGCYSPVYgiN0TgRHKVPSgENIQINADNQIYVLKWD gi 1163188 I L VKAELRLQSRVGCYSPVYgiNjjTgRHKVPSgENIQINADNQIYVLK D
260 270 280 290 300
CRF2-13-EX ANWAFLVLPSLLILLLVIAAGG VIWKTLMG
Figure imgf000010_0001
gi I 432 I YPYENATFQAQWLRAFFKKIPGNHSDKWKQIPNCE VTSTHCVFPREVSS gi 1163188 | YPYENATFQAQWLRAFFKKIPG HSDK KQIPNCENVTSTHCVFPREVSS
310 320 330 340 350
CRF2-13-EX -NPWFQgAKMPP LDF@GHTHPVAτSQBsRPESVNDLFLCBQKEIiTRGVR gi 11521282 — BBG|J25S SSGS @E|^-TPRSTJJ^E0KB DEΞSMNITQENG
Figure imgf000010_0002
gi 11521282
Figure imgf000010_0003
gi I 432 I GlSEvSSiSSNGNGTgFEE-EKEfN EMKjilB Ft35SlSVKSifiTD gi 1163188 I RGlBΪVJ2SSSSNGNGTIFillE- EKE[|NTE KJil0 FJ55Bl SVKSJJTD
360 370 380 390 400
CRF2 -13 -EX PTPRVRAPATQQTR KKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQE gi 1 1521282 SLJJVILHAPNLPYRYQ KElφ^IEDYgEgLøRJjFIINJJjSLEKJjjQ gi 1 1521283 AKGL gi 1 1521282 SL0VILHAPNLPYRYQ KEKJ^IED SEøLgJRJSFII-^S EKllQ gi I 432 I DSJJHVSVGASE ESEJSM|VNQ 0P2IBEJ5IFWE;BJTSNAER gi 1 163188 | DSJjHVSVGASE ESEJJjMf NQLøpøløEBlFWEiJiTSN gR
410 420 430 440 450
CRF2 - 13 -EX HQAPGHSEAGGVDSGR@RAPLVPSEGSSAWDSSDRSWASTVDSSWDR- gi 1 1521282 jg^ gGAHRAVEIEAJj BHSSS^VgJEIYQP gi 1 1521283
Figure imgf000011_0001
gi 143 1 [S5L|3KRTN-FIFPD2KJ3LTVEBKSRALIENDRRNKGSSFSDTVCEKTKP gi 1 1631881 |2SLJ3KRTN-FIFPDJJKJ3LTVBSEK0RALIENDRRNKGSSVSDTVCEKTKP
460 470 480 490 500
CRF2-13-EX AGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT gi 1 1521282 gi 1 1521283 gi 1 1521282 gi I 432 1 GNTSKT LIVGTCTALFSIPWIYWSVFLRCVKYVFFPSSKPPSSVDEY gi 1 163188 | GNTSKTWLIVGTCTALFSIPWIYWSVFLRCVKYVFFPSSKPPSSVDEY
510 520 530 540 550
CRF2-13-EX WGTLPPEPNJJVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWG
Figure imgf000011_0002
gi 1 1521283
Figure imgf000011_0003
gi I 432 I FSDQPLRNL|jLST|EEQT|3ggFI0ENASIITEIEETDEIDEVHKKYSSQT gi 1 163188 | FSDQPLRNLjjLSTgEEQTJ^FløENASIITEIEETDEIDEVHKKYSSQT
560 570
CRF2-13-EX AESTQRTEDRGRTLGHYMAR
Figure imgf000011_0004
gi I 432 I SQDSG YSNEDENSGSKISEEFPQQDSV gi|l63188| SQDSG YSNEDENSGSKISEΞFPQQDSV
The presence of identifiable domains in the protein disclosed herein was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the ProDom or Interpro number by crossing the domain match (or numbers) using either the Interpro website (http:www.ebi.ac.uk interpro/) or the ProDom database (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/jj/prodomsrchjj.html). Tables 3C-3E list the domain descriptions from DOMAIN analysis results of CRF2-13 polypeptide using Pfam (Table 3C) and ProDomain (Tables 3D and 3E). This indicates that the CRF2-13 protein sequence has properties similar to those of other proteins known to contain these domains.
Table 3C Domain Analysis of CRF2-13 Protein gnl |Pfam|pfam01108 Tissue_fac, Tissue factor (SEQ ID NO: XX) CD- Length = 293 residues, 61.1% aligned Score = 37.0 bits (84), Expect = 0.003
Query: 9 PLLL--CLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRR 66
Sbjct: 19 TLLLGWLLAQVAGAAGTTEKAYNLTWKSTNFKTILEWEP KPINHVYT —QISTRSG 73
Query: 67 RWREVEECAGTKELLCSMMCLKKQDLYNKFKGRV RTVSPSSKSPWVES-EYL 117 1+ +1 . .I + I. + +1.+ + I.I. +1 + 1+ I 1+
Sbjct: 74 N K--NKCFYTTDTECDLTDEIVKDVTQTYLARVLSYPARNDQTTGSGEEPPFTNSPEFT 131
Query: 118 DYL FEVEPAPPVLVLTQTEEILSANATYQLPPCMPPLDLKYEVAFWK-E 165
II II + + ++ I 1 + + 11 1 + +11 Sbjct: 132 PYLDTNLGQPTIQSFEQVGT LNVTVQDARTLVRR GTFLSLRDVFGKDL YTLYYWKAS 191
Query: 166 GAGNKT 171
Sbjct: 192 STGKKT 197
Table 3D Domain Analysis of CRF2-13 Protein
PD338678 (Q9UHF4_HUMAN 36-246) COAGULATION FACTOR III PALMITATE TISSUE LIPOPROTEIN SIGNAL GLYCOPROTEIN TRANSMEMBRANE PRECURSOR (SEQ ID NO: XX)Score = 101 (43.3 bits). Expect = 0.003 Identities = 33/118 (27%), Positives = 50/118 (41%) Query: 24 LAPPQNVTLLSQNFSVYLTWLPGLG-NPQDVTYFVAYQSSPTRRR REVEECAGTKELLC 82
I M+MM I I I I ill I I +++I II I
Sbjct: 37 LPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIY-GQKKWLNKSECRNINRTYC 95
Query: 83 SMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILS 140 + + I +++ +I+ + + I I H j + j i + i i |+ +|
Sbjct: 96 DLSA-ETSDYEHQYYAKVKAIWGTKCSK AESGRFYPFLETQIGPPEVALTTDEKSIS 152 Table 3E Domain Analysis of CRF2-13 Protein
PD008555 (INRl_MOUSE 19-216) RECEPTOR TRANSMEMBRANE GLYCOPROTEIN PRECURSOR CHAIN SIGNALINTERFERON-ALPHA/BETA IFN-ALPHA-REC (SEQ ID NO: XX) Score = 98 (42.1 bits), Expect = 0.007 Identities = 46/207 (22%), Positives = 88/207 (42%)
Query: 14 LLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRR REVEE 73
+1 +1 I I 11+1+ + + + I I + 11+ I++ +1 +1 I
Sbjct: 19 VLPSAAGGENLKPPENIDVYIIDDNYTLK SSHGESMGSVTFSAEYRTK-DEAK LKVPE 77
Query: 74 CAGTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSP VESEYLDYLFEVEPAPPVLVLT 33
I I I I ++I I + III +1 I I I + + ' +11 + I
Sbjct: 78 CQHTTTTKCEFSLLDT-NVYIKTQFRVRAEEGNSTSSWNEVDPFIPFYTAHMSPPEVRLE 36
Query: 134 QTEEILSANATYQLPP CMPPLDLKYEVAF KEGAGNKTLFPVTPHGQPVQITL 86
++ + + + | | + 1 + 1 ++ + + 1 | + + + i
Sbjct: 137 AEDKAILVHIS PPGQDGNM ALEKPSFSYTIRIWQKSSSDKKTINSTYYVEKIP-EL192
Query: 187 QPAASEHHCLSARTIYTFSVPKYSKFS 213
I + +11 + 1+ 1+ l+l +1
Sbjct: 193 LPETT—YCLEVKAIHP-SLKKHSNYS 216
Growth factors such are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Cytokines (e.g., lymphokines; interleukin and interferon) are a unique family of growth factors. A number of receptors for lymphokines, hematopoeitic growth factors and growth hormone-related molecules share common domains, and can be divided into families. The cytokine receptor class 2 family includes interleukin- 10 receptor; interferon-gamma receptor; interferon- alpha/beta receptor; and tissue factor (Konigsberg et al, Nature 380:41-46, 1996). The presence of regions of CRF2-13 polypeptide related to domains found on tissue factor and coagulation factor III palmitate tissue lipoprotein signal glycoprotein transmembrane precursor are consistent with the localization of CRF2-13 polypeptide to the plasma membrane and the assignment of CRF2-13 polypeptide to the cytokine receptor superfamily. The presence of a region of CRF2-13 polypeptide related to interferon 1 receptor transmembrane glycoprotein precursor signal chain interferon alpha/beta EFN-alpha receptor reinforces this assignment. The nucleotide sequence shown in Table 1 was identified as part of the genomic DNA sequence shown in Table 4:
Table 4
1 GAAAGAGAGA GAAAAAAGAA GGAAGGAAGG AAGGAAGGAA GGAAGGAAGG
51 AAGGAAAGAA AGAAAGAAAG AAAGAAAGAA AGAAAGAAAG AAAGAAAGAA
101 AGAGAGAGAA AGGAAGGAAG GAAGGAGAAA AGAAAGTCAA CAGTCAACAT
151 TTCAGAGATC CCAAGATACC AACACTGACC GTGCCTGCTG CTCTTCCATC
201 CTCCTCCACC CTGCGCCTTT GAGGTGGAAT TGCGTCCTCT GTGAGCAGGG
251 CTTTGTTAAG AGATCCTAAT TAAGGCCAGG CACAGTGGCT CATGCCTGTA
301 ATCCCAGCAC TTTGGGAGGC TGAGGTCACC TGAGGTCAGG AGTTCAAGAC
351 CAGCCTGCCC AACATGGTGA AACCCCATCT CTACAAAAAT TAGCTGAGCA
401 TGATGGCAGG TGCCTGTAAT CCCAACTACT TGGGAGGCTG AAGTGAGAAA
451 A AGCTTGAA CCCAGGAGGC GGGGTTGCAG TGAGCCAAGA TCACACTATT
501 GCATTCCAGC CTGGGCGACA GAGCTTTTGT CTAAAAAAAA AAAAAGAAAA
551 AAAATCCTGA TTAAGCAGAA GCCTTGATGC TAGTCCCAGA AGCATCCTGA
601 AATTTCCAAA AGAAATTTCC CCCGCGGTTA AACTCAGAGC AACTTTTGGA
651 CCCACCAAGC TCTGTGAAAA TCATTTTCTC TTCCAAAAAC TGATGGGACC
701 AAAGCTGATC CCAGTTTCAA ATAATTATCA AAAAATTGGA AACGAAATAT
751 GATCAGAAAA GAAGAAAGTT GAAAAAGAAA ATCCTTATCA CCCAAAGACA
801 ACAACCATTA ATATTTTGGT AATTATTATT ACAAATATCT TTCTATGCAT
851 ACAGACAGAC TCACACACAC ACACACACAC ACACACACAC ACTTTTTTTT
901 TTTTTTTTGA AACTGAGTTT CACTCTGTCG CCCAGGCTGG AGTGCAGTGG
951 CGCGATCTCG GCTCACTGCA ACCTCCGCCT CCTGGGTTCA AGCGATTCTC
1001 CTGCCTCAGC CTCCCTGATA GCTGGGATTA CAGGTGAATG CCACCACGCC
1051 CGGCTGATTT TCTGTATTTT TAGTAGAGAC GGGGTTTCAC CATGTTGGCC
1101 AGGCTTGTCT CCAACTCCTG ACCTCAGGCG ATCCACCCGC CTCACCCTCC
1151 CAAAGTGCTG GGATTACAGG CGTGAGCCAC CGCGCCCGGC TACACACACA
1201 CTTTTTTAAT GGGCCTATGT TTTAGCACTC GCTTTTCTGT TTCTCAGTGT
1251 GTTGCAAACA CCTCGGTGTC GATACACACC ATTCGGCAAC GTCCTCCTAA
1301 AGGGCCGCAT AATATTGCGC GTCGTGGCGT GTGCCTTACT GGGAAGCTAC
1351 TGCTGTCCAG GTGAACACCA CAGCCTTCGG GGTCAGAAAG ACAGCTTTCC
1401 CCAGAACAAG CACCTGAAGC TCTGGGGCCT GCCGCTCCCC GGGTAGAGAA
1451 GTACGTGGAG AAGGGCAGC CGGATCCGCC GGGATCCCCG GGGGCATTAA
1501 AGGGAATCGC GTGTGTAAGG CGCGGAGCTC AGCATCCGGC TCAGAAACGC
1551 GCTCGGATCC CGCCAATGGC ATTGAGGCCG CGTAGCCAAA CCGGCCTTGA
1601 ACTCTCCCTA ATCCTGCCAA AATGGCCCGT CCTGGAGCAC TGGACTGGCC
1651 GTGGGTTATT GATCATCAGC CGGTTTCTTC CCCTCCCCTG CCCTTCCCCC
1701 GTGCACGGAT TTACTGATTT TTTTTTCCGG GAATTGAGTA AAACAAAACT
1751 AAGTGCAGAT GAAGCAGAGG TACGGGCGAG TTTCGAGCGC GGGGACCGGC
1801 GCGCTCCCCC CCCCCTCCCC CCGCGGCGGG GCTGTCCCCA GGGACCTTCT
1851 CAGTGAATCC TAGGCGGCAG GGACGGGCCC GCGGCTCTGC GGGCCATTGG
1901 CTGCCGACTG CGTCACCTGC CCGCGGTGGG CTAGGAGACG GGAGGCGGGA
1951 GGCGGGAGGC GGGGACCTGG GTCCGGGCGG GGACGCCGCG GCAGGAAGGC
2001 CATGGCGGGG CCCGAGCGCT GGGGCCCCCT GCTCCTGTGC CTGCTGCAGG
2051 CCGCTCCAGG TAAGGGCGCG GGGCCGCGGG AGGGAGGGGG AAGAGGGCTC
2101 CCCGGGCCGG GCCGCGCCTA CCCTCGGACC CAGAGCTCCT GGGACAGGCA
2151 CGGGGTCCGC AGCCACCCGA GCCGGGTGCG AATCGGCCCT GCCTACGCGC
2201 CCCCAGTTTG CTTCTTCCCA GGACTGAACA GAACCGGGTC TTTGATATTC 2251 CTCTCCCGCA GGAAACGAAT CCAGTTTCCT AATGCTTCCA GCTTCAGGAG
2301 AACTGGAGAA AAAAGACAGC GGCAGTTTGA TACTGCATAT TTTTTAATAA 2351 AGTGCTTTTT AATGTTTCCT AAAGAAAGCA CTGATCCCTG CGTGAAAACC 2401 ACACTTGACC CTAAAGTGTG GACAGCAGGG AAAGTGGGAC CGATTGATGT 2451 CCCTTCCCGT TCCTGCCAGG CCTCTGGTGG GACGGAGCTC TGGTCGCCTG 2501 TGCCCTGCTT TCTAACAAGA CGGCTTTCTT TTGGTGGTGG TTGTTGTTTT 2551 GTTGTTGTTT TGTTGTTGTT GTTGTTGTTG TTGTTTTCCC ACCTCTACTG 2601 ATGAGTAAGG TGTCAGGTAC AAAATTCCTC GCCGTAGGAC CCAACCACCA 2651 AACCTCACCG CCCACGACTC CAACCGAAGC AGGGAAGAGA AGGTCCAGAA 2701 ATCGCCCCCA GGATATTTTC CTAGTCTTGG ACTCACAGTT TAAAGAGCTG 2751 TAAAGGTCCC TGGGCATAAT CCAATCATCA TAAAAGCCTA TATTTATTCA 2801 GCAACTTCTT TGTGCCAGGC ACCGCATTAT TCTGGAAGCC TCACGACCCA 2851 GCCATCCTAG GAGGTAGATA TTATTTTTAC TTTTCCGATG GGAAAACTGA 2901 GGCTCAGAGC AATTCAGGGA ATTCCTCAAG AAGGACGGCA GAGGTGAGGC 2951 ACACAGAAGA GAGAAGAGGG GCTAAAGCAA GCCTGGCTAG CTTTTGCCTC 3001 CAGGGTAGGC ACGTGGGACA GGCTGTCCAT CCACTGGGTC ACTAGGCCAG 3051 CCAGGGATGC TCCAGCCCCC AGTGCCCACA GCAGCGTTCT CTGTGGCTGA 3101 TGAGGGACCG TGTACCTGTG TGTGGAGGGA GGGTGGGGTC TTCTGTTCCC 3151 CTTTCACTGT CAAGCCCAGA CCTTCTTGTA CTTTCACCTG ATAAGTATTT 3201 AATATACACA ACACTAACTA TGGTGTGATG ATTTAGGAGT AAGTACAGCC 3251 AGATCTAAGT TCAAATACTG GCTCCCACAC AAACTGACTG TGTAGCCTCA 3301 GGCAAGTTAG TTAGCATCTG TCTCTGAGCC TAGCGCCCTT TCCATGGAAG 3351 CAGAATGAAT GACACCTACC CCATAGGGTG GTCTGTCCCA AGGGTGATTG 3401 AGGTTTTACA TGTAAAGAGC CAAACTAGTG CCTGGCATCC TTTGAAGGCT 3451 TCATAGAGGA AAGTTGCTCT AGCTGCTGTT TTTCTCATGT GACCTAGCTC 3501 GAATCTGGGG ACTGTCCTGC CCATAGGATA CCTTACAAGT GGCTTGCAGA 3551 CAGCCTGGTC TCCTGCTGGT CACCCGTTAG GAAGTCCAGA AGCTGGGAGT 3601 AGTAATAGCA CTAGCCTCGT GGTGATACAG TCCCAGCTAG AGGACACAGG 3651 ATGAGGTGGA AGCAGGCACC CACTTTTGGG TCTAAAAGGT GATGGGTAGG 3701 CAGCCGAGGC TGGGGACAGC CATCCACAGA ACTGGACCCT CCCTCCCTGA 3751 TGCCATTTTG CAACCCGTAT GGATTTCCAT CATGGCACAT GGGACACTTC 3801 AGGACCCTGA ATTCTCCATG GGACCATGAG CTCCTATAGG GCAGGAATGA 3851 AGTTGTGTTC TTCTTTGAAA CCCCTGGCAC ACCGTGGTCA ACAGATCTTG 3901 TTTGACTCGT AGTGGTCAAT AGATGGAATA GTTGGAATCA TAAAGCTCAA 3951 TAGACCCCAT GAGAACCTAG AAGACAAAGT ACAGTCAAGA GCTCGGACTT 4001 TGGAGTTGGC TAGGCCTGGA CTGAATCTGA TTCTACAACT TAATAGCTGA 4051 GAGGGCCTTG GTTTTCCCAT CTGTAAAGAT TATAATTATT ATAATGAATA 4101 CCTACCTCCT AGGGATGTAA TGAGGATTAA AAGAGAAAGT GCAGGTAAAC 4151 TGTTTAACAC AGAACCTGGC TCATAGAACA CAATACACAT TAGCTGCTAT 4201 TATTATTATT ATTATTTTAT TTATTTATTT TGAGACAGAG TCTCACTCTG 4251 TCACCCAGGC TGGAGTGCAG TGGCGCAATC TCGGCTCACT GCAACCTCCA 4301 CCTATCGGGT TCAAGCAATT CTCGTGTCTC AGCCTCCCAA GTAGCTGAGA 4351 TGACAGGCGT GTGCCACCAT GCCCAACTAA TTTTTGTATT TTTAGAAGAG 4401 ACGTGGTTTC ACCATGTTGG CCAGGCTGGT CTCAAACTCC TGACCTCAGG 4451 TGATTTGCCT ACCTCTGCCT CCCAAAATGC TGGGATCACA GGGGTGAGTT 4501 ACCATGCCCG GCCTTAGCTG CTATTATTAT CATCATCGTT ATCATCATCA 4551 TCATCACCTC GTAGATATGT CAAGGAAGAT TCCCTGGAGG AAGTGACATT 4601 TGAATCAAGT ATTTCAAAGA CTAGATGGTG AATACCAGGC AGTCAAAGAC 4651 ACCTGGGTTT AAAAACATCC AGAAGAATGC AGTGGCTTGG CAACATCGAG 4701 CAGGAAGATT GCCTGATGAG CCTGTAGGGT AGCTGTTGGG GAGAGAGCAG 4751 CAAGACGGCC TGGCCAGGCC AGGCCAGGCC ACGTCAGGCA GGGCCTCACA 4801 AACCTCAATA ACAAATGTGG ACTTTATTCT GAGGCCAAGG AAAGGGCATG 4851 AAACTGGGGA GTGGTGTAAT CAGATGCGTA TTTCAGAAGA TGAAGATTAA 4901 CAGTGAGAAG GAAAATGTGC CACAGAGGGG AATAGAGGTC AGTTAAAGGG 4951 AGTCAGGGAA AGTGTCCTCG AGACAGTGAC ATCAAAGGAA TGTGAAAACA 5001 GCAAAGGAGT GAGCCAGGTG GATATCCAGG GGCAGAACTG TTAAGGCAGA 5051 GGGAACAGCA TGAGGGAACA GCGTGTGCAA AGGCCTGGAG TTGGGAGTGT 5101 GGCTGGGGTG CTCCAGGAAG GGCAAAAAGT CCTGTGTGGA TGGAGATATG 5151 GGAGCAAGGG AGGAGTGGTG GGTCAGATTG GGTAGGGCCT TGGTGGTGAT 5201 TGTAAAGACT TTGGAGTTTA GACCAGGCAC AGTGGCTCAG GCCTGTAATC 5251 CCAGCACTTT GAGAGGCCAA GGTGGGCGGA TCACCTGAGG TCAGGAGTTC 5301 GAGACCAGCC TGTAATCCCA GCTACTCTGG AGGCTGAGGC AGGAGAATCG 5351 CTTGAACCCG GAAGGTGGAG GTTGCAGTGA GCTGAGATTG TGCCACTGTA 5401 CTCCAGCCTG GGTGGCAGCA TAAGACTCTG CCTCAAAATA AAATAAAAAT 5451 AATAAAGACT TTTGAGTTTC CCTGGAGTGA GAGGAAAGCC TTAGAGGGCT 5501 TTAGCAGAAG ATGAACATGA TCTGATTTTC ATTTTTAATC CTTCCCTGCT 5551 AATGTGGAGA ATGGACTGAA GGCAAGGTGT TTTGTATATT TGTCTGTTTC 5601 GTAGAGACAG GGTCTTGCTC TGTTGGCCAG ACTGAAGTGC AGTGGCACAA 5651 TCACGGCAGC CTTGAACTCC TGGGCTCAGG CGAAACTCCC ACCTCAGCCT 5701 CCTTACTCTC ACCATTGTGC CCTGCTAATT TTTTAAAAAA TTTATTTTGT 5751 AGAGATGTGG TCTCACTATG TTGCCTAGGC AAGTCTTAAA TTCCTGGTCT
5801 CAAATGATTC TCCTGCCTCG ATGTCCCAAA GTGCTGGGAT TACAGGTGTC
5851 AGCTGCCATG CCCGACCTGT ATTTTTTTTT TTAATGGGGA AAAAGCCTTT
5901 TAATAGTATG AGGTGTTTTC TGGTGTTTCT ACCATAAAGC TCTTCTGTAA
5951 ATCAAAATGA GAATGTAATT ATTGATAGAG CAATGACCTT AGACTACAGT
6001 GCAGACTTTT CATCTTACAT TTGGGCTCAT GAATTTTAGT ATAACTGATT
6051 ATGACAGTGT TTTTTACATA GTTATGATCT AGAGCAGAAC TGAAAACAAA
6101 ATAACACATA CTCTACATCA ATATATTCGT TCAGTAATAT CTGGGCTTGG
6151 ATGAACCTGC AGAAGTAGGT AAAGCTGTCA GATATTTTCT TAAACCAACA
6201 GAAAAGAAAT GTATATGACA GATGTTGTGT TTACTTACTT ATTTATTTAT
6251 TTATTTATTT ATTTGAGATG GAGTCTCACT GTGTCACCAG GCTGGAGTAC
6301 AGTGGTGTGA TCTCTGCTCA CTGCAACCTC CACCTCCCGG ATTCAAGCGA
6351 TTCTCCTGCC TCAGCCTCCT GAGTAGCTGG GATTACAGGC GTGCACCACC
6401 ACGCCTGGCT AATTTTTGTG TTTTTAGTAG AGACAGGGTT TCACCATGTT
6451 GGTCAGGCTG GTCTCGAACT CCTGACCTCG GGATCTGCCC ACATCAGCCT
6501 CCCAAAGTAC TGGGATTACA GGCATGAACC ACCACGCCCA GCCTGTATTT
6551 ATTTTTTTAC CACTATGGAG TCCAATATGA AATTCTCACA ACTATGCATA
6601 TACATTATTA ACATGTAAGC ACACCTAGGT ATAAATATGC ACATAGTCCA
6651 TTAATTACAT CAGGGGAATT AAAAACATAC TTTCAAGTTA AAATGAATTT
6701 TCAGGAAAAA AACTGCATTC ACAAATCTGA AATGTGAATA CAAAAATGAA
6751 ATTGTGAAAT AAATAATGAA TATAGGTGTC ACCTAAACTT CCATAGTAAC
6801 ATGCCTCCAA ATGTGGATTT AGTGATCATC CACCTTGGGA CAAGGGCTTT
6851 TGAGAGCCTC CAGCTAAATT AGGGTTCCAG TAGCAGAGTG GCTGGCAAGC
6901 CTGCCCTAAT GAATAATGCC AGCGAGCTGG GCGTGGGTAC TTACAGTGTG
6951 CCCTTCATGG AATACTTTTT TTTTTTTTTT TGGAATGGAG TCTCGCCCTG
7001 TTGCCCAGGC TGGAATGCAG TGGCACAATC TCAGCTCACT GCAACCTCGT
7051 CCTCCTGGGT TCAAGCAATT CTCGTGCCTC AGCCTCCCAG GTAGCTGAGA
7101 CTACAGCCCT GTGCCATCAT GTTCTGCTAA TTTTTGCATT TTTAGTAGAG
7151 ACGAGGTTTC ACCAAGTTGG CCAAGACTGG TCTTGAATTC CTGACCTCAG
7201 GTGATCTGCC CACCTTGACC TCCCAAAGTG CTGGGATTAC AGGCTTGAGC
7251 CACTGCGCCC GGCCCATGAA ATACTTCTTA CCTGGCGGAC AGCCTAATAG
7301 CCTAGCTGTC TAACCCATGG CTGGGGGTCC TTCACACTTG TTTATACTGG
7351 CAGACGTCCC TGTGACTCTT GTCTGATCCA TGTCCAAGTT TATGCCTGTC
7401 TGACCATTGC TCTGGCGCTG GGAGCCAGAC TGTGTTCCCA GCAACCCAGG
7451 GAAAACCAGG CCTGGGCTGG GCCTGGGTTC CTGAGATGGA AGGTGCAAAT
7501 TCAGTACACC ACCTCAATGC AAAACAAGTT CAAAGGCTTA TTACTTACAG
7551 ATCCTGAGCA GGGAAGGTGC AATGAGTAGG GAGGGTCATC CTCCATCCTG
7601 GGCTACATGA AGCGGGAATG AAGAGTCAGG CAAAAAGAAA GTGAGAGCTT
7651 GTGGCAATGA GAAGTATATT ATGTAAGGGA CTAGGGTGTG GGTCAGGTTA
7701 AGTTTGAGGG CAAATGCTTG AATGATCCCT TTAAAGGAAT GGGTGGGAAG
7751 TGGGGAGCCC AGTTTGCCGG GAGGGAGAGA TGCCTCGAAG TTCTTATCTC
7801 TGGCCACTGG CTTGGGCCAT CTGAGTGTGG CATCTACTTC TAATGCCTAG
7851 GCAGCAACCT TTGCTGTGTC ATCTCCCTTA CACAAGGTTG GAAGCAGGGA
7901 GACCGGTCAG GAAGCCTTTG GTGTAACCCA TGTTATTGTA A ATTCATTC
7951 ATTTACTCAA CAGATGTTTA TTGTGCACCT ACTATGTGCT GAGGCCATGG
8001 CAGGCAGGCT CTGGGGATGT GGCTGAGAAC AGGACAGAGC CCCTGGTCCT
8051 TGATATCCTC AAGGATGCTC CCTCCTGGAG GCCATTAGGT TCCTGTTCCA
8101 TGGTGTTCTG CTGGAACCCT CCGGTCCCAG AGTGTGCAGG AGCCTCCCCT
8151 CCTGGCAAAG GGTCTTCTCT CATGGCACAA GGGCTGCAGT ACAGCCAGTC
8201 AGTGGCTCCT GGTTCCTCAA ACTCAGTGAG CACTTGCCTG CCCTTCGTGC
8251 TGCCCCTCAG CTTGGGATGG CCTGAGTCAA GACCAGCCAG GAGCTCCAGG
8301 CTTCATGACC CCTTTCTTTC CCCCAGGGAG GCCCCGTCTG GCCCCTCCCC
8351 AGAATGTGAC GCTGCTCTCC CAGAACTTCA GCGTGTACCT GACATGGCTC
8401 CCAGGGCTTG GCAACCCCCA GGATGTGACC TATTTTGTGG CCTATCAGAG
8451 GTAGAGGAGA CTCTCTCGGC TGGTGGATGG GAAGACTGAG GGGGTGGGTG
8501 GGGGCTTGGA GGGGCTTCTC TGGGACAGCT GCACCCAGTG TGGGCAGCAC
8551 TGGCTAGCTC TCTGGGCCCT ACGGGAGATG GCATGTGGCC GGCATTTGGA
8601 GAGGGGCTTT TGATAAAGGT CTGGAGGTGG GGAAGATGTT GAATGAAGAG
8651 CAGTGTACAG GTGACCAGTC TGCCGGGGCG GGGGTAAGTC TTTGAGGAAA
8701 GTTGGTGTGG GGCATGGATG TAGCTGTGGG GGCCAGAGGA TGAAATTCTC
8751 AAGTGGCTGG ATGAGGTGCT TGGAGCTGTC CCAGCTGATC AGTGAGGCAA 8801 CTAGGTACAC GGCAGAGGAG CTGTTACCTG GGCAATTAGG CATCCCTCAA
8851 TGATCACACT TTTTTTCTCT r r rpr rprπrprpr r TTTTTGAGAC AGAGTCTTGG
8901 TCTGTCACCC AAGCTGGAGT GCAGTGGCTT GATCTCGGCT CACTGCAACC
8951 TCCACCTCCT GGGTTCAAGT GATTCTCCTG CCTCAGCCTC CAGAGTAGCT
9001 GGGATTACAG GCATATGCCA CCACATCTGG CTAATTTTTG TATTTTTAAT
9051 ACAGACGAGG TTTCTCCATG TTGCCCACGC TGGTCTCGAA CTCCTGAGCT
9101 CAGGTGATCC ACCCACCTCA GCCTCCCAAA GTGTTGGGAT TACAGGCGTA
9151 AGCCACCGCG CTTGGCCAAA TGGTCACACT TTTCCCGATG GGATCATTCT
9201 CAATTTGGAA GCCCAGGCAG CCACAGCGAA TCCAGAGAAA TCTGACAATG
9251 GAAGCAGATC CACCATCTTC GAACATAGAT GGGAATCGTT CAGAGTTCTT
9301 TAGCAGGACA GTGAGATGAT AGAAGCAGAA GCTCGGGAGG ATTCACCTGG
9351 AGTTGGTGAG GAGGGGAAAG CAGGAAGAGG AGGGGACCCA CCGTGTCCTC
9401 AGGACCCGTC CTGTGCCAGG CCAAGTGCTA AGGGCCCTAC GTGAATATTT
9451 CACTTCCTTC TCCCAATGTG ACCAGGCAGG CTCTGTGTTT TCCCCATTCT
9501 AGAGGTGAGG GGGATTGAGC ACTGTGTCAA CACATGTAAT GAACTTAATC
9551 TCACAGCAGC TCTCTGAGGA CAAGTTCAGT ACGCCTCTTT ACAGAGGAGG
9601 AGACTGAAGC ACCAAGGGTG CATGTTGCTC AAAGTCACAC AGCTGGGCGT
9651 AGTATGGCTG GAATAAATTT ATTAAGGAGT TGAAAGTCTA TCCTCTAGGA
9701 CCAAGCATGG TGGCTTACAT CTGTAATCCC AGCACTTTGG GAGGCCGAGG
9751 - TGGGTGGGGA GATTGCTTGA GTCCAAGAGT TCCACACCAT CCTGGGTAAC
9801 ATGGTGAAAC CCTGTCTCTA CAAAAAAAAA AAATACAAAA AATTAGTGAA
9851 GTGTAGTAGC ATGTGCCTGT GTTCCCAGCT ACTTGGGAGG CTGAGGTGGG
9901 AAGGATCACT TGAGCCCAGG AGATGGAGGT TGCAGTAACA AAGATCACAC
9951 CACTGCACTC CAACATAACA ACAGAGCAAG ATCAAAAGGG TTTTTAGCTC
10001 CCACTGAACG CCNCGTCATA NCCTTAGGTN NNNNNNNNNN NNNNNNNNNN
10051 NNNNNNNNNN I^NNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN
10101 N NN NN NNNNNNNNNN NNNNNNNNNG AACAACAGAG CAAGATCCTA
10151 AAAAGAAAGA AAGTCTATCC TCTGAACTTC TATGATATTT TTCATGTCTT
10201 TTATACATTA GAATGGTGAT ATTCTAATTA TATAATTTTT TTCATTTGTT
10251 AGTTGGAATT ATTTTATAAA GAGATGTATC CTCTCATCTG GTATTTGATA
10301 TCCAGTCATA CTATTCAAAT AGGCAAGAGA GGATAAATGC TTAATTTTTT
10351 TCCTTTATCA ATTTTCAAGA TAATGAATTG GTTCCTTATC ATCTCCCAAA
10401 GGTGATTGCT AGTTTATTAT TATCATTATG AACTCAGGCA TTTAAACACA
10451 TTTGGTGGTT TCAGTCTATT GCGACGTACT CTGCTCATTG AAGCTTGAAT
10501 TGCCTCATCT CTGTCCAGTG GGAGTCTCAT CAAGTTTGCT CCTGAGTCCT
10551 TTTAACTTGA CCCTAGTGGT CAAGTTAAAT CTTTCCAGAT TTAACAGATA
10601 CCTTTCCAGC TGTCCATTAC GACAAGATGT TCCAGGTCCC TCTGGTACAA
10651 TTCCTGACCT AAAACCTGCA GTCAGCCATT TCTCCATTTA GTAAGAAATG
10701 GTTATAAAGA CTATAATCTG CATGCTAGCT ATGCTGATCA CTACTTAGCT
10751 ATTGCTTTTG GTGTTTTCAG TGAACAGAGT GATGTGTGTA TACCACATAG
10801 ACACACACAT GTACATACTT TTTTTTTTTA GACAGAGCTT CACTCTGTCA
10851 CCCAGGCCAG AGTGCAGTGG CATGATCTCG GCTCACTGCA ACCTCCACCT
10901 CCTGGGTTCA AGAGATTATC CTGCCTCAGC CTACTAAGTA GTTGGGATTA
10951 CAGGCGCCCA CCACCATACC CGGCTAATTT TTGTATTTTT AGTAGAGACG
11001 GGGTTTCACC ATGTTGGCCA GGCTGGTGTC GAACTCCTGA CCTCAAGTGA
11051 TCTGCCCCCC TCGGCCTCCC AAAATGCTGG GATTACAGGC ATGAGCCATC
11101 GCACCCAGCC TACATGTACA TAATTTTTAA GATAAAATGC CTAATGAGTT
11151 ATACGGGTGC TTCCCATCTA AATTTAGTTC CTTAGGATTT TTACCTGACT
11201 TCTATGGTAC ATCTATATTT TCTTTCTTTC ACACTGAGAA TCCTGTTTCT
11251 CAAGGACAGG GGACATGATA GAACTAGAAT GACCCATAAT TACTCATTTT
11301 CTTTATCCCA AAACATACAT ACTTGCCTCT TAATAGTTTC TTGCTCTTTT
11351 CGCCCAAAGG GTTTGTGATG GTCAATATTA GGTGTCAACT TAATTGGGTT
11401 GAAGGATGCC TAGATGGCTG TTAAAGTTTT GTTTCTGGGG GTGTCTGTGA
11451 GGGTGTTGCC AGAGGAGACT GACATTTGAG TCAGTGGACT GGGAATGGAA
11501 GACTCGTCCT CACTCAGTGT GGGTGGGCAC AACCCAACTG GCTGCCAGGC
11551 TGGCTGGAAA GCAGGTGGCA GATGGTGGGA TAGCTTCACT TGCTGGGTCT
11601 TCCAGCTTCC TTCTTTCTCC CGTGCGGGAT GCTTCCTTCT GCTCCTCCTG
11651 CCCTTGAACA TCACACTCCG GGTTTTTTGG CCTTTAGACT CTTGGACTTA
11701 AGTTAGTGGT TTGCTGGGGG CTCTCGGATC TTTGGTCACA GACTGAAGGC
11751 TGCACTTTCA GCTTCCCTGG TTTTGAGGGT TTCAGATTCG GACTGAGTCA
11801 CTATGGCTTC TTTCTTTCCC ACCTTGCTGA CGGCCTATCG TGGGACTTCG 11851 CCTTGTGATC GTGTGAGCCA ATTCTCCTTA ATAAACTCCC TTTCATATAT 11901 ACGTATAACC TATTAGTTCT GTTCCTCTGG AGAACCCTGA CTAATAAAGG 11951 GTTGTTGCTT TTTCTTTAAA ATCTAGTAAT TTTATTTGAC TGTGTGTTGG 12001 TATTGCTCAT TCATTCTGAG TTGATATTTT TAGGCACTCA ATATTCTCAC 12051 TTAATACATG GTTCCAAGGC ATTTTTATTT TAGGAAGGTT TTCTTAAATT 12101 ATAGTTTTAG TATTTGTTCT ATTCTCTTGT TTTGATTTTC TTCTTTAGGG 12151 ACTCATATCA CTTGTATGTT GGATCTTCTT TTTCTGTGTT CAGTATTTGT 12201 CTTTTGGGCA CAGAGACTCA CACCTATAAT TCCAAGACTT TGTGAGGCAT 12251 AGGTAGGAGG ATCGCTTGAG CCCAGGAGTT TGAGACCAGC CTGGGCAACA 12301 TGGTGAGGCC CTGTCTCAAA TTAAAGAAAA AGGAGAGAAT ACTTGTCTTT 12351 TTCTTTCAAA TGCCTTTTAT CTGTCTGTCT ATCTACTATT CTGCTCTCTA 12401 AATGAAATAG GTTTCACTCT TGAGTTTTTA AAAAACTGTG TGCTTCCATG 12451 TGTGAGATTA TTCAACATCT TATTTGTAAT CTTTCTCTTG GTTACATTTA 12501 TTTTTCCTGA AAACTCTAGT CTGCTTTTAG CTGACATGTT TGTAGCTAAG 12551 AGCGCACATT TCTTATCATA GCTTGCCGTG CTGAATTAAT TCCAATTTTC 12601 TTTTAAAACC AACATTATTG AGTTAAAATG TATATAGAAT AAACTGTTCC 12651 CATTTTAAAG TATACAATTT GATGAGTTTT GACAAAAGTG GGCACCCACG 12701 TACCCACCAC CACAATCAAG ATGTAAGACG TTCTCTATCA CCCCAGAAAG 12751 TTCCCTCATC CACTTTGCAT TCAGGCCTCC AGATCTAGGC AACCACAGAT 12801 CTGCTTTCTG ACACTGTGGA TTAAACTTTG CCTGTTCCAG AATTTCATAT 12851 AAATGGATGT GTATAGTATG TACCCTTTCG TGTCTGGCTC CTTTCCCTCA 12901 GCATAATGTT TCTGAAATTC ACCCACATTG TTACATGTAT CAGTAGTTAA 12951 TTCCTTTTTA TTGCTGAGTA GTAATGCCAT TGTATGACTA TGTATGACAT 13001 TTGTTAATCC ATTTTCCCGT CAGTGGATAT TTGGGTTGCT TCCAGTTCTG 13051 GGCAGGTATT CATTTGCTAG GGCTGCCATA TGCTTGCCCT CTGGCCTCCC 13101 AAAATTTGTG TCCTTTTCAT ATGCAAAATA CATTCACCCC CTCCCAACAG 13151 CCCCAAAACT CTCTTTTTTT TTTTTTTTTG AAACAGAGTT TTGCTCTTGT 13201 TGCCCAAGCT GGAGTGCAAT GGTGTGATCT CGGCTCACTG CAACCTCTGC 13251 CTCCCGGGTT CAAGAGATTC TCCTGCCTCA GCCTCCTGAG TAGCTGGGAT 13301 TACAGGCATG CGCCACCACG CCTGGCTAAT TTTTTATATT TTTAGTAGAA 13351 ATGGGGTTTC ACCGTGTTAG CCAGGCTGGT CTTGAACTCC TGACCTCAGG 13401 TGATCCGCCT GCCTTGGCCT CCCAAAGGGC TGGGATTACA GGCATGAGCT 13451 ACTGCACCTG GCTAGCCCCA AAACTCTTAA CCCATTTCAG CATCTACTCT 13501 AAGTCCAAAG TCTCATCTAA ATCAGGTATG GGTGTGACTG GAGGTGTTAC 13551 TCATCCTGAG GCCAAATTCC TCTCCACTTA TGAACCTGTG AAACCAGACA 13601 GGTTATGTGC TTTGAAAATA AAGTGATGGG ACATGCATGG GATAGACTTT 13651 CCCATTCCAA AAGAGAAAAA TAGGAAAGAA GGAAAGAGTG ACAGGTCCCA 13701 AGCAAGTCTA AAACCTCGCA GGGCAAATTC CATTAGATTT TAAGTTTCAA 13751 GAATAGCCCT CTTTGGCTCA GTGCTCTGCC CTTTGGGCCC ACTGGGGCGG 13801 CAGCCCTATC CCCTTTGCCC TGGGTGGTGA CCCTACCCTC GAGTCACTGG 13851 TTAGCAGCAG CCTAGCCTGC TGAAACTAAG GAGGGGACAG TGTTGCCTCC 13901 AGGTCTTTGG TGGCAGTGAC AACCCTGCTG ATCTCTGAAT CATCTTCCAG 13951 GAAATTTTTC CCTATACTTG AAGGATATTG CGTGTTCACA GCCAAATAGC 14001 TCCAGCTCTT GTCCCTTTCT TTAGAATCCC AGAAGTCCAA CAGCCTTCCT 14051 TCATTCTGTC CCATCTCTGT CCCCTTTAGT CAAAGCTGGA AGTGCCTCTG 14101 CTGGTATAAT CCCATCAGTA TGTCTAATTT CTGCTTAAAT GGCTGATTAA 14151 GTCTATGAGT TGCACCTCTG ATCTCTTTAT CAAAAGGTTG TTCTAGCCAC 14201 AACCTTAGTG TCCTCCCCAG AACATGCTTT CTCATTTTTT TTTTTGCAAT 14251 GTGGATAGGC TGAAAATTTT CCAAAGCTTC AAGTTCTAGT TCCTTTTGGC 14301 TTACCAATTC TTTTCATATA TCTCTTCTCT CACATTTTAC TATAAGCAGT 14351 AAGAAGAAAC CAGGTTGTAC CTTCAGCACT TTGCTTAGAA ATCTCTTCTG 14401 CTAAGCATCC AAGTTTATGT CTTTTAAATT ATCTTTTTGT TATTTATTTT 14451 ATATTATCAT TTTTGAGATG GCTAGCCAAT GATCTTTTAA CTTCTAATTT 14501 CTGCAAAACA CTAGAAGACA ATTCAACCAG TTCTTTGCCA CTTTATAACA 14551 AGGATCACCT TTCCTCCAGT TTCCAATAAC ACATTCCTCT TTTCCACCTG 14601 AGACCTCACC AGAATCACCT TTAATGTCTA TATTCCTACC AATAGTCTTT 14651 TTAAGGCAAT ATAGGCTTTC TCTAACATGC ACTTCAAACT TCAAGATTCT 14701 ACCCATTATG CAATTCCAAA GCCACTTCCA CATTTTTAGG TATTGATTAC 14751 CTCAGCACCT CATTTCTGGT GCCCAAATCT GCACTGGTTT GCTAGGGCTG 14801 CCATAACAAA GTACGACAGT CTGGGTAAAC AACAGAATTT TATTTTCTCA 14851 AAATTCTGGA GGTTGGAAGT CCAAGGTCAA GGCGTTGCTA GGTTTAGTTT 14901 CTCCTGAAGC : CTCTCTCCTT ' GGCTAGCAGA . TGGCTGCCTT 1 CTTGCTGTGT
14951 CCTCACGTGG i CTTTTTCTCT ' GTGTGTGTTC 1 ACTCTGGTAT ' CTCTTCCTCT
15001 TCTTACAAGT ACACCAGTCC TACTGGATTA GGGCCCCAGC CTTATTACTT
15051 CATTTAACCA TAATTACCTC TTTAAAGCTC TTATCTCAAA ACACAATACC
15101 ACTGGGGATG AGGTCTTCAA CATATGAATT TTGGGGGAAC TCAATTCGTC
15151 CATAATAGGG CTATTATGAA TTAAGCTGCT GTGAACATTC ATGTACAAGT
15201 CTTTGTGTGG ATATGTTTTC ATTTCTCTTA GATAAAGATC TAGGAGTATC
15251 AGCCTGGGCA ACATAGTGAG ACCCCATCTT TACAAAAAAT TTTCAAAATT
15301 AGCCAGGCAT GGTGGCGTAC ACCTGTAGCC CTGCCATCTC AGGAGGCTGA
15351 GGTGGGAGGA TCCCTTGAGC CCAGGGGTTT TAGACTGCAG TGAACTATGA
15401 TTGCACCACT GCACCCCAGC CTGGGTGACA GAGTGAGACT CTGTCTCTAA
15451 AAAAAAGAGA GAGAGGGGAG GAAGGAAAGA AGAAAGAGAG GGAGGGAAGG
15501 AGGGAGGGAG GGAGGGAGAA GAAAAATGGA TCTAGGGTTA AGATTTAGGA
15551 GATTAGGTAA TGAATGTGTA CTATTACAGG GAACTGTCGA GCTGTTTCCA
15601 AAGTGACTGT ACCATTGTTC ATTGCCACCA ACAATACATG AGAGTTCTAG
15651 TTACTCCATG TGCTTGTTAC ACTTAGTATT ATCAGTCTTT TTCATTTTAA
15701 CCATTCTAGT GAGTATGTAG TAGTATTTTA TTATGGCTTT AATTTACAAC
15751 TCCCTAATGA TGAATGATGT TGAACATCTT TTCATGTGCT TATTGGCCAT
15801 TCATATATCT TTTGTGAAGT GACTATTCAA ATATTTTTCC ACTTTTTATT
15851 AGGTCATTTA TTTTCTTATT ATTGAGTTAT CTATGAATAC AAATCCTTTA
15901 TCAGTGTATG TATTGTGATT TTTTTCCCCA GTGGCTGGCC TTTTCATTTT
15951 CGTTAGGCTT TTTTGGTGGG TTTTTTTGGA AGAGAAAAAT
16001 ATTTTAATTT GATAAAATCC AGTATATCAG GTGTTATAGA CTGAATTATA
16051 CTCTACCCCA CAAATTCATA TGTTGAAGCC CTAACCTCTA AGTGACTATT
16101 TGGAGATGAG CCTTTAAGGA GGTAATTAAA GTAAAATGAG ATCATAAGGG
16151 TGGGCCCTAA TCTAATAGGA CTGGTGTCTT TATAAGAAGA GGAAGACACC
16201 AAGAGCGCAT GCACACAGAA GAACGGCCTT GTGAGGACAC AGCAAGATGA
16251 CGGCCATCTG CAAGCCAAGG AGAGAGGCCT CAGTAGAAAC CAAACCTGCT
16301 GATGCCTTGA TCTTGGACTT CCAGCCTCCA GATTTCTGTT GCTGAAGCCA
16351 CCCTGCCTGT GGTGTCTTAC CATGGCAGCC CTCACAGACT AATATATCAG
16401 ATTTTTTTCC TTCAACAGTT AACGCTTTTG GTGTCCTAAG CAATATTCGC
16451 CTGACCCAGG GTCATGAAGA TTTTTCTTCT ATGCTTTCTT CTGGAAGTTC
16501 TATAATTTTA GCTTTTACAT ATTTTTTTAA CTTTCCTTCT TCTTGCCTTC
16551 TGTTTCTTTT AAGGCATCAT CTATTGTGTT AATTTGTTCT TGTATTCCTT
16601 CTGATTTATT CTTCACTTCT GAAATGAATT TTGCTTTTTA AAAATATATA
16651 TAATTCTTTT CTGTGTCTGA GTTTTTCTAA TTAGGTTTTA TGTGGTTTTT
16701 TCTTGTCCTG CATCACTTTT TACTGTCTTT TGCCCATTTT GAAGTATCAG
16751 GTTCCAGTTT TGATCTGTTC ATGGATATGT TTTTGTGACA TGTTTCTTCT
16801 GGCTTCTTAT CATTTATTGC TTAGCTTATT AATTTCTATT CTTTCTTATT
16851 TTCTATTATA AGTATTTAAA GCTATATGTT TTCCTCTAAG TATTACTTAG
16901 CTGTCTTATA CGTTTTCATT TGTGTTATTT GGTGATCATT CACTTTCAGC
16951 TATTTATTAA TTTCCATTAT AATTCTTTCA TCTATGGGTT GTTTTAAAAA
17001 ATATTTTTAA GGCCAGGTGT GGTGACTCAC ATCTGTAATC ACAGCACTTA
17051 GGGAGGCTGA GGTGGGAGGA TTGCTTGAGG CCAGAAGTTT GAGACCGGCC
17101 TAGGCAACAA AGTGAGACCC CCTCTCTACA GAATATTTTT TTAAAATTAG
17151 CTGGGCCAGG CGTGGTGGCT CATCCCAGCA CCTGTAATAC CAGCACTTTG
17201 GGAGGCCAAG GCAGATGGAT CACCTGAGGT CAGGAGTTCG AGACCAGCCT
17251 GGGCAACATG GTAAAACCCC ATCTCTACTA AAATATAAAA ATTAGCCAGG
17301 TGTGGTGATA GGTGCCTGTA ATCCCAGCTA CTTGGGAGGC TGAGGCAGGA
17351 GAATTCTTTG AACCCAGGAG GAGGAGTTTG CAGTGAGCCG AGATTGCACC
17401 ACTGCACTCC AGCCTGGATG ACAGAGCGAG ACTCTGTCTC AAAAAAAAAA
17451 AGAAAAGAAA ATTAGCTGGG TGTAGTGGCA GGTACCTGTG GTCCCAGTGA
17501 CTCAGAGACT GAGGCAGGAG GATCACCTGA GCCCAGGAGT AGAGGCTGCA
17551 GTGAGCTATG TTTGTGCCAC TGCACTCCAG CCTGTGCAAC AGAGCAAGAC
17601 GCTGTCTCAA AAAATATATA TTTTTTTAAA TTTTCAAACT TCCTTTAGTT
17651 CTCTTTTTGT TATTAACTTT TAACTGAATG TTTTGCAATC AGAAGAAATA
17701 CTTTATGAGA TACCTATTCT TTAAAATTTC TTAAGAATTG CTTTGTGTTA
17751 ATATTTTGTT AATAGTTCAC ATGTGGTTCA ACCAATTTGT TTAGTTAGTT
17801 CTGTATATGT TCATTAGACC AACTTGATAA CTGTGTTGTT CTTTATTTAT
17851 TTATGTATTT ATTTTTCTTT GTCTATTCAT CAATTGCTGG GTGAGATGTA
17901 TTAAAATTTC TTGTTGTAAG TGTGGCTGTT CACTTTCTAC CTGTAGTTTG 17951 TCTGTTTGCT TTATAGAGGG TGAAGTTGTT TAGTAGGCAC ACATAAGTTA
18001 GAATTTTTCT GTCTTCCTGG TGAATGGAAT CATTTATCAT TATCTAATGT
18051 TCTTTTCATC TTTAGTATTG CTTTGGACTT GGAAGTCTGT ATTTTGTCTC
18101 CTGTTAATAT AACTACACTG GTTCCTTTGG TGTGAATATT TGCATAGTAT 18151 AACATTTTCC ATGAAGAAAC AAAACAGAGG AATTGGTTCT TTCTCAAAAT
18201 CTGATCTTTG TGTCAGCCCC CATCTCAGCC TTCTCCATTC ATCCTTGGTC
18251 ACTCCCCAAA CCCAGGAGCA ATCCTTGATT CTCCTTTTCC CCACATTCTA
18301 CATCCAATCC GTTAGCAAGT TCTATTAGTT CTATTATTAC CTCCAAAATA
18351 GATATTGAAT CCAGCCCTTT CTCACTGTCT CCACCATCAT CCTGTCTCAC 18401 ATCCCTACCA TGGCCTCCTT GCTGGTTGAC CAGAGTGATC TTGTAAAAAC
18451 ATGTTAGGCC AGGCACGGTG GCTCCTGCCT GTAATCCCAA CACTTTGGGA
18501 GGCCAAGCGG GTGGGTCACC TGAGGTCAGG AGTTGGAGAC CAGCCTGGCC
18551 GACATGGTGA AACCCTGTCT CTACTAAAAA TACAAAATTA GCCAGGTGTG
18601 GTTATGCTGG CCTGTAATCC CATCTACTCG GGAGGCTGAG GCAGGAGAAT 18651 CACTTGAACC CAGGAGGCGG AGGTTGCAGT GAGCCAAGAT CATGCCACTG
18701 CACCCCAGCC TGGGCAACAG AACAAGACTC CATCTCAAAA AATAAAAATT
18751 AAAATAAAAT GTTAGGCTCC CTGGGTCTCT GGCTTAGTCC ATTTGTACTG
18801 CTTTAACAAA ATACCTTAGA ATGGTGTAAT TCTAATAATT GCTATTAATA
18851 AATAATAGCA ATTAATAAAT AATAGCAATT TCCTTCTCAC AGTTCTAGAG 18901 GCTGGGAAGT TCAGGGTCAA GGTGGCACCT GACTCCGTTC TGGTAAGGGC
18951 GGCTCTCTGC TTCCAAGATG GTGCCTTCTC GCTGCGTCTT CGCATAGCGG
19001 AAGGGCAAAC ACTGTGTCCT CACGTGGCAG AAGAGATAGA AGGGCCAGGC
19051 AGCTCTCTGA AGTATCCAGG TTGGAGTCAT GGACCTGCAT GTTCCCCTCT
19101 GACATCCACA GAGTACCTAT CATGGTCCTT GGCATGCAGC AGGTGGCCCA 19151 TAAACGCCTG AATGAACAAA CATATAGTAA TGGTCGCTAG TACTAGGAAT
19201 AGCAGCCACC GCAACAGTCC TGTGAGGGAG GCATTACAGA TGAGGAAACT
19251 GAGGTTTAGG GGCAAGGACC TGCCCATGGT CCCAAAGCTA GGGAGGGACA
19301 GGGCTGGGAT TCCCACTCCC ATCCATCTGG CTCCAGAACC TGAGCTCCTG
19351 ACCAGGCTGT TCTTATCCTG TCTCAGCCAG TGGCTGCCTG TCTGGACGGA 19401 TGGACCTAAA -GTCAGTCCAG CCAAACAGAG GGAAGCATGA TCAACTGTTC
19451 TCTAAGTTCC GTGACCCGGA GAGGCTGAGT CCATGGCCCA AGCTCTCCTC
19501 TCTCCTCCCC CAGCTCTCCC ACCCGTAGAC GGTGGCGCGA AGTGGAAGAG
19551 TGTGCGGGAA CCAAGGAGCT GCTATGTTCT ATGATGTGCC TGAAGAAACA
19601 GGACCTGTAC AACAAGTTCA AGGGACGCGT GCGGACGGTT TCTCCCAGCT 19651 CCAAGTCCCC CTGGGTGGAG TCCGAATACC TGGATTACCT TTTTGAAGGT
19701 AGGTCTGTGG GTAAGGGACT GAGTGGAAGG CTGTCCATCC CATCGGGGAG
19751 CTGTGCTCAG TGCTCAGTGG TTCTGTTCTC CTGACCATCT GTCTCCCACT
19801 TCCCCAAAGC AGAGGGCAGC TCCCTGGGCC AGGCCCTTTG AGATGGGGTG
19851 TGGGACCAGC AACAGCGAGG GACCATGTCT GGTAGCCTGT CAGGGAGTTA 19901 GGGGAGCTCC AGCCAGCACC AGCAATCTCA CGTGCACCCT CTGCTAACAA
19951 TGTTCATTAT TTTCAGTTGA GCACCATTTT GGTCATGGAC TACACAAGGC
20001 ACTTTATATG CTTATTCCTA TTTTTTTATG TTCAGCTTCT CTCCTTAAAA
20051 ACAATGTTTA AAACCAATTC TGGGCCAGGC GTGGTGGCTC ACGCCTGTAA
20101 TCCCAGCACT TTGGGAGGCC AAGGCAGGTG GATCACCTGA GGTCAGGAGT 20151 TTGAGACCAC CCTGGCCAAC ATGGCAAAAC CCCGTCTTTA CTAAAAATAC
20201 AAAAATTAGC CAGGCTTGGT GGCAGGCACC TGTAATCCCA GCTACTCGGG
20251 AGGCTGAGGC AGGAGAATCG CTTGAACCC GGAGGCGGAG GTTGCAGTGA
20301 GCCAAGATCA CGCCCCTGCA CTCCAGCCTG GGCGACAGAG CGTCTCAAAA
20351 GAAAAAAATT AATAAACAAA GAAAAAAAAA CAAATTCTGT TTGCAAAAGT 20401 ATTTTCTATA CACTGTAGAA ATTTGTGGGG TGTGGGGGGG TAAAGATGAT
20451 AGAAAAAAAA ATGTCCCATG CTTACTGGC GAAATCATGT ATTGACATTG
20501 GGTGAGGAGG GCACTTTTTT TTTTTCAGTC TATTTTTAAT CTTCACAGCA
20551 AACTTGTGAG GTTCATTTCC ATCAACCTGA GACTCACAGA AGCTAAGAAA
20601 CTTGATACCG CTAGTAACCA GTGGACTTGA TACCGCTAGT AACCGGTGGA 20651 CATAGATGTG AACTGGATCT TTCTGACCTC GGGCAGGGCC GGGTAACAAG
20701 GGGAGGATAA ATGCCCAGAC AGTGTCCTCA GAGAGCTGAG AGCTGTAACT
20751 TGCTGCCCGG GCTTCTCACA GTGTTCAAGG ACAAAATAAG GCTTTAAGAG
20801 AGAAGAGGGA CAGACTGATT GCAGGGCAGC AGGAAGAGAT GGTAGAGAAG
20851 GAAGAAGAGA TGATTCGTGT GGAAAGAAGC TGGCTCGGTG GATGGATAAA 20901 AGAAGGGAAG GACAGATGGG TAAGAAGAAA GGGAGGATGG AGGGGATGGA
20951 GGAGGAAGCA ATGGAAAAAT GGGAAGGAAG GAGGTTGGAT GGAAGGATAG 21001 ATGCCTATTA GGAAGGAAAT ATGTGTGGAT AGAGAGATGG AGGATAGGAA 21051 GTATGTTAGT CAAGGTTCTC CAGAGAAACT GAACCAATAG GATATATACA 21101 GATACACTAA GAGGAGGCCA GCCGGGCGCG GTGGCTCAAG CTTGTAATCC 21151 CAGCACTTTA GGAGGCCGAG GCGGGCGGAT CACGAGGTCA GGAGATCAAG 21201 ACCATCCTGG CTAACACAGT GAAACCCCGA CTCTACTAAA AATACAAAAA 21251 AAAATTAGTT GGGCGTGATG ATGTGCGCCT GTAGTCCCAG CTGCTGGGGA 21301 GGCTAAGGCA GGAGGATGGC GTGAACCCAG GAGGCAGAGC TTGCAGTGAG 21351 CTGAGATCGT GCCACTGCAC TTCAGCCTGG GTGACAGAGC AAGACTCCGT 21401 CTCAAAATAA ATAAATAAAT AAATAAAAAG AGGCCAGCCA TGGTGGCTCA 21451 CACCTGTAAT CTGAGCACTT TGGGAGGCCG AGGCGGATGG ATCATTTGAG 21501 ATCAGGAGTT CAAGACCAGC CTGGCCAACA TGGTGAAACC CTGTCTCTAC 21551 TAAAAATACA AAAGTTACCC GTGTGTGGTG GCACACACCT GTAGTCCCAG 21601 CTACTCAGGA GGCTGAGGCA GGAGAATTGC TTGAACTTGG GAAGCAGAGG 21651 TTGCAGTGAG CTGAGATCAC GACACTGCAC TCCAGCCTGG GTGACAGAGC 21701 AAGACTTTGT CTCAAAAAAA AAAAATTTAT AATAAGAGGA GATTTATTAT 21751 GGGAATTGGC TCATGCAATC ACAGACACAA AAATGTCCCC CAGCATGCAG 21801 TCATGGGCTG GACAACCAGG AAAGCTTGTG GTGTGATTCT GTCTGAGTCT 21851 GAAGGCCCAA GGCCAGGGGA GCAGTGGTGT AACCCCCAGT CCGAGGCCAC 21901 AGGCCCGACA ATCAGAGGGG CCACTGATAT AAGTCCCAGA GTCCAAATGC 21951 CGGAGAACAG GAAGCTCCAA CGTCCAAGGA CAGGAGAAGT TGATGTGCCA 22001 GCTCAGGAAG AGAGAATGTG AATGTGCCAT TCCTCCTCCA TTTTTTGTTC 22051 TCTTTGGGCC GTCAGTGGAT TGGATGATGC CTGCCCACAC TGGTGAGGAC 22101 AGATCATCAC CAAATCTGCC GATTAAAATG TTAATCTCTT CTGGAAAAAT 22151 CCTCACAGAT GGGCCCAGAA ATAATGTTTT ACTGTCTACC TGGGTATCCC 22201 TTAGTGCAGC TAAATTGACA CATAAACTTA ACCATCACAG GCCAGGCACT 22251 GTGGCTCACA CCTGTAATCC CATCACTTTG GGAGGCCAAG GTGGGAAGAT 22301 CCTTTGAGGA TGAGGTAGGC AGATCACTTG AGCCTAGGAG TTCAAGACCA 22351 GCCTAGGCAA CATAGGGAGA CCTCGTCTCT ACAAAAAAAA AAAAAATTTA 22401 AATTCGCTGG GTACGGTGGT GGGCACCTGT GGTCCCAGCT ATCTGGGAGG 22451 CCAAGGTAGG AGGATGACTT GAGCCCAGGA GGTCAAGGCT GCAGTGAGCC 22501 ATGATTGTTC CATTGAATTC CAGCCTCGGT GACAGAGCAA CACCCTGTCT 22551 TAAAGAAAGA AAAAATTTAA CCATCACAGA AGGCAGAAGA AAAGGCAGAT 22601 GGGTGGATGA GATGGGTGGG TAGATAGTAT AGAAGAAAAG CGGGACATCC 22651 AGGCAGGGAA GGAAGGGCTG GAGCGAAGGA GAAGCAAGGA AGGAAGGAAG 22701 GAGAGACAAG AAGGAAGGAT GTGTAGAAAG GTGGAAGAGA AAAGAAGAAT 22751 GGATGTATGG GAAGAATGGA TGAGTAGGTT AGAAGGCTCA CTGGCTAGAT 22801 AAAAGGTGAG AAGTATAAAT GAATAATAAG AAAGGAGGCA TAGGAAGAAA 22851 AAAATATTGG TTAGAAAGGA TGATTGAGAA GAAAGGGTGG TTGGGAAGGA 22901 AGGAAGGAAG GATGGATGGA TGGATGGATG GATGGGAAGG AAAGGAAGGA 22951 TAAGAAGGCA GACAGGAAGG CTCTCTGGCT AGAAGAATGG CAGACAAACC 23001 ACAATAATTG CTGAATGGGT AGGAATAAGA CATTAGAAGA ATAAAGGGAA 23051 AGACACAAAG ATATTTAAAA TGTTTTCATT AATTTTTTGC CTCCTCCCTG 23101 AATTTCTCCT GATTCTTCAG CCCCACATCC CAAGCCAGGG TGATCCTTCC 23151 TGCCTTTACA CTCCCTCCAC ACTTTTTCTG CTCTCATATG TGGCCGTGGT 23201 CACTTTCTTT TGGTAGTTTG CATATTTCAT TTACCCCAAA CTTTCAGCTC 23251 CTGAAGGTCA GGATACAAGG AGGCCTCATC TCCGCATTCC CCTCAGCTCC 23301 CTTCCTGAAG CTTGATACCT AGTCAGTACC CAGTGGATGT TTCCTAAACA 23351 TGTAAGTAAT GACATCATGA AGAAGCCACA TGTTTACCTT GACCACAAAC 23401 ACAGGGCAAA GGTGACTAGT GTGGTCAGAG ATCCCTGCTG GCTGGGAATC 23451 AGGGAAGGCT GCATGGAAGA AGTGGCATTT TAGTTAGAAC TTGAAAGGTG 23501 GTGTATTTAG TTTTCTCTGG CTGCCATATT CCTTGTCACA TTGCCCTCTC 23551 CATCTTCAAG CCACTGGGCA AGGCTAGAAG GCCCTCAACA GACTATCGGT 23601 AGGAATGTGG AAGTTGAAGA CTCAGAGTGC AGAAAGAAAC AAGTAGCATT 23651 TTAGAGAAAA GCTAAATCCC CTCCAAGAAT ACCTCAATCA TCGTGAAGAG 23701 CCTGTTAGTA GACGCACTAA CACTCAAGGC ACTGCTTCAC AAGGTAAGGA 23751 ACGTGTAATT GAAAACTTGA GAAAGGAAGA AACTTGTTCT GTACTGGCAG 23801 AAAGCTTAGC AGAATTGTGT CCTGCAGTCA TATGGGACAC AGAGCTTGTA 23851 AATGATGAAT TTGAATGCTT ATCCGAGAAG GTTTCCAAAT AAAATGTGGA 23901 AGGCACGGCC TGGTTTCTTC CTGCCTCTTA TAGTAAAATG CAAGAGGAGA 23951 GAGAGAAAAT GAGGGAAGAA CTTAAACAGA AAGGAACCAG GACTTGATGA 24001 TTTGGGAGGT TCTCAACCTA TGCAAAAAAC AATAAAATTA AGAGATTGTA 24051 GCTGGGCACA GTGGCTCATG CCTGTAATCC CAGCACTTTG AGAGTCCGAG
24101 GCGAGCAGAT CACCTGAGGT CAGGAGTTTG AGACCAGCCT GGCCAATGTG
24151 GGGAAACTCC GTCTCTACTA AAAATACAAA AATTAGCTGG GTGTGGTGGC
24201 GGGCACCTGT AATCCCAGCT ACTCAGGAGG CTGAGGTGGG AGGATCACTT 24251 GAACCCAAGA GGCGGAGGTT GCAGTGAGCC AAGATCATGC CACTGCACTC
24301 CAGCCTGGGT GGGTGACAGA GCAAGACTCC ATCTCAAAAA AAAAAAAAAA
24351 AAGAGATTGC TCCCAAAAGT GTGACATAGA GAAACAGCCA AGTATGTGAT
24401 TATACCAAAC TTCAGGAAGA TAAAAGATCA AAGTACTCAG TCGCTCAAAA
24451 GGCTCTTTGA AGAGATTAAG ATTATAACTC ACAGTCCCCT TCAATCAAAC 24501 CAGGGGACTT CTAGGAAGCT GAACAGCATT GTCCCTCAGC CATATCAGCT
24551 GGAGCCAAAA GTAGAGAAGG GCTTATCTGA AAAAAGGATC TGTGGACCTG
24601 GCTTTTATCT AATAATGCAG TGGATTCCCC CATGACATCC ATAGGAGACC
24651 CGTAAAGTTC CTGAGACGTT TACATCCACA GAAACACTGT TAGCTTGGAT
24701 TAAATGGAAC ACAGAGAGTA TGAAATCAAA GAAGGCTGTT GGACTCTCCA 24751 GTTTCTACTG TTGAGATGCA GACTGGTAAA ACTACTTAGC TGCAAACACC
24801 TGCTACCTTT AGTGAAAAGG AAGGATATCT CAGACGGTGA AACCAGAAGC
24851 TCAAAGGGCA GTGCTAAGAG CGAAAGAGAA TTCTTCCCAG GCCTTGAAAC
24901 CTAATGGAGT TTTCTTGGCT GGATTTTCAA ACTGCATTGG ACCATGACCT
24951 GATTGTCCCT TTCATGTCCC CATGCTTGAG CCAGATTGTC TGCAACTGTT 25001 ATCCTGTGCC TGTCCCACAT TTTATGTTGG GAGCAGAAAA CTTTAGTTTT
25051 GCTGGCCCAC AGATAGAGAG AAACTGTACC CCGAGAGTTG TACTGACTGG
25101 ACTATGCCCA GAGTCTATTT GACTCTGACT TAGATACTGT TGATTTGGGA
25151 ATTTGAGTTG ATGCTGTAAT GAGATGAGAC TTTGGGGGAC ATTGGGATGG
25201 AGTGAATGGA TTTTGCATTT GAAAGAGATG TGGGTTGGGT AATCCTAGCC 25251 CACACCTGTA ATCCCAGCAC TTTGGGAGGC CGAGGCAGGC AGATCACCTG
25301 AGGTCGGCAG TTCGAGACCA GCCTGACCAC CATGGAGAAA CCCCATCTCT
25351 ACTAAAAATA CAAAATTAGC CAAGCATGGT AGCACATGCC TATAATCCCA
25401 GCTACTCGGG AGGCTGAGGC AGTAGAATCG CTTGAACCCG GGAGGCAGAG
25451 GTTGCGGTGA GCCGAGATCA CGCCATTGCA CTCCAGCCTG GGCAACAAGA 25501 GTGAAACTCC ATCTAAAAAA AAAAAAAAAG AAAGAAAGAG ATGTGGATTT
25551 TGGGTGGGGG ACAGAGGGAA GACCATGGTA GGCAGAATGA TCCTCTAAAG
25601 GTGCTCTGCC CTAATCCCCA GAAGCTAAGA ATATGTTAGA TGTCAGTATT
25651 GCGTGGCAGT AGGAATCTTA ATTAACGTTA TAGACTGTTA TGGTTTGAAT
25701 GTCCCCTCTA AAACTCCTGT TGACATTTAA TCATCATTGT GATTGCATTA 25751 AGAAGTGGCC CTGTTAAAAG GTGATTTAGT CCTTAAGAAC GCTGCCCCCG
25801 TGAATAGATT AAGGTCAGTC TTGCGGGAGT GTGTTTATCA AGAATGGATT
25851 GTTAAAAAGT GAGTTCTGGC CAGGGGCAGT GGCTTATGCC ACTCAGCACT
25901 TTGCGGGGCC AAGACTTGAA GTCAGTTGTT TGAGACCAGC CTGGCCAACA
25951 TGGTGAAAGT CTGTCTCTAC TAAAAAATAC AAAAAGTGTC CGGGAGTGGT 26001 GGCGGGCGCC TGTAATCCCA GCTGCTCAGG AGGCCGAAGC AGGAGGATCG
26051 CATGAATCCG GGAGGCAGAG GTTGCAGTGA GCTGAGATCG CCCCGTTGCA
26101 CTCCAGCCTG GGTGATAGAG CAAGACTCTG TCTCAAAAAA ANNNNNNNNN
26151 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NAAAGAAAGA
26201 AAGAAAAGAA AAGAAAAGTG AGTTCTGCCC TCTCTTGCTG GCTTACTCTC 26251 ACCCTCTCTT GCCCTTCCAC CTGCCACCAT GGGATGACAC AGCACAAAGG
26301 CCCTCACCAG ATGCCAGTGC CATGCTCTTG GACTTCCAAG TCTCCAGAAA
26351 CATGAGCCAA ATACACTTCT GTTCATTATA AATTACCCAG CCTGTGATAT
26401 TCTGTAATAA CAACACAAAA TAGACTGAGA CATAGATCTT CAAATAGTGA
26451 GGTTATCCTG GATAATCCAG ATGGGCCCAA TCTAATCCCA TGAGCCTTTA 26501 AAACTTTCTC CAGATGGAGG CAGAAGAGAA GTGGCAGAAG GGGAAGTCAG
26551 AGAGATTTGA AGCATAAACA GGACTCCATG GTGCCGTTTC TGGTTTGACG
26601 ATGGAGTGGT AACGTGATGA AAAATGTGGG TGCCTTCCGG AGCTGAGAGG
26651 CTCCCACTAA CAATCGGCCA GGAAACAGGG ACCACAGCCC TACAGCCACA
26701 AAGAACTAAG TTTTGCTGAC AACCCAAGGG GGCTTGGAAG TGTCTTCTCC 26751 CCCATCGGTT CCAGATGTGA GACCCAGAGC GAAGGAACCA GCTGAGCCCA
26801 CCTGGACTTC TGACCTAGAG AACTGTGAGA TAATAAGTTT GTATCATTTT
26851 TAAGGCACTG TGTGTGTGGT AATTTGTTAT GACAGCAATA GAAAATGAAT
26901 CCAGATGGGC AGGATCTGCC AGGCCAGTGA CATGTGGAGG GCACCCAGGC
26951 GGATGGGATG GCATGAGAGA AGGCAGGTCA GCAATGAGCT TGCCCAGGTC 27001 ACCTCTCCTC TCTAAGCCTC AGTTTTCCTC TCTATGAAAT GAGAGTAGTG
27051 ATATCTCCCT CCCAGGGTCA GTGCAAGGCT GAAATAACAG ATTATAAGGT 27101 GCTAGGTGCA CAAGAAGTGT TTGAAACATG CTAGTTGCTT TTCCATTTCC
27151 AAGAGAGCTC TCTGGTCTTG GGGGATGGAG GCAGTGCGGC CCCTCGGGAT
27201 TACTGACAGG TCCTGCTCTG TTTCTGCAGT GGAGCCGGCC CCACCTGTCC
27251 TGGTGCTCAC CCAGACGGAG GAGATCCTGA GTGCCAATGC CACGTACCAG 27301 CTGCCCCCCT GCATGCCCCC ACTGGATCTG AAGTATGAGG TGGCATTCTG
27351 GAAGGAGGGG GCCGGAAACA AGGTGGGAAG CTCCTTTCCT GCCCCCAGGC
27401 TAGGCCCGCT CCTCCACCCC TTCTTACTCA GGTTCTTCTC ACCCTCCCAG
27451 CCTGCTCCTG CACCCCTCCT CCAGGAAGTC TTCCCTGTAC ACTCCTGACT
27501 TCTGGCAGTC AGCCCTAATA AAATCTGATC AAAGTATGAT GACCTACAGG 27551 AGGCCTGCTT GCCAAGTCAA CAGATTCAGT ACAGAAAAAC TGAAAAATAC
27601 AGATAAGCTC TAAGAAGCAG ACCAAAAGTA CCCAGAGATG ACCGCACATC
27651 ACTCTGGTGT ATATCCAATT TCAGATTTGT TTTCTGTGTA TGCATGTGTG
27701 TA AGCTGCA TTTATTTATG GCAAGGGCTG GCAGACTTTC CCGAAGAAGG
27751 CCAGATAGTC GATATGTTTG GCTTCATGGG CCGTATGTTC GCTCAGGACT 27801 ACTCAACGCT GCAGTTATAG CACAAAAGGA GCCGTAGCCT ATACGTAAAT
27851 GAATGGGCAT CGCTGGGTTC CAGTAAAACT GTTTACAGGC CAGGTGCGGT
27901 GGCTCATGCC TGTAATCTCA GTACTTTGGG AGGCCGAGGT GGTGGGAGGA
27951 TTACCTTAGC CCAGGAGTTC AAGACCAGCC TGGGGAACAT GGTGAAACAT
28001 TATCCCTACA AAAAAAAAAA AAGCTGGGTG TGGTGATGCA TGCTTGTGGT 28051 CCCAGCTGCT TGGGATGCTG AGGCAGGAGG ATCGCTCGAG CCCAGGAAGC
28101 AAGGCCACAG TGAGCCATGA TCGCACCACT GCACTTTAGT CTGGGCAACA
28151 GAGTGAGACC TTGTCTCAAA AAAAACAAAA AATAAAACTT TTTACATAAA
28201 CAAGTGGCCA ACCAGACTTG GTCCCTGGGC CTCTGCTCTT GAATGTTCTT
28251 GCTTCCACTA AAGTAACATT CACACTCCCG ATTTTTGCAT ACTCTGGGTT 28301 CTGGGGAATA TAGATCCGAA TCCAGCGTGG TTCCTGCCTT CAAGAACCTC
28351 ACAAATATTC TAGACCAGCA CTGCCCAATA GAAAGAAATA TAATGCAAGC
28401 CACATGTGCA GTTTTAAGTG TTCCATGTTA AATTAAGTAA AAAGAGACGG
28451 GTAAATCGAA TTTTAATAAC AGATTTTACT TCATCCAATT GAATGGTATC
28501 ATTTCAATGA GCAATTCTGA TAGTGATTGA GATCTTTTAC ATTCTTTTTC 28551 ACTACGTCTT TAAAATCTGA TGTGTGTTTT GTACTTGGAA CACTTCTCAG
28601 TGTGGACCAG ATGCATTTCA CATACTCAGT AGTCACGCGT GGCCAGTGCC
28651 TTCCATACCA CACAGTGCAG CATCTGTAGA GGTTTCCTCC ACTGCTGATA
28701 GACTAGGAGA CCCCAAGATG GAAAGCCTGA AGAATCTGCT CCTTGAAGTA
28751 GGGACCTTAA TGGGGTGCAC GCCAGGGCGA CCCCAAGTGG TAGGCTGCTT 28801 TTGAACCATG GCTATCCCTA CCTCTAGACT CAGCTGAAAA GAACTCAGGT
28851 AGTCTTGGGA AGTGCTTCCT CAATGCTTAA ACTTTAATGC AGGAAAAGAA
28901 TAGAAAGTTC AGGCAAGGAG GGAGGATCAC TTGAGGCTGG GAGTTCGAGA
28951 CCAGCCTGGG CAACAGCAAG ACCTTGCCTA TACAAAAAAT AATTTTAAAA
29001 AATTACCCAG GTATGGTGGT GTGGATCTGT AGTCCCTAGT TACTTGGAGA 29051 GCTGAGGTAG GAGGATCGCT TGAGCCCAGG AGTTTGAGGC TGCAGTGAGC
29101 TGTGATCACA CCACTGCACT TTGGCCTGGG TGACAGAACC AAACCCTATC
29151 CCCTACAAAA AAACAAAAAA AAAAAACAAA AAAAAACACC CTACCATGTC
29201 TGCCAACCCC ACTCTGTCCT GGCTGTGTGA AACCAGTCCC CACAGCAGCT
29251 CTGCCACTCT CTGCTTCTTT TCCAAACAGA CCCTATTTCC AGTCACTCCC 29301 CATGGCCAGC CAGTCCAGAT CACTCTCCAG CCAGCTGCCA GCGAACACCA
29351 CTGCCTCAGT GCCAGAACCA TCTACACGTT CAGTGTCCCG AAATACAGCA
29401 AGTTCTCTAA GCCCACCTGC TTCTTGCTGG AGGTCCCAGG TGGGTATCAA
29451 GTGGTGCAGA AGGAGAAACT TTCCCTCTGG GCCTTGGGAG CTTCGTGACA
29501 CAGTGGTTAA GAACATGAGC CTAGAGATAG ACTCGCCTGG ATTAAAACCA 29551 CACTCATTGT GTGTCTTTGG GCAGCTTACA TAATGCCCCG AACCTTGGTT
29601 TGCACAGTCT GCAGGATGGG TTTATTCTTG TGAGGATTAA ATAGGGTCAT
29651 GTATGTGAAG CACTCGGCAC AGGTGCAGTT GTAGACAAGA GCCATTGTTG
29701 TTTCTCTCAT TGTTATTTTT CCTTCCTTAG AAGCCAACTG GGCTTTCCTG
29751 GTGCTGCCAT CGCTTCTGAT ACTGCTGTTA GTAATTGCCG CAGGGGGTGT 29801 GATCTGGAAG ACCCTCATGG GGAACCCCTG GTTTCAGCGG GCAAAGATGC
29851 CACGGGCCCT GGTATAGCAA ATCTGGGGGT GTGCGGCAGG TGGGGAGGGG
29901 TTGAGAGTAA GGGAGTGGGG CTGGAGCTAT GAGTTGTTCA GATAGAATAT
29951 CAAGATGGTC CAGACTCTTG GACCAAAACA TCTATCTTTG TGTCTGAATT
30001 TCCACCATTA GTAATGCATT CATTTAGTCC TGAATAAAAT GGCAAACAGG 30051 CCCTGGAGGG AGCAGTGCCT TAAGTTCCTT TGAGATAAAT AACTTCACCT
30101 CTGCTAAGGA TGTGTCAGCT GCTGAGAGCA GAGCCCCTGG CCTTGGACCT 30151 CAGGAGAGAC ACTCAAAAGG GGAGGAGAGG AGGCACCAAA GGGGACATCT
30201 TAAAAGAGTT CCAATTTTTA GTTCACACTT TAACCCAGGA TAAGCTGTGT
30251 CCTGGCTGAC CTTGGAGTTT CTTCCCTGGT CTGCTGGGTC TCTCCCTTAG
30301 AACCTAGGGG CGAGCTGGGG CAGGGGAAGC CCAGGAGGTG ATATAGGTCG
30351 GCCCTGTTCA GATGAGGGCT GGCAGGGGCA GCTTGGGCAT ATGCGAGGCT
30401 CCGATGGGCA TGGGGGCTTT GAGGATGGAT TCTGAGTGTC CCTGCATCGT
30451 GGCAGGGTGG CAAAGGGAGC ATTTCCAAAT TTCCTGGCTC CAGGATCTGT
30501 GGGAGAATCC CACTAACTGT CAGGGTGACA ACCTCGGGTA GACATGTCTG
30551 TGCCCTGCCC CGTGCCCTCA GCCTTCCTGT TAAGAGCACA CCAGCTGGAT
30601 TTGCAACTCC CAGCGCCTGC ACCCAATGGG CTTTCTCTGG CCTCTGGAGC
30651 CCACATTGCC CCTGCATGTG GCAGGCTGCA AGTGTCACAG CCACCAGCTC
30701 TTCCATTCCT CAACAATGAC TGTGGGTAAA TAGCCCAGGA GCGTCCCCCT
30751 CCTGGGATGG TTCTGAGGTG CGTGTGCCCA GTGGCTCCCT GAGTTGCCAG
30801 CAGGATTAAG TGCCAGTAGC CCTAGTGGTC AGCTGCTTGA TAACACCCTG
30851 CTTCCTGGCT GCTCCCCCAG TCCCATCTGG TGTGTTCTGG GATCATCTCC
30901 CAAAGAAACT GCTTACACTT GAAGCCTTGT CTGAGGTCTG TTTCTAGGGG
30951 AATTCAGATG ACGATAATTA TGCTTCAGGA AAGCCTAAAT TTTCTGCTTT
31001 TCTCTCCCCT ACCCAAATCA GGACTTTTCT GGACACACAC ACCCTGTGGC
31051 AACCTTTCAG CCCAGCAGAC CAGAGTCCGT GAATGACTTG TTCCTCTGTC
31101 CCCAAAAGGA ACTGACCAGA GGGGTCAGGC CGACGCCTCG AGTCAGGGCC
31151 CCAGCCACCC AACAGACAAG ATGGAAGAAG GACCTTGCAG AGGACGAAGA
31201 GGAGGAGGAT GAGGAGGACA CAGAAGATGG CGTCAGCTTC CAGCCCTACA
31251 TTGAACCACC TTCTTTCCTG GGGCAAGAGC ACCAGGCTCC AGGGCACTCG
31301 GAGGCTGGTG GGGTGGACTC AGGGAGGCCC AGGGCTCCTC TGGTCCCAAG
31351 CGAAGGCTCC TCTGCTTGGG ATTCTTCAGA CAGAAGCTGG GCCAGCACTG
31401 TGGACTCCTC CTGGGACAGG GCTGGGTCCT CTGGCTATTT GGCTGAGAAG
31451 GGGCCAGGCC AAGGGCCGGG TGGGGATGGG CACCAAGAAT CTCTCCCACC
31501 ACCTGAATTC TCCAAGGACT CGGGTTTCCT GGAAGAGCTC CCAGAAGATA
31551 ACCTCTCCTC CTGGGCCACC TGGGGCACCT TACCACCGGA GCCGAATCTG
31601 GTCCCTGGGG GACCCCCAGT TTCTCTTCAG ACACTGACCT TCTGCTGGGA
31651 AAGCAGCCCT GAGGAGGAAG AGGAGGCGAG GGAATCAGAA ATTGAGGACA
31701 GCGATGCGGG CAGCTGGGGG GCTGAGAGCA CCCAGAGGAC CGAGGACAGG
31751 GGCCGGACAT TGGGGCATTA CATGGCCAGG TGAGCTGTCC CCCGACATCC
31801 CACCGAATCT GATGCTGCTG CTGCCTTTGC AAGGACTACT GGGCTTCCCA
31851 AGAAACTCAA GAGCCTCCGT ACCTCCCCTG GGCGGCGGAG GGGCATTGCA
31901 CTTCCGGGAA GCCCACCTAG CGGCTGTTTG CCTGTCGGGC TGAGCAATAA
31951 GATGCCCCTC CCTCCTGTGA CCCGCCCTCT TTAGGCTGAG CTATAAGAGG
32001 GGTGGACACA GGGTGGGCTG AGGTCAGAGG TTGGTGGGGT GTCATCACCC
32051 CCATTGTCCC TAGGGTGACA GGCCAGGGGG AAAAATTATC CCCGGACAAC
32101 ATGAAACAGG TGAGGTCAGG TCACTGCGGA CATCAAGGGC GGACACCACC
32151 AAGGGGCCCT CTGGAACTTG AGACCACTGG AGGCACACCT GCTATACCTC
32201 ATGCCTTTCC CAGCAGCCAC TGAACTCCCC CATCCCAGGG CTCAGCCTCC
32251 TGATTCATGG GTCCCCTAGT TAGGCCCAGA TAAAAATCCA GTTGGCTGAG
32301 GGTTTTGGAT GGGAAGGGAA GGGTGGCTGT CCTCAAATCC TGGTCTTTGG
32351 AGTCATGGCA CTGTACGGTT TTAGTGTCAG ACAGACCGGG GTTCAAATCC
32401 CAGCTCTGCT CTTCACTGGT TGTATGATCT TGGGGAAGAC ATCTTCCTTC
32451 TCTGCCTCGG CTTCCTCATC TGCAGCTACG CCTGGGTGTG GTGAGGGTTC
32501 TAGGGGATCT CAGATGTGTG TAGCACGGAG CCTGCTGTGT CCTGGGTGCT
32551 CTCTACGTGG TGGCCGGTAG AATTCTCCAT CTATCCAGGC TCCAGGAGAC
32601 CCCTGGGCAT CTCCCACCTG TGGCCCCTAA ACCCAGAGTG ACTGAGAGCA
32651 CTTACCATTC AGCTTGTCTC ATCCCCAGTC TACCTCCTTC CTTCTACCCT
32701 CACTGCCTCC CAGTCAGGAG AGTGAGCTCT CAGAAGCCAG AGCCCCACCC
32751 AAGGGGACCC TGGTCTCTCC GCCTTCACCT AGCAATGGGA ACCCTGCTTC
32801 CCAGGGGAGG AACCAACTGC TCCACCTTCT AGGGACCCAG TTTGTTGGAG
32851 TAGGACAGTA ACATGGCAGG AATCGGACTT CTGGGCCTGT AATCCCAGTT
32901 TGGATGGCAC GTTAGACTCT TGGTTGACCG TTGTGGTCCT TAGAAGTCCC
32951 ATTCTCCCTT CCAGTTATGA GAAACCAATG CCTTCTAGAT TCAGGTGACT
33001 ATCCTTACCT GGGGGTGCTG ATGCATCCTC AGTTAACCTA CACCCACCTG
33051 AATATAGATG AGCGTAGCTG AGTTTTCACC CGTAGGACCG AAGTGTTTTG
33101 TGGTGGAGTA TCTGAACAAC CTTGGCTCTG TGGCCATTCA ACCTGCCAGG
33151 ACTAACATTT CTGGATTTGT GAAGAAGGGA TCTTCAAAGC CATTGAACCC 33201 ACAGAGCTGT GTTGCTTTAA AGCCACCACA AGGGTACAGC ATTAAATGGC 33251 AGAACTGGAA AAGCTTCTTA GGGCATCTCA TCCAGGGATT CTCAAACCAT 33301 GTCCCCCAGA GGCCTTGGGC TGCAGTTGCA GGGGGCGCCA TGGGGCTATA 33351 GGAGCCTCCC ACTTTCACCA GAGCAGCCTC ACTGTGCCCT GATTCACACA 33401 CTGTGGCTTT CCACGTGAGG TTTTGTTTAG AGGGATCCAC TACTCAAGAA 33451 AAAGTTAGCA AACCACTCCT TTTGTTGCAA AGGAGCTGAG GTCAAGGGTG 33501 GCAAAGGCAC TTGTCCAAGG TCGCCCAGCA GTGCTGCTCT GATGACTTGT 33551 GCACATCCCC AAGGGTAAGA GCTTCGATCT CTGCACAGCC GGGCCAACCT 33601 CTGACCCCTT GTCCATGTCA GTAAAATATG AAGGTCACAG CCAGGATTTC 33651 TAAGGGTCAG GAGGCCTTCA CCGCTGCTGG GGCACACACA CACACATGCA 33701 TACACACATA CGACACACAC CTGTGTCTCC CCAGGGGTTT TCCCTGCAGT 33751 GAGGCTTGTC CAGATGATTG AGCCCAGGAG AGGAAGAACA AACAAACTAC 33801 GGAGCTGGGG AGGGCTGTGG CTTGGGGCCA GCTCCCAGGG AAATTCCCAG 33851 ACCTGTACCG ATGTTCTCTC TGGCACCAGC CGAGCTGCTT CGTGGAGGTA 33901 ACTTCAAAAA AGTAAAAGCT ATCATCAGCA TCATCTTAGA CTTGTATGAA 33951 ATAACCACTC CGTTTCTATT CTTAAACCTT ACCATTTTTG TTTTGTTTTG 34001 TTTTTTTGAG TCGGAGTTTT GTTCTTGTTG CCTAGGCTGG AGTGCAGTGG 34051 TGCGATCTCG GCTCACTGCA ACCTCCACCT CCCGGGTTCA AGTGATTCTC 34101 CTGCCTCAGC CTCCCAAGTA GCTGGGATTA CAGGCACCCG CCACCACACC 34151 TGGCTAATTT TTTTGTATTT TTAGTAGAGA TGGGGTTTCA CCATGTTGGC 34201 CAGGCTGGTC TCGAACTCCT GACCTCAGGT GATCCGCCCG CCTCGGCCTC 34251 CCAAAGTGCT GGGATTACAG GCGTGAGCCA CCGCGCCCAG CCAAACCTTA 34301 CTATTTTTTT AAAGAATTTT TTCCAGAGTT TAATTTCTGA CATAGCTTAA 34351 GTTTTCCAGT AACTCTAAAC TCCATCTCCT TTATCGTCAT TAAGTCATTC 34401 ACAAAAAGCC AGGAGAAGCA TTTGGAAAGG GCATGATAAT CAGTATAATA (SEQ ID NO: 3)
Table 5 presents a correlation between the genomic sequence shown in Table 4 and the location ofthe corresponding regions ofthe cDNA sequence shown in Table 1.
Table 5
Figure imgf000025_0001
Figure imgf000026_0001
Several sequence polymorphisms have been identified in the sequence shown in Table 4. These are summarized in the Table 6:
Table 6
Figure imgf000026_0002
A CRF2-encoding nucleic acid is also present in the genomic nucleic acid sequence shown in Table 7:
Table 7
AGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAAGAAAGAAAGAAAGAAAGAAAGAAAGA AAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAGAAAGGAAGGAAGGAAGGAGAAAA GAAAGTCAACAGTCAACATTTCAGAGATCCCAAGATACCAACACTGACCGTGCCTGCTGC TCTTCCATCCTCCTCCACCCTGCGCCTTTGAGGTGGAATTGCGTCCTCTGTGAGCAGGGC TTTGTTAAGAGATCCTAATTAAGGCCAGGCACAGTGGCTCATGCCTGTAATCCCAGCACT TTGGGAGGCTGAGGTCACCTGAGGTCAGGAGTTCAAGACCAGCCTGCCCAACATGGTGAA ACCCCATCTCTACAAAAATTAGCTGAGCATGATGGCAGGTGCCTGTAATCCCAACTACTT GGGAGGCTGAAGTGAGAAAATAGCTTGAACCCAGGAGGCGGGGTTGCAGTGAGCCAAGAT
CACACTATTGCATTCCAGCCTGGGCGACAGAGCTTTTGTCTAAAAAAAAAAAAAGAAAAA AAATCCTGATTAAGCAGAAGCCTTGATGCTAGTCCCAGAAGCATCCTGAAATTTCCAAAA GAAATTTCCCCCGCGGTTAAACTCAGAGCAACTTTTGGACCCACCAAGCTCTGTGAAAAT CATTTTCTCTTCCAAAAACTGATGGGACCAAAGCTGATCCCAGTTTCAAATAATTATCAA AAAATTGGAAACGAAATATGATCAGAAAAGAAGAAAGTTGAAAAAGAAAATCCTCATCAC
CCAAAGACAACAACCATTAATATTTTGGTAATTATTATTCCAAATATCTTTCTATGCATA CAGACAGACTGACACACACACACACACACACACACACACACACACACACACTTTTTTTTT TTTTTTGAAACTGAGTTTCACTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCGATCTCGGC TCACTGCAACCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCTGATAGC TGGGATTACAGGTGAATGCCACCACGCCCGGCTGATTTTCTGTATTTTTAGTAGAGACGG
GGTTTCACCATGTTGGCCAGGCTTGTCTCCAACTCCTGACCTCAGGCGATCCACCCGCCT CACCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGCTACACACACACT TTTTTAATGGGCCTATGTTTTAGCACTCGCTTTTCTGTTTCTCAGTGTGTTGCAAACACC TCGGTGTCGATACACACCATTCGGCAACGTCCTCCTAAAGGGCCGCATAATATTGCGCGT CGTGGCGTGTGCCTTACTGGGAAGCTACTGCTGTCCAGGTGAACACCACAGCCTTCGGGG
TCAGAAAGACAGCTTTCCCCAGAACAAGCACCTGAAGCTCTGGGGCCTGCCGCTCCCCGG GAGAGAAGTACGTGGAGAAGGGCAGCACGGATCCGCCGGGATCCCCGGGGGCATTAAAGG GAATCGCGTGTGTAAGGCGCGGAGCTCAGCATCCGGCTCAGAAACGCGCTCGGATCCCGC CAATGGCATTGAGGCCGCGTAGCCAAACCGGCCTTGAACTCTCCCTAATCCTGCCAAAAT GGCCCGTCCTGGAGCACTGGACTGGCCGTGGGTTATTGATCATCAGCCGGTTTCTTCCCC
TCCCCTGCCCTTCCCCCGTGCACGGATTTACTGATTTTTTTTTCCGGGAATTGAGTAAAA CAAAACTAAGTGCAGATGAAGCAGAGGTACGGGCGAGTTTCGAGCGCGGGGACCGGCGCG CTCCCCCCCCCCCCTCCCCCCGCGGCGGGGCTGTCCCCAGGGACCTTCTCAGTGAATCCT AGGCGGCAGGGACGGGCCCGCGGCTCTGCGGGCCATTGGCTGCCGACTGCGTCACCTGCC CGCGGTGGGCTAGGAGACGGGAGGCGGGAGGCGGGAGGCGGGGACCTGGGTCCGGGCGGG GACGCCGCGGCAGGAAGGCCATGGCGGGGCCCGAGCGCTGGGGCCCCCTGCTCCTGTGCC TGCTGCAGGCCGCTCCAGGTAAGGGCGCGGGGCCGCGGGAGGGAGGGGGAAGAGGGCTCC CCGGGCCGGGCCGCGCCTACCCTCGGACCCGGAGCTCCTGGGACAGGCACGGGGTCCGCA GCCACCCGAGCCGGGTGCGAATCGGCCCTGCCTACGCGCCCCCAGTTTGCTTCTTCCCAG GACTGAACAGAACCGGGTCTTTGATATTCCTCTCCCGCAGGAAACGAATCCAGTTTCCTA
ATGCTTCCAGCTTCAGGAGAACTGGAGAAAAAAGACAGCGGCAGTTTGATACTGCATATT TTTTAATAAAGTGCTTTTTAATGTTTCCTAAAGAAAGCACTGATCCCTGCGTGAAAACCA CACTTGACCCTAAAGTGTGGACAGCAGGGAAAGTGGGACCGATTGATGTCCCTTCCCGTT CCTGCCAGGCCTCTGGTGGGACGGAGCTCTGGTCGCCTGTGCCCTGCTTTCTAACAAGAC GGCTTTCTTTTGGTGGTGGTTGTTGTTTTGTTGTTGTTTTGTTGTTGTTGTTGTTGTTGT TGTTTTCCCACCTCTACTGATGAGTAAGGTGTCAGGTACAAAATTCCTCGCCGTAGGACC CAACCACCAAACCTCACCGCCCACGACTCCAACCGAAGCAGGGAAGAGAAGGTCCAGAAA TCGCCCCCAGGATATTTTCCTAGTCTTGGACTCACAGTTTAAAGAGCTGTAAAGGTCCCT GGGCATAATCCAATCATCATAAAAGCCTATATTTATTCAGCAACTTCTTTGTGCCAGGCA CCGCATTATTCTGGAAGCCTCACGACCCAGCCATCCTAGGAGGTAGATATTATTTTTACT
TTTCCGATGGGAAAACTGAGGCTCAGAGCAATTCAGGGAATTCCTCAAGAAGGACGGCAG AGGTGAGGCACACAGAAGAGAGAAGAGGGGCTAAAGCAAGCCTGGCTAGCTTTTGCCTCC AGGGTAGGCACGTGGGACAGGCTGTCCATCCACTGGGTCACTAGGCCAGCCAGGGATGCT CCAGCCCCCAGTGCCCACAGCAGCGTTCTCTGTGGCTGATGAGGGACCGTGTACCTGTGT GTGGAGGGAGGGTGGGGTCTTCTGTTCCCCTTTCACTGTCAAGCCCAGACCTTCTTGTAC TTTCACCTGATAAGTATTTAATATACACAACACTAACTATGGTGTGATGATTTAGGAGTA AGTACAGCCAGATCTAAGTTCAAATACTGGCTCCCACACAAACTGACTGTGTAGCCTCAG GCAAGTTAGTTAGCATCTGTCTCTGAGCCTAGCGCCCTTTCCATGGAAGCAGAATGAATG ACACCTACCCCATAGGGTGGTCTGTCCCAAGGGTGATTGAGGTTTTACATGTAAAGAGCC AAACTAGTGCCTGGCATCCTTTGAAGGCTTCATAGAGGAAAGTTGCTCTAGCTGCTGTTT
TTCTCATGTGACCTAGCTCGAATCTGGGGACTGTCCTGCCCATAGGATACCTTACAAGTG GCTTGCAGACAGCCTGGTCTCCTGCTGGTCACCCGTTAGGAAGTCCAGAAGCTGGGAGTA GTAATAGCACTAGCCTCGTGGTGATACAGTCCCAGCTAGAGGACACAGGATGAGGTGGAA GCAGGCACCCACTTTTGGGTCTAAAAGGTGATGGGTAGGCAGCCGAGGCTGGGGACAGCC ATCCACAGAACTGGACCCTCCCTCCCTGATGCCATTTTGCAACCCGTATGGATTTCCATC ATGGCACATGGGACACTTCAGGACCCTGAATTCTCCATGGGACCATGAGCTCCTATAGGG CAGGAATGAAGTTGTGTTCTTCTTTGAAACCCCTGGCACACCGTGGTCAACAGATCTTGT TTGACTCGTAGTGGTCAATAGATGGAATAGTTGGAATCATAAAGCTCAATAGACCCCATG AGAACCTAGAAGACAAAGTACAGTCAAGAGCTCGGACTTTGGAGTTGGCTAGGCCTGGAC TGAATCTGATTCTACAACTTAATAGCTGAGAGGGCCTTGGTTTTCCCATCTGTAAAGATT
ATAATTATTATAATGAATACCTACCTCCTAGGGATGTAATGAGGATTAAAAGAGAAAGTG CAGGTAAACTGTTTAACACAGAACCTGGCTCATAGAACACAATACACATTAGCTGCTATT ATTATTATTATTATTTTATTTATTTATTTTGAGACAGAGTCTCACTCTGTCACCCAGGCT GGAGTGCAGTGGCGCAATCTCGGCTCACTGCAACCTCCACCTATCGGGTTCAAGCAATTC TCGTGTCTCAGCCTCCCAAGTAGCTGAGATGACAGGCGTGTGCCACCATGCCCAACTAAT TTTTGTATTTTTAGAAGAGACGTGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCT GACCTCAGGTGATTTGCCTACCTCTGCCTCCCAAAATGCTGGGATCACAGGGGTGAGTTA CCATGCCCGGCCTTAGCTGCTATTATTATCATCATCGTTATCATCATCATCATCACCTCG TAGATATGTCAAGGAAGATTCCCTGGAGGAAGTGACATTTGAATCAAGTATTTCAAAGAC TAGATGGTGAATACCAGGCAGTCAAAGACACCTGGGTTTAAAAACATCCAGAAGAATGCA GTGGCTTGGCAACATCGAGCAGGAAGATTGCCTGATGAGCCTGTAGGGTAGCTGTTGGGG AGAGAGCAGCAAGACGGCCTGGCCAGGCCAGGCCAGGCCACGTCAGGCAGGGCCTCACAA ACCTCAATAACAAATGTGGACTTTATTCTGAGGCCAAGGAAAGGGCATGAAACTGGGGAG TGGTGTAATCAGATGCGTATTTCAGAAGATGAAGATTAACAGTGAGAAGGAAAATGTGCC ACAGAGGGGAATAGAGGTCAGTTAAAGGGAGTCAGGGAAAGTGTCCTCGAGACAGTGACA TCAAAGGAATGTGAAAACAGCAAAGGAGTGAGCCAGGTGGATATCCAGGGGCAGAACTGT TAAGGCAGAGGGAACAGCATGAGGGAACAGCGTGTGCAAAGGCCTGGAGTTGGGAGTGTG GCTGGGGTGCTCCAGGAAGGGCAAAAAGTCCTGTGTGGATGGAGATATGGGAGCAAGGGA GGAGTGGTGGGTCAGATTGGGTAGGGCCTTGGTGGTGATTGTAAAGACTCTGGAGTTTAG ACCAGGCACAGTGGCTCAGGCCTGTAATCCCAGCACTTTGAGAGGCCAAGGTGGGCGGAT CACCTGAGGTCAGGAGTTCGAGACCAGCCTAGCCAACATGGTGAAACCTCGTCTCAACTA AAAATACCCAAATTAACCAGGTGTGGTGGCACAAACCTGTAATCCCAGCTACTCTGGAGG CTGAGGCAGGAGAATCGCTTGAACCCGGAAGGTGGAGGTTGCAGTGAGCTGAGATTGTGC CACTGTACTCCAGCCTGGGTGGCAGCATAAGACTCTGCCTCAAAATAAAATAAAAATAAT AAAGACTTTTGAGTTTCCCTGGAGTGAGAGGAAAGCCTTAGAGGGCTTTAGCAGGAGATG AACATGATCTGATTTTCATTTTTAATCCTTCCTGCTATGTGGAGAATGGACTGAAGGCAA GGTGTTTTGTATATTTGTCTGTTTCGTAGAGACAGGGTCTTGCTCTGTTGGCCAGACTGA AGTGCAGTGGCACAATCACGGCAGCCTTGAACTCCTGGGCTCAGGCGAAACTCCCACCTC AGCCTCCTTACTCTCACCATTGTGCCCTGCTAATTTTTTAAAAAATTTATTTTGTAGAGA TGTGGTCTCACTATGTTGCCTAGGCAAGTCTTAAATTCCTGGTCTCAAATGATTCTCCTG CCTCGATGTCCCAAAGTGCTGGGATTACAGGTGTCAGCTGCCATGCCCGACCTGTATTTT TTTTTTTAATGGGGAAAAAGCCTTTTAATAGTATGAGGTGTTTTCTGGTGTTTCTACCAT AAAGCTCTTCTGTAAATCAAAATGAGAATGTAATTATTGATAGAGCAATGACCTTAGACT ACAGTGCAGACTTTTCATCTTACATTTGGGCTCATGAATTTTAGTATAACTGATTATGAC AGTGTTTTTTACATAGTTATGATCTAGAGCAGAACTGAAAACAAAATAACACATACTCTA CATCAATATATTCGTTCAGTAATATCTGGGCTTGGATGAACCTGCAGAAGTAGGTAAAGC TGTCAGATATTTTCTTAAACCAACAGAAAAGAAATGTATATGACAGATGTTGTGTTTACT TACTTATTTATTTATTTATTTATTTATTTGAGATGGAGTCTCACTGTGTCACCAGGCTGG AGTACAGTGGTGTGATCTCTGCTCACTGCAACCTCCACCTCCCGGATTCAAGCGATTCTC CTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGTGCACCACCACGCCTGGCTAATTT TTGTGTTTTTAGTAGAGACAGGGTTTCACCATGTTGGTCAGGCTGGTCTCGAACTCCTGA CCTCGGGATCTGCCCACATCAGCCTCCCAAAGTACTGGGATTACAGGCATGAACCACCAC GCCCAGCCTGTATTTATTTTTTTACCACTATGGAGTCCAATATGAAATTCTCACAACTAT GCATATACATTATTAACATGTAAGCACACCTAGGTATAAATATGCACATAGTCCATTAAT TACATCAGGGGAATTAAAAACATACTTTCAAGTTAAAATGAATTTTCAGGAAAAAAACTG CATTCACAAATCTGAAATGTGAATACAAAAATGAAATTGTGAAATAAATAATGAATATAG GTGTCACCTAAACTTCCATAGTAACATGCCTCCAAATGTGGATTTAGTGATCATCCACCT TGGGACAAGGGCTTTTGAGAGCCTCCAGCTAAATTAGGGTTCCAGTAGCAGAGTGGCTGG CAAGCCTGCCCTAATGAATAATGCCAGCGAGCTGGGCGTGGGTACTTACAGTGTGCCCTT CATGGAATACTTTTTTTTTTTTTTTTGGAATGGAGTCTCGCCCTGTTGCCCAGGCTGGAA
TGCAGTGGCACAATCTCAGCTCACTGCAACCTCGTCCTCCTGGGTTCAAGCAATTCTCGT GCCTCAGCCTCCCAGGTAGCTGAGACTACAGCCCTGTGCCATCATGTTCTGCTAATTTTT GCATTTTTAGTAGAGACGAGGTTTCACCAAGTTGGCCAAGACTGGTCTTGAATTCCTGAC CTCAGGTGATCTGCCCACCTTGACCTCCCAAAGTGCTGGGATTACAGGCTTGAGCCACTG CGCCCGGCCCATGAAATACTTCTTACCTGGCGGACAGCCTAATAGCCTAGCTGTCTAACC CATGGCTGGGGGTCCTTCACACTTGTTTATACTGGCAGACGTCCCTGTGACTCTTGTCTG ATCCATGTCCAAGTTTATGCCTGTCTGACCATTGCTCTGGCGCTGGGAGCCAGACTGTGT TCCCAGCAACCCAGGGAAAACCAGGCCTGGGCTGGGCCTGGGTTCCTGAGATGGAAGGTG CAAATTCAGTACACCACCTCAATGCAAAACAAGTTCAAAGGCTTATTACTTACAGATCCT GAGCAGGGAAGGTGCAATGAGTAGGGAGGGTCATCCTCCATCCTGGGCTACATGAAGCGG
GAATGAAGAGTCAGGCAAAAAGAAAGTGAGAGCTTGTGGCAATGAGAAGTATATTATGTA AGGGACTAGGGTGTGGGTCAGGTTAAGTTTGAGGGCAAATGCTTGAATGATCCCTTTAAA GGAATGGGTGGGAAGTGGGGAGCCCAGTTTGCCGGGAGGGAGAGATGCCTCGAAGTTCTT ATCTCTGGCCACTGGCTTGGACCATCTGAGTGTGGCATCTACTTCTAATGCCTAGGCAGC AACCTTTGCTGTGTCATCTCCCTTACACAAGGTTGGAAGCAAGGAGACCGGTCAGGAAGC CTTTGGTGTAACCCATGTTATTGTAATATTCATTCATTTACTCAACAGATGTTTATTGTG CACCTACTATGTGCTGAGGCCATGGCAGGCAGGCTCTGGGGATGTGGCTGAGAACAGGAC AGAGCCCCTGGTCCTTGATATCCTCAAGGATGCTCCCTCCTGGAGGCCATTAGGTTCCTG TTCCATGGTGTTCTGCTGGAACCCTCCGGTCCCAGAGTGTGCAGGAGCCTCCCCTCCTGG CAAAGGGTCTTCTCTCATGGCACAAGGGCTGCAGTACAGCCAGTCAGTGGCTCCTGGTTC
CTCAAACTCAGTGAGCACTTGCCTGCCCTTCGTGCTGCCCCTCAGCTTGGGATGGCCTGA GTCAAGACCAGCCAGGAGCTCCAGGCTTCATGACCCCTTTCTTTCCCCCAGGGAGGCCCC GTCTGGCCCCTCCCCAGAATGTGACGCTGCTCTCCCAGAACTTCAGCGTGTACCTGACAT GGCTCCCAGGGCTTGGCAACCCCCAGGATGTGACCTATTTTGTGGCCTATCAGAGGTAGA GGGGACTCTCTCGGCTGGTGGATGGGAAGACTGAGGGGGTGGGTGGGGGCTGGAGGGGCT
TCTCTGGGACAGCTGCACCCAGTGTGGGCAGCACTGGCTAGCTCTCTGGGCCCTACGGGA GATGGCATGTGGCCGGCATTTGGAGAGGGGCTTTTGATAAAGGTCTGGAGGTGGGGAAGA TGTTGAATGAAGAGCAGTGTACAGGTGACCAGTCTGCCGGGGCGGGGGTAAGTCTTTGAG GAAAGTTGGTGTGGGGCATGGATGTAGCTGTGGGGGCCAGAGGATGAAATTCTCAAGTGG CTGGATGAGGTGCTTGGAGCTGTCCCAGCTGATCAGTGAGGCAACTAGGTACACGGCAGA
GGAGCTGTTACCTGGGCAATTAGGCATCCCTCAATGATCACACTTTTTTTCTCTTTTTTT TTTTTTTTTGAGACAGAGTCTTGGTCTGTCACCCAAGCTGGAGTGCAGTGGCTTGATCTC GGCTCACTGCAACCTCCACCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCAGAGT AGCTGGGATTACAGGCATATGCCACCACATCTGGCTAATTTTTGTATTTTTAATACAGAC GAGGTTTCTCCATGTTGCCCACGCTGGTCTCGAACTCCTGAGCTCAGGTGATCCACCCAC CTCAGCCTCCCAAAGTGTTGGGATTACAGGCGTAAGCCACCGCGCTTGGCCAAATGGTCA CACTTTTCCCGATGGGATCATTCTCAATTTGGAAGCCCAGGCAGCCACAGCGAATCCAGA GAAATCTGACAATGGAAGCAGATCCACCATCTTCGAACATAGATGGGAATCGTTCAGAGT TCTTTAGCAGGACAGTGAGATGATAGAAGCAGAAGCTCGGGAGGATTCACCTGGAGTTGG TGAGGAGGGGAAAGCAGGAAGAGGAGGGGACCACCGTGTCCTCAGGACCCGTCCTGTGCC
AGGCCAAGTGCTAAGGGCCCTACGTGAATATTTCACTTCCTTCTCCCAATGTGACCAGGC AGGCTCTGTGTTTTCCCCATTCTAGAGGTGAGGGGATTGAGCTCAGAGGGTGCTGTGTCT TGTCTGAGGAAGGACGTCATGGAGCCAGAAGGGGAACTCGGGTCCGACTCCAACATTTGT GCCCTTCCTGTTGCATCACGTCATCCTTCCATGTGTGGAATCCACATGTGAGTGATGGGA GCCTGGCTTGAGCAGGGACAGACTGCAAGAGAGCTTTCAAAAGCAAGAGCGTTATCAGGT
GCCAGAAAACACCTAATATTTACTGTGTGGCTGGCACTGTGTCAACACATGTAATGAACT TAATCTCACAGCAGCTCTCTGAGGACAAGTTCAGTACGCCTCTTTACAGAGGAGGAGACT GAAGCACCAAGGGTGCATGTTGCTCAAAGTCACACAGCTGGGCGTAGTATGGCTGGAATA AATTTATTAAGGAGTTGAAAGTCTATCCTCTAGGACCAAGCATGGTGGCTTACATCTGTA ATCCCAGCACTTTGGGAGGCCGAGGTGGGTGGGGAGATTGCTTGAGTCCAAGAGTTCGAG
ACCAGCCTGGGTAACATGGTGAAACCCTGTCTCTACAAAAAAAAAAAATACAAAAAATTA GTGAAGTGTAGTAGCATGTGCCTGTGTTCCCAGCTACTTGGGAGGCTGAGGTGGGGAGGA TCACTTGAGCCCAGGAGATGGAGGTTGCAGTGAGCTGAGATCACACCACTGCACTCCAAC CTGAACAACAGAGCAAGATCCTAAAAAGAAAGAAAATCTATCCTCTGAACTTCTATGATA TTTTTCATGTCTTTTATACATTAGAATGGTGATATTCTAATTATATAATTTTTTTCATTT
GTTAGTTGGAATTATTTTATAAAGAGATGTATCCTCTCATCTGGTATTTGATATCCAGTC ATACTATTCAAATAGGCAAGAGAGGATAAATGCTTAATTTTTTTCCTTTATCAATTTTCA AGATAATGAATTGGTTCCTTATCATCTCCCAAAGGTGATTGCTAGTTTATTATTATCATT ATGAACTCAGGCATTTAAACACATTTGGTGGTTTCAGTCTATTGCGACGTACTCTGCTCA TTGAAGCTTGAATTGCCTCATCTCTGTCCAGTGGGAGTCTCATCAAGTTTGCTCCTGAGT
CCTTTTAACTTGACCCTAGTGGTCAAGTTAAATCTTTCCAGATTTAACAGATACCTTTCC AGCTGTCCATTACGACAAGATGTTCCAGGTCCCTCTGGTACAATTCCTGACCTAAAACCT GCAGTCAGCCATTTCTCCATTTAGTAAGAAATGGTTATAAAGACTATAATCTGCATGCTA GCTATGCTGATCACTACTTAGCTATTGCTTTTGGTGTTTTCAGTGAACAGAGTGATGTGT GTATACCACATAGACACACACATGTACATACTTTTTTTTTTTAGACAGAGCTTCACTCTG TCACCCAGGCCAGAGTGCAGTGGCATGATCTCGGCTCACTGCAACCTCCACCTCCTGGGT TCAAGAGATTATCCTGCCTCAGCCTACTAAGTAGTTGGGATTACAGGCGCCCACCACCAT ACCCGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGT GTCGAACTCCTGACCTCAAGTGATCTGCCCCCCTCGGCCTCCCAAAATGCTGGGATTACA GGCATGAGCCATCGCACCCAGCCTACATGTACATAATTTTTAAGATAAAATGCCTAATGA
GTTATACGGGTGCTTCCCATCTAAATTTAGTTCCTTAGGATTTTTACCTGACTTCTATGG TACATCTATATTTTCTTTCTTTCACACTGAGAATCCTGTTTCTCAAGGACAGGGGACATG ATAGAACTAGAATGACCCATAATTACTCATTTTCTTTATCCCAAAACATACATACTTGCC TCTTAATAGTTTCTTGCTCTTTTCGCCCAAAGGGTTTGTGATGGTCAATATTAGGTGTCA ACTTAATTGGGTTGAAGGATGCCTAGATGGCTGTTAAAGTTTTGTTTCTGGGGGTGTCTG
TGAGGGTGTTGCCAGAGGAGACTGACATTTGAGTCAGTGGACTGGGAATGGAAGACTCGT CCTCACTCAGTGTGGGTGGGCACAACCCAACTGGCTGCCAGGCTGGCTGGAAAGCAGGTG GCAGATGGTGGGATAGCTTCGCTTGCTGGGTCTTCCAGCTTCCTTCTTTCTCCCGTGCGG GATGCTTCCTTCTGCTCCTCCTGCCCTTGAACATCACACTCCGGGTTTTTTGGCCTTTAG ACTCTTGGACTTAAGTTAGTGGTTTGCTGGGGGCTCTCGGATCTTTGGTCACAGACTGAA
GGCTGCACTTTCAGCTTCCCTGGTTTTGAGGGTTTCAGATTCGGACTGAGTCACTATGGC TTCTTTCTTTCCCACCTTGCTGACGGCCTATCGTGGGACTTCGCCTTGTGATCGTGTGAG CCAATTCTCCTTAATAAACTCCCTTTCATATATACGTATAACCTATTAGTTCTGTTCCTC TGGAGAACCCTGACTAATAAAGGGTTGTTGCTTTTTCTTTAAAATCTAGTAATTTTATTT GACTGTGTGTTGGTATTGCTCATTCATTCTGAGTTGATATTTTTAGGCACTCAATATTCT
CACTTAATACATGGTTCCAAGGCATTTTTATTTTAGGAAGGTTTTCTTAAATTATAGTTT TAGTATTTGTTCTATTCTCTTGTTTTGATTTTCTTCTTTAGGGACTCATATCACTTGTAT GTTGGATCTTCTTTTTCTGTGTTCAGTATTTGTCTTTTGGGCACAGAGACTCACACCTAT AATTCCAAGACTTTGTGAGGCATAGGTAGGAGGATCGCTTGAGCCCAGGAGTTTGAGACC AGCCTGGGCAACATGGTGAGGCCCTGTCTCAAATTAAAGAAAAAGGAGAGAATACTTGTC
TTTTTCTTTCAAATGCCTTTTATCTGTCTGTCTATCTACTATTCTGCTCTCTAAATGAAA TAGGTTTCACTCTTGAGTTTTTAAAAAACTGTGTGCTTCCATGTGTGAGATTATTCAACA TCTTATTTGTAATCTTTCTCTTGGTTACATTTATTTTTCCTGAAACTCTAGTCTGCTTTT AGCTGACATGTTTGTAGCTAAGAGCGCACATTTCTTATCATAGCTTGCCGTGCTGAATTA ATTCCAATTTTCTTTTAAAACCAACATTATTGAGTTAAAATGTATATAGAATAAACTGTT CCCATTTTAAAGTATACAATTTGATGAGTTTTGACAAAAGTGGGCACCCACGTACCCACC ACCACAATCAAGATGTAAGACGTTCTCTATCACCCCAGAAAGTTCCCTCATCCACTTTGC ATTCAGGCCTCCAGATCTAGGCAACCACAGATCTGCTTTCTGACACTGTGGATTAAACTT TGCCTGTTCCAGAATTTCATATAAATGGATGTGTATAGTATGTACCCTTTCGTGTCTGGC TCCTTTCCCTCAGCATAATGTTTCTGAAATTCACCCACATTGTTACATGTATCAGTAGTT
AATTCCTTTTTATTGCTGAGTAGTAATGCCATTGTATGACTATGTATGACATTTGTTAAT CCATTTTCCCGTCAGTGGATATTTGGGTTGCTTCCAGTTCTGGGCAGGTATTCATTTGCT AGGGCTGCCATATGCTTGCCCTCTGGCCTCCCAAAATTTGTGTCCTTTTCATATGCAAAA TACATTCACCCCCTCCCAACAGCCCCAAAACTCTCTTTTTTTTTTTTTTTTGAAACAGAG TTTTGCTCTTGTTGCCCAAGCTGGAGTGCAATGGTGTGATCTCGGCTCACTGCAACCTCT
GCCTCCCGGGTTCAAGAGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCA TGCGCCACCACGCCTGGCTAATTTTTTATATTTTTAGTAGAAATGGGGTTTCACCGTGTT AGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCGCCTGCCTTGGCCTCCCAAAGG GCTGGGATTACAGGCATGAGCTACTGCACCTGGCTAGCCCCAAAACTCTTAACCCATTTC AGCATCTACTCTAAGTCCAAAGTCTCATCTAAATCAGGTATGGGTGTGACTGGAGGTGTT
ACTCATCCTGAGGCCAAATTCCTCTCCACTTATGAACCTGTGAAACCAGACAGGTTATGT GCTTTGAAAATAAAGTGATGGGACATGCATGGGATAGACTTTCCCATTCCAAAAGAGAAA AATAGGAAAGAAGGAAAGAGTGACAGGTCCCAAGCAAGTCTAAAACCTCGCAGGGCAAAT TCCATTAGATTTTAAGTTTCAAGAATAGCCCTCTTTGGCTCAGTGCTCTGCCCTTTGGGC CCACTGGGGCGGCAGCCCTATCCCCTTTGCCCTGGGTGGTGACCCTACCCTCGAGTCACT
GGTTAGCAGCAGCCTAGCCTGCTGAAACTAAGGAGGGGACAGTGTTGCCTCCAGGTCTTT GGTGGCAGTGACAACCCTGCTGATCTCTGAATCATCTTCCAGGAAATTTTTCCCTATACT TGAAGGATATTGCGTGTTCACAGCCAAATAGCTCCAGCTCTTGTCCCTTTCTTTAGAATC CCAGAAGTCCAACAGCCTTCCTTCATTCTGTCCCATCTCTGTCCCCTTTAGTCAAAGCTG GAAGTGCCTCTGCTGGTATAATCCCATCAGTATGTCTAATTTCTGCTTAAATGGCTGATT
AAGTCTATGAGTTGCACCTCTGATCTCTTTATCAAAAGGTTGTTCTAGCCACAACCTTAG TGTCCTCCCCAGAACATGCTTTCTCATTTTTTTTTTTGCAATGTGGATAGGCTGAAAATT TTCCAAAGCTTCAAGTTCTAGTTCCTTTTGGCTTACCAATTCTTTTCATATATCTCTTCT CTCACATTTTACTATAAGCAGTAAGAAGAAACCAGGTTGTACCTTCAGCACTTTGCTTAG AAATCTCTTCTGCTAAGCATCCAAGTTTATGTCTTTTAAATTATCTTTTTGTTATTTATT TTATATTATCATTTTTGAGATGGCTAGCCAATGATCTTTTAACTTCTAATTTCTGCAAAA CACTAGAAGACAATTCAACCAGTTCTTTGCCACTTTATAACAAGGATCACCTTTCCTCCA GTTTCCAATAACACATTCCTCTTTTCCACCTGAGACCTCACCAGAATCACCTTTAATGTC TATATTCCTACCAATAGTCTTTTTAAGGCAATATAGGCTTTCTCTAACATGCACTTCAAA CTTCAAGATTCTACCCATTATGCAATTCCAAAGCCACTTCCACATTTTTAGGTATTGATT
ACCTCAGCACCTCATTTCTGGTGCCCAAATCTGCACTGGTTTGCTAGGGCTGCCATAACA AAGTACGACAGTCTGGGTAAACAACAGAATTTTATTTTCTCAAAATTCTGGAGGTTGGAA GTCCAAGGTCAAGGCGTTGCTAGGTTTAGTTTCTCCTGAAGCCTCTCTCCTTGGCTAGCA GATGGCTGCCTTCTTGCTGTGTCCTCACGTGGCTTTTTCTCTGTGTGTGTTCACTCTGGT ATCTCTTCCTCTTCTTACAAGTACACCAGTCCTACTGGATTAGGGCCCCAGCCTTATTAC
TTCATTTAACCATAATTACCTCTTTAAAGCTCTTATCTCAAAACACAATACCACTGGGGA TGAGGTCTTCAACATATGAATTTTGGGGGAACTCAATTCGTCCATAATAGGGCTATTATG AATTAAGCTGCTGTGAACATTCATGTACAAGTCTTTGTGTGGATATGTTTTCATTTCTCT TAGATAAAGATCTAGGAGTATCAGCCTGGGCAACATAGTGAGACCCCATCTTTACAAAAA ATTTTCAAAATTAGCCAGGCATGGTGGCGTACACCTGTAGCCCTGCCATCTCAGGAGGCT
GAGGTGGGAGGATCCCTTGAGCCCAGGGGTTTTAGACTGCAGTGAACTATGATTGCACCA CTGCACCCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCTAAAAAAAAGAGAGAGAGGGG AGGAAGGAAAGAAGAAAGAGAGGGAGGGAAGGAGGGAGGGAGGGAGGGAGAAGAAAAATG GATCTAGGGTTAAGATTTAGGAGATTAGGTAATGAATGTGTACTATTACAGGGAACTGTC GAGCTGTTTCCAAAGTGACTGTACCATTGTTCATTGCCACCAACAATACATGAGAGTTCT
AGTTACTCCATGTGCTTGTTACACTTAGTATTATCAGTCTTTTTCATTTTAACCATTCTA GTGAGTATGTAGTAGTATTTTATTATGGCTTTAATTTACAACTCCCTAATGATGAATGAT GTTGAACATCTTTTCATGTGCTTATTGGCCATTCATATATCTTTTGTGAAGTGACTATTC AAATATTTTTCCACTTTTTATTAGGTCATTTATTTTCTTATTATTGAGTTATCTATGAAT ACAAATCCTTTATCAGTGTATGTATTGTGATTTTTTTCCCCAGTGGCTGGCCTTTTCATT
TTCGTTAGGCTTTTTTGGTGGGTTTTTTTTTTTTTTTTTGGAAGAGAAAAATATTTTAAT TTGATAAAATCCAGTATATCAGGTGTTATAGACTGAATTATACTCTACCCCACAAATTCA TATGTTGAAGCCCTAACCTCTAAGTGACTATTTGGAGATGAGCCTTTAAGGAGGTAATTA AAGTAAAATGAGATCATAAGGGTGGGCCCTAATCTAATAGGACTGGTGTCTTTATAAGAA GAGGAAGACACCAAGAGCGCATGCACACAGAAGAACGGCCTTGTGAGGACACAGCAAGAT GACGGCCATCTGCAAGCCAAGGAGAGAGGCCTCAGTAGAAACCAAACCTGCTGATGCCTT GATCTTGGACTTCCAGCCTCCAGATTTCTGTTGCTGAAGCCACCCTGCCTGTGGTGTCTT ACCATGGCAGCCCTCACAGACTAATATATCAGATTTTTTTCCTTCAACAGTTAACGCTTT TGGTGTCCTAAGCAATATTCGCCTGACCCAGGGTCATGAAGATTTTTCTTCTATGCTTTC TTCTGGAAGTTCTATAATTTTAGCTTTTACATATTTTTTTAACTTTCCTTCTTCTTGCCT
TCTGTTTCTTTTAAGGCATCATCTATTGTGTTAATTTGTTCTTGTATTCCTTCTGATTTA TTCTTCACTTCTGAAATGAATTTTGCTTTTTAAAAATATATATAATTCTTTTCTGTGTCT GAGTTTTTCTAATTAGGTTTTATGTGGTTTTTTCTTGTCCTGCATCACTTTTTACTGTCT TTTGCCCATTTTGAAGTATCAGGTTCCAGTTTTGATCTGTTCATGGATATGTTTTTGTGA CATGTTTCTTCTGGCTTCTTATCATTTATTGCTTAGCTTATTAATTTCTATTCTTTCTTA
TTTTCTATTATAAGTATTTAAAGCTATATGTTTTCCTCTAAGTATTACTTAGCTGTCTTA TACGTTTTCATTTGTGTTATTTGGTGATCATTCACTTTCAGCTATTTATTAATTTCCATT ATAATTCTTTCATCTATGGGTTGTTTTAAAAAATATTTTTAAGGCCAGGTGTGGTGACTC ACATCTGTAATCACAGCACTTAGGGAGGCTGAGGTGGGAGGATTGCTTGAGGCCAGAAGT TTGAGACCGGCCTAGGCAACAAAGTGAGACCCCCTCTCTACAGAATATTTTTTTAAAATT
AGCTGGGCCAGGCGTGGTGGCTCATCCCAGCACCTGTAATACCAGCACTTTGGGAGGCCA AGGCAGATGGATCACCTGAGGTCAGGAGTTCGAGACCACCCTGGGCAACATGGTAAAACC CCATCTCTACTAAAATATAAAAATTAGCCAGGTGTGGTGATAGGTGCCTGTAATCCCAGC TACTTGGGAGGCTGAGGCAGGAGAATTCTTTGAACCCAGGAGGAGGAGTTTGCAGTGAGC CGAGATTGCACCACTGCACTCCAGCCTGGATGACAGAGCGAGACTCTGTCTCAAAAAAAA
AAAGAAAAGAAAATTAGCTGGGTGTAGTGGCAGGTACCTGTGGTCCCAGTGACTCAGAGA CTGAGGCAGGAGGATCACCTGAGCCCAGGAGTAGAGGCTGCAGTGAGCTATGTTTGTGCC ACTGCACTCCAGCCTGTGCAACAGAGCAAGACGCTGTCTCAAAAAATATATATTTTTTTA AATTTTCAAACTTCCTTTAGTTCTCTTTTTGTTATTAACTTTTAACTGAATGTTTTGCAA TCAGAAGAAATACTTTATGAGATACCTATTCTTTAAAATTTCTTAAGAATTGCTTTGTGT
TAATATTTTGTTAATAGTTCACATGTGGTTCAACCAATTTGTTTAGTTAGTTCTGTATAT GTTCATTAGACCAACTTGATAACTGTGTTGTTCTTTATTTATTTATGTATTTATTTTTCT TTGTCTATTCATCAATTGCTGGGTGAGATGTATTAAAATTTCTTGTTGTAAGTGTGGCTG TTCACTTTCTACCTGTAGTTTGTCTGTTTGCTTTATAGAGGGTGAAGTTGTTTAGTAGGC ACACATAAGTTAGAATTTTTCTGTCTTCCTGGTGAATGGAATCATTTATCATTATCTAAT GTTCTTTTCATCTTTAGTATTGCTTTGGACTTGGAAGTCTGTATTTTGTCTCCTGTTAAT ATAACTACACTGGTTCCTTTGGTGTGAATATTTGCATAGTATAACATTTTCCATGAAGAA ACAAAACAGAGGAATTGGTTCTTTCTCAAAATCTGATCTTTGTGTCAGCCCCCATCTCAG CCTTCTCCATTCATCCTTGGTCACTCCCCAAACCCAGGAGCAATCCTTGATTCTCCTTTT CCCCACATTCTACATCCAATCCGTTAGCAAGTTCTATTAGTTCTATTATTACCTCCAAAA
TAGATATTGAATCCAGCCCTTTCTCACTGTCTCCACCATCATCCTGTCTCACATCCCTAC CATGGCCTCCTTGCTGGTTGACCAGAGTGATCTTGTAAAAACATGTTAGGCCAGGCACGG TGGCTCCTGCCTGTAATCCCAACACTTTGGGAGGCCAAGCGGGTGGGTCACCTGAGGTCA GGAGTTGGAGACCAGCCTGGCCGACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAT TAGCCAGGTGTGGTTACGCTGGCCTGTAATCCCATCTACTCGGGAGGCTGAGGCAGGAGA
ATCACTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCCAAGATCATGCCACTGCACCCCAG CCTGGGCAACAGAACAAGACTCCATCTCAAAAAATAAAAATTAAAATAAAATGTTAGGCT CCCTGGGTCTCTGGCTTAGTCCATTTGTACTGCTTTAACAAAATACCTTAGAATGGTGTA ATTCTAATAATTGCTATTAATAAATAATAGCAATTAATAAATAATAGCAATTTCCTTCTC ACAGTTCTAGAGGCTGGGAAGTTCAGGGTCAAGGTGGCACCTGACTCCGTTCTGGTAAGG
GCGGCTCTCTGCTTCCAAGATGGTGCCTTCTCGCTGCGTCTTCGCATAGCGGAAGGGCAA ACACTGTGTCCTCACGTGGCAGAAGAGATAGAAGGGCCAGGCAGCTCTCTGAAGTATCCA GGTTGGAGTCATGGACCTGCATGTTCCCCTCTGACATCCACAGAGTACCTATCATGGTCC TTGGCATGCAGCAGGTGGCCCATAAACGCCTGAATGAACAAACATATAGTAATGGTCGCT AGTACTAGGAATAGCAGCCACCGCAACAGTCCTGTGAGGGAGGCATTACAGATGAGGAAA
CTGAGGTTTAGGGGCAAGGACCTGCCCATGGTCCCAAAGCTAGGGAGGGACAGGGCTGGG ATTCCCACTCCCATCCATCTGGCTCCAGAACCTGAGCTCCTGACCAGGCTGTTCTTATCC TGTCTCAGCCAGTGGCTGCCTGTCTGGACGGATGGACCTAAAGTCAGTCCAGCCAAACAG AGGGAAGCATGATCAACTGTTCTCTAAGTTCCCTGACCCGGAGAGGCTGAGTCCATGGCC CAAGCTCTCCTCTCTCCTCCCCCAGCTCTCCCACCCGTAGACGGTGGCGCGAAGTGGAAG
AGTGTGCGGGAACCAAGGAGCTGCTATGTTCTATGATGTGCCTGAAGAAACAGGACCTGT ACAACAAGTTCAAGGGACGCGTGCGGACGGTTTCTCCCAGCTCCAAGTCCCCCTGGGTGG AGTCCGAATACCTGGATTACCTTTTTGAAGGTAGGTCTGTGGGTAAGGGACTGAGTGGAA GGCTGTCCATCCCATCGGGGAGCTGTGCTCAGTGCTCAGTGGTTCTGTTCTCCTGACCAT CTGTCTCCCACTTCCCCAAAGCAGAGGGCAGCTCCCTGGGCCAGGCCCTTTGAGATGGGG TGTGGGACCAGCAACAGCGAGGGACCATGTCTGGCAGCCTGTCAGGGAGTTAGGGGAGCT CCAGCCAGCACCAGCAATCTCACGTGCACCCTCTGCTAACAATGTTCATTATTTTCAGTT GAGCACCATTTTGGTCATGGACTACACAAGGCACTTTATATGCTTATTCCTATTTTTTTA TGTTCAGCTTCTCTCCTTAAAAACAATGTTTAAAACCAATTCTGGGCCAGGCGTGGTGGC TCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGTGGATCACCTGAGGTCAGGA
GTTTGAGACCACCCTGGCCAACATGGCAAAACCCCGTCTTTACTAAAAATACAAAAATTA GCCAGGCTTGGTGGCAGGCACCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAT CGCTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCCAAGATCACGCCCCTGCACTCCAGCC TGGGCGACAGAGCGTCTCAAAAGAAAAAAATTAATAAACAAAGAAAAAAAAACAAATTCT GTTTGCAAAAGTATTTTCTATACACTGTAGAAATTTGTGGGGTGTGGGGGGGTAAAGATG
ATAGAAAAAAAAATGTCCCATGCTTACTGGCAGAAATCATGTATTGACATTGGGTGAGGA GGGCACTTTTTTTTTTTCAGTCTATTTTTAATCTTCACAGCAAACTTGTGAGGTTCATTT CCATCAACCTGAGACTCACAGAAGCTAAGAAACTTGATACCGCTAGTAACCAGTGGACTT GATACCGCTAGTAACCGGTGGACATAGATGTGAACTGGATCTTTCTGACCTCGGGCAGGG CCGGGTAACAAGGGGAGGATAAATGCCCAGACAGTGTCCTCAGAGAGCTGAGAGCTGTAA
CTTGCTGCCCGGGCTTCTCACAGTGTTCAAGGACAAAATAAGGCTTTAAGAGAGAAGAGG GACAGACTGATTGCAGGGCAGCAGGAAGAGATGGTAGAGAAGGAAGAAGAGATGATTCGT GTGGAAAGAAGCTGGCTCGGTGGATGGATAAAAGAAGGGAAGGACAGATGGGTAAGAAGA AAGGGAGGATGGAGGGGATGGAGGAGGAAGCAATGGAAAAATGGGAAGGAAGGAGGTTGG ATGGAAGGATAGATGCCTATTAGGAAGGAAATATGTGTGGATAGAGAGATGGAGGATAGG AAGTATGTTAGTCAAGGTTCTCCAGAGAAACTGAACCAATAGGATATATACAGATACACT AAGAGGAGGCCAGCCGGGCGCGGTGGCTCAAGCTTGTAATCCCAGCACTTTAGGAGGCCG AGGCGGGCGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCTAACACAGTGAAACCCC GACTCTACTAAAAATACAAAAAAAAATTAGTTGGGCGTGATGATGTGCGCCTGTAGTCCC AGCTGCTGGGGAGGCTAAGGCAGGAGGATGGCGTGAACCCAGGAGGCAGAGCTTGCAGTG
AGCTGAGATCGTGCCACTGCACTTCAGCCTGGGTGACAGAGCAAGACTCCGTCTCAAAAT AAATAAATAAATAAATAAAAAGAGGCCAGCCATGGTGGCTCACACCTGTAATCTGAGCAC TTTGGGAGGCCGAGGCGGATGGATCATTTGAGATCAGGAGTTCAAGACCAGCCTGGCCAA CATGGTGAAACCCTGTCTCTACTAAAAATACAAAAGTTACCCGTGTGTGGTGGCACACAC CTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATTGCTTGAACTTGGGAAGCAGA GGTTGCAGTGAGCTGAGATCACGACACTGCACTCCAGCCTGGGTGACAGAGCAAGACTTT GTCTCAAAAAAAAAAAATTTATAATAAGAGGAGATTTATTATGGGAATTGGCTCATGCAA TCACAGACACAAAAATGTCCCCCAGCATGCAGTCATGGGCTGGACAACCAGGAAAGCTTG TGGTGTGATTCTGTCTGAGTCTGAAGGCCCAAGGCCAGGGGAGCAGTGGTGTAACCCCCA GTCCGAGGCCACAGGCCCGACAATCAGAGGGGCCACTGATATAAGTCCCAGAGTCCAAAT
GCCGGAGAACAGGAAGCTCCAACGTCCAAGGACAGGAGAAGTTGATGTGCCAGCTCAGGA AGAGAGAATGTGAATGTGCCATTCCTCCTCCATTTTTTGTTCTCTTTGGGCCGTCAGTGG ■ATTGGATGATGCCTGCCCACACTGGTGAGGACAGATCATCACCAAATCTGCCGATTAAAA TGTTAATCTCTTCTGGAAAAATCCTCACAGATGGGCCCAGAAATAATGTTTTACTGTCTA CCTGGGTATCCCTTAGTGCAGCTAAATTGACACATAAACTTAACCATCACAGGCCAGGCA
CTGTGGCTCACACCTGTAATCCCATCACTTTGGGAGGCCAAGGTGGGAAGATCCTTTGAG GATGAGGTAGGCAGATCACTTGAGCCTAGGAGTTCAAGACCAGCCTAGGCAACATAGGGA GACCTCGTCTCTACAAAAAAAAAAAAAATTTAAATTCGCTGGGTACGGTGGTGGGCACCT GTGGTCCCAGCTATCTGGGAGGCCAAGGTAGGAGGATGACTTGAGCCCAGGAGGTCAAGG CTGCAGTGAGCCATGATTGTTCCATTGAATTCCAGCCTCGGTGACAGAGCAACACCCTGT
CTTAAAGAAAGAAAAAATTTAACCATCACAGAAGGCAGAAGAAAAGGCAGATGGGTGGAT GAGATGGGTGGGTAGATAGTATAGAAGAAAAGCGGGACATCCAGGCAGGGAAGGAAGGGC TGGAGCGAAGGAGAAGCAAGGAAGGAAGGAAGGAGAGACAAGAAGGAAGGATGTGTAGAA AGGTGGAAGAGAAAAGAAGAATGGATGTATGGGAAGAATGGATGAGTAGGTTAGAAGGCT CACTGGCTAGATAAAAGGTGAGAAGTATAAATGAATAATAAGAAAGGAGGCATAGGAAGA
AAAAAATATTGGTTAGAAAGGATGATTGAGAAGAAAGGGTGGTTGGGAAGGAAGGAAGGA AGGATGGATGGATGGATGGATGGATGGGAAGGAAAGGAAGGATAAGAAGGCAGACAGGAA GGCTCTCTGGCTAGAAGAATGGCAGACAAACCACAATAATTGCTGAATGGGTAGGAATAA GACATTAGAAGAATAAAGGGAAAGACACAAAGATATTTAAAATGTTTTCATTAATTTTTT GCCTCCTCCCTGAATTTCTCCTGATTCTTCAGCCCCACATCCCAAGCCAGGGTGATCCTT
CCTGCCTTTACACTCCCTCCACACTTTTTCTGCTCTCATATGTGGCCGTGGTCACTTTCT TTTGGTAGTTTGCATATTTCATTTACCCCAAACTTTCAGCTCCTGAAGGTCAGGATACAA GGAGGCCTCATCTCCGCATTCCCCTCAGCTCCCTTCCTGAAGCTTGATACCTAGTCAGTA CCCAGTGGATGTTTCCTAAACATGTAAGTAATGACATCATGAAGAAGCCACATGTTTACC TTGACCACAAACACAGGGCAAAGGTGACTAGTGTGGTCAGAGATCCCTGCTGGCTGGGAA TCAGGGAAGGCTGCATGGAAGAAGTGGCATTTTAGTTAGAACTTGAAAGGTGGTGTATTT AGTTTTCTCTGGCTGCCATATTCCTTGTCACATTGCCCTCTCCATCTTCAAGCCACTGGG CAAGGCTAGAAGGCCCTCAACAGACTATCGGTAGGAATGTGGAAGTTGAAGACTCAGAGT GCAGAAAGAAACAAGTAGCATTTTAGAGAAAAGCTAAATCCCCTCCAAGAATACCTCAAT CATCGTGAAGAGCCTGTTAGTAGACGCACTAACACTCAAGGCACTGCTTCACAAGGTAAG
GAACGTGTAATTGAAAACTTGAGAAAGGAAGAAACTTGTTCTGTACTGGCAGAAAGCTTA GCAGAATTGTGTCCTGCAGTCATATGGGACACAGAGCTTGTAAATGATGAATTTGAATGC TTATCCGAGAAGGTTTCCAAATAAAATGTGGAAGGCACGGCCTGGTTTCTTCCTGCCTCT TATAGTAAAATGCAAGAGGAGAGAGAGAAAATGAGGGAAGAACTTAAACAGAAAGGAACC AGGACTTGATGATTTGGGAGGTTCTCAACCTATGCAAAAAACAATAAAATTAAGAGATTG TAGCTGGGCACAGTGGCTCATGCCTGTAATCCCAGCACTTTGAGAGTCCGAGGCGAGCAG ATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAATGTGGGGAAACTCCGTCTCTAC TAAAAATACAAAAATTAGCTGGGTGTGGTGGCGGGCACCTGTAATCCCAGCTACTCAGGA GGCTGAGGTGGGAGGATCACTTGAACCCAAGAGGCGGAGGTTGCAGTGAGCCAAGATCAT GCCACTGCACTCCAGCCTGGGTGGGTGACAGAGCAAGACTCCATCTCAAAAAAAAAAAAA
AAAAGAGATTGCTCCCAAAAGTGTGACATAGAGAAACAGCCAAGTATGTGATTATACCAA ACTTCAGGAAGATAAAAGATCAAAGTACTCAGTCGCTCAAAAGGCTCTTTGAAGAGATTA AGATTATAACTCACAGTCCCCTTCAATCAAACCAGGGGACTTCTAGGAAGCTGAACAGCA TTGTCCCTCAGCCATATCAGCTGGAGCCAAAAGTAGAGAAGGGCTTATCTGAAAAAAGGA TCTGTGGACCTGGCTTTTATCTAATAATGCAGTGGATTCCCCCATGACATCCATAGGAGA
CCCGTAAAGTTCCTGAGACGTTTACATCCACAGAAACACTGTTAGCTTGGATTAAATGGA ACACAGAGAGTATGAAATCAAAGAAGGCTGTTGGACTCTCCAGTTTCTACTGTTGAGATG CAGACTGGTAAAACTACTTAGCTGCAAACACCTGCTACCTTTAGTGAAAAGGAAGGATAT CTCAGACGGTGAAACCAGAAGCTCAAAGGGCAGTGCTAAGAGCGAAAGAGAATTCTTCCC AGGCCTTGAAACCTAATGGAGTTTTCTTGGCTGGATTTTCAAACTGCATTGGACCATGAC
CTGATTGTCCCTTTCATGTCCCCATGCTTGAGCCAGATTGTCTGCAACTGTTATCCTGTG CCTGTCCCACATTTTATGTTGGGAGCAGAAAACTTTAGTTTTGCTGGCCCACAGATAGAG AGAAACTGTACCCCGAGAGTTGTACTGACTGGACTATGCCCAGAGTCTATTTGACTCTGA CTTAGATACTGTTGATTTGGGAATTTGAGTTGATGCTGTAATGAGATGAGACTTTGGGGG ACATTGGGATGGAGTGAATGGATTTTGCATTTGAAAGAGATGTGGGTTGGGTAATCCTAG CCCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCAGATCACCTGAGGTCGGC AGTTCGAGACCAGCCTGACCACCATGGAGAAACCCCATCTCTACTAAAAATACAAAATTA GCCAAGCATGGTAGCACATGCCTATAATCCCAGCTACTCGGGAGGCTGAGGCAGTAGAAT CGCTTGAACCCGGGAGGCAGAGGTTGCGGTGAGCCGAGATCACGCCATTGCACTCCAGCC TGGGCAACAAGAGTGAAACTCCATCTAAAAAAAAAAAAAAAGAAAGAAAGAGATGTGGAT TTTGGGTGGGGGACAGAGGGAAGACCATGGTAGGCAGAATGATCCTCTAAAGGTGCTCTG CCCTAATCCCCAGAAGCTAAGAATATGTTAGATGTCAGTATTGCGTGGCAGTAGGAATCT TAATTAACGTTATAGACTGTTATGGTTTGAATGTCCCCTCTAAAACTCCTGTTGACATTT AATCATCATTGTGATTGCATTAAGAAGTGGCCCTGTTAAAAGGTGATTTAGTCCTTAAGA ACGCTGCCCCCGTGAATAGATTAAGGTCAGTCTTGCGGGAGTGTGTTTATCAAGAATGGA TTGTTAAAAAGTGAGTTCTGGCCAGGGGCAGTGGCTTATGCCACTCAGCACTTTGCGGGG CCAAGACTTGAAGTCAGTTGTTTGAGACCAGCCTGGCCAACATGGTGAAAGTCTGTCTCT ACTAAAAAATACAAAAAGTGTCCGGGAGTGGTGGCGGGCGCCTGTAATCCCAGCTGCTCA GGAGGCCGAAGCAGGAGGATCGCATGAATCCGGGAGGCAGAGGTTGCAGTGAGCTGAGAT CGCCCCGTTGCACTCCAGCCTGGGTGATAGAGCAAGACTCTGTCTCAAAAAAAAAAAAAA AAGAGGAAAGAAAGAAGAAAGAAAGAGAAAGAAAGAAAAGAAAGAAAAGGAAGGAAGGAA GGAAGGAAGGAAGGAAGGAAGGAAGGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAA AGAAAGAAAGAAAGAAAGAAAGAAAGAAAAGAAAGAAGAAAAAAAGAAAGAAAAAAGAAA GAAAGAAAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAAGAAAAGAAA AGTGAGTTCTGCCCTCTCTTGCTGGCTTACTCTCACCCTCTCTTGCCCTTCCACCTGCCA CCATGGGATGACACAGCACAAAGGCCCTCACCAGATGCCAGTGCCATGCTCTTGGACTTC CAAGTCTCCAGAAACATGAGCCAAATACACTTCTGTTCATTATAAATTACCCAGCCTGTG ATATTCTGTAATAACAACACAAAATAGACTGAGACATAGATCTTCAAATAGTGAGGTTAT CCTGGATAATCCAGATGGGCCCAATCTAATCCCATGAGCCTTTAAAACTTTCTCCAGATG GAGGCAGAAGAGAAGTGGCAGAAGGGGAAGTCAGAGAGATTTGAAGCATAAACAGGACTC CATGGTGCCGTTTCTGGTTTGACGATGGAGTGGTAACGTGATGAAAAATGTGGGTGCCTT CCGGAGCTGAGAGGCTCCCACTAACAATCGGCCAGGAAACAGGGACCACAGCCCTACAGC CACAAAGAACTAAGTTTTGCTGACAACCCAAGGGGGCTTGGAAGTGTCTTCTCCCCCATC GGTTCCAGATGTGAGACCCAGAGCGAAGGAACCAGCTGAGCCCACCTGGACTTCTGACCT AGAGAACTGTGAGATAATAAGTTTGTATCATTTTTAAGGCACTGTGTGTGTGGTAATTTG TTATGACAGCAATAGAAAATGAATCCAGATGGGCAGGATCTGCCAGGCCAGTGACATGTG GAGGGCACCCAGGCGGATGGGATGGCATGAGAGAAGGCAGGTCAGCAATGAGCTTGCCCA GGTCACCTCTCCTCTCTAAGCCTCAGTTTTCCTCTCTATGAAATGAGAGTAGTGATATCT CCCTCCCAGGGTCAGTGCAAGGCTGAAATAACAGATTATAAGGTGCTAGGTGCACAAGAA GTGTTTGAAACATGCTAGTTGCTTTTCCATTTCCAAGAGAGCTCTCTGGTCTTGGGGGAT GGAGGCAGTGCGGCCCCTCGGGATTACTGACAGGTCCTGCTCTGTTTCTGCAGTGGAGCC GGCCCCACCTGTCCTGGTGCTCACCCAGACGGAGGAGATCCTGAGTGCCAATGCCACGTA CCAGCTGCCCCCCTGCATGCCCCCACTGGATCTGAAGTATGAGGTGGCATTCTGGAAGGA GGGGGCCGGAAACAAGGTGGGAAGCTCCTTTCCTGCCCCCAGGCTAGGCCCGCTCCTCCA CCCCTTCTTACTCAGGTTCTTCTCACCCTCCCAGCCTGCTCCTGCACCCCTCCTCCAGGA AGTCTTCCCTGTACACTCCTGACTTCTGGCAGTCAGCCCTAATAAAATCTGATCAAAGTA TGATGACCTACAGGAGGCCTGCTTGCCAAGTCAACAGATTCAGTACAGAAAAACTGAAAA ATACAGATAAGCTCTAAGAAGCAGACCAAAAGTACCCAGAGATGACCGCACATCACTCTG GTGTATATCCAATTTCAGATTTGTTTTCTGTGTATGCATGTGTGTATAGCTGCATTTATT TATGGCAAGGGCTGGCAGACTTTCCCGAAGAAGGCCAGATAGTCGATATGTTTGGCTTCA TGGGCCGTATGTTCGCTCAGGACTACTCAACGCTGCAGTTATAGCACAAAAGGAGCCGTA GCCTATACGTAAATGAATGGGCATCGCTGGGTTCCAGTAAAACTGTTTACAGGCCAGGTG CGGTGGCTCATGCCTGTAATCTCAGTACTTTGGGAGGCCGAGGTGGTGGGAGGATTACCT TAGCCCAGGAGTTCAAGACCAGCCTGGGGAACATGGTGAAACATTATCCCTACAAAAAAA AAAAAAGCTGGGTGTGGTGATGCATGCTTGTGGTCCCAGCTGCTTGGGATGCTGAGGCAG GAGGATCGCTCGAGCCCAGGAAGCAAGGCCACAGTGAGCCATGATCGCACCACTGCACTT TAGTCTGGGCAACAGAGTGAGACCTTGTCTCAAAAAAAACAAAAAATAAAACTTTTTACA TAAACAAGTGGCCAACCAGACTTGGTCCCTGGGCCTCTGCTCTTGAATGTTCTTGCTTCC ACTAAAGTAACATTCACACTCCCGATTTTTGCATACTCTGGGTTCTGGGGAATATAGATC CGAATCCAGCGTGGTTCCTGCCTTCAAGAACCTCACAAATATTCTAGACCAGCACTGCCC
AATAGAAAGAAATATAATGCAAGCCACATGTGCAGTTTTAAGTGTTCCATGTTAAATTAA GTAAAAAGAGACGGGTAAATCGAATTTTAATAACAGATTTTACTTCATCCAATTGAATGG TATCATTTCAATGAGCAATTCTGATAGTGATTGAGATCTTTTACATTCTTTTTCACTACG TCTTTAAAATCTGATGTGTGTTTTGTACTTGGAACACTTCTCAGTGTGGACCAGATGCAT TTCACATACTCAGTAGTCACGCGTGGCCAGTGCCTTCCATACCACACAGTGCAGCATCTG TAGAGGTTTCCTCCACTGCTGATAGACTAGGAGACCCCAAGATGGAAAGCCTGAAGAATC TGCTCCTCGAAGTAGGGACCTTAATGGGGTGCACGCCAGGGCGACCCCAAGTGGTAGGCT GCTTTTGAACCATGGCTATCCCTACCTCTAGACTCAGCTGAAAAGAACTCAGGTAGTCTT GGGAAGTGCTTCCTCAATGCTTAAACTTTAATGCAGGAAAAGAATAGAAAGTTCAGGCAA GGAGGGAGGATCACTTGAGGCTGGGAGTTCGAGACCAGCCTGGGCAACAGCAAGACCTTG
CCTATACAAAAAATAATTTTAAAAAATTACCCAGGTATGGTGGTGTGGATCTGTAGTCCC TAGTTACTTGGAGAGCTGAGGTAGGAGGATCGCTTGAGCCCAGGAGTTTGAGGCTGCAGT GAGCTGTGATCACACCACTGCACTTTGGCCTGGGTGACAGAACCAAACCCTATCCCCTAC AAAAAAACAAAAAAAAAAAACAAAAAAAAACACCCTACCATGTCTGCCAACCCCACTCTG TCCTGGCTGTGTGAAACCAGTCCCCACAGCAGCTCTGCCACTCTCTGCTTCTTTTCCAAA
CAGACCCTATTTCCAGTCACTCCCCATGGCCAGCCAGTCCAGATCACTCTCCAGCCAGCT GCCAGCGAACACCACTGCCTCAGTGCCAGAACCATCTACACGTTCAGTGTCCCGAAATAC AGCAAGTTCTCTAAGCCCACCTGCTTCTTGCTGGAGGTCCCAGGTGGGTATCAAGTGGTG CAGAAGGAGAAACTTTCCCTCTGGGCCTTGGGAGCTTCGTGACACAGTGGTTAAGAACAT GAGCCTAGAGATAGACTCGCCTGGATTAAAACCACACTCATTGTGTGTCTTTGGGCAGCT
TACATAATGCCCCGAACCTTGGTTTGCACAGTCTGCAGGATGGGTTTATTCTTGTGAGGA TTAAATAGGGTCATGTATGTGAAGCACTCGGCACAGGTGCAGTTGTAGACAAGAGCCATT GTTGTTTCTCTCATTGTTATTTTTCCTTCCTTAGAAGCCAACTGGGCTTTCCTGGTGCTG CCATCGCTTCTGATACTGCTGTTAGTAATTGCCGCAGGGGGTGTGATCTGGAAGACCCTC ATGGGGAACCCCTGGTTTCAGCGGGCAAAGATGCCACGGGCCCTGGTATAGCAAATCTGG
GGGTGTGCGGCAGGTGGGGAGGGGTTGAGAGTAAGGGAGTGGGGCTGGAGCTATGAGTTG TTCAGATAGAATATCAAGATGGTCCAGACTCTTGGACCAAAACATCTATCTTTGTGTCTG AATTTCCACCATTAGTAATGCATTCATTTAGTCCTGAATAAAATGGCAAACAGGCCCTGG AGGGAGCAGTGCCTTAAGTTCCTTTGAGATAAATAACTTCACCTCTGCTAAGGATGTGTC AGCTGCTGAGAGCAGAGCCCCTGGCCTTGGACCTCAGGAGAGACACTCAAAAGGGGAGGA
GAGGAGGCACCAAAGGGGACATCTTAAAAGAGTTCCAATTTTTAGTTCACACTTTAACCC AGGATAAGCTGTGTCCTGGCTGACCTTGGAGTTTCTTCCCTGGTCTGCTGGGTCTCTCCC TTAGAACCTAGGGGCGAGCTGGGGCAGGGGAAGCCCAGGAGGTGATATAGGTCGGCCCTG TTCAGATGAGGGCTGGCAGGGGCAGCTTGGGCATATGCGAGGCTCCGATGGGCATGGGGG CTTTGAGGATGGATTCTGAGTGTCCCTGCATCGTGGCAGGGTGGCAAAGGGAGCATTTCC AAATTTCCTGGCTCCAGGATCTGTGGGAGAATCCCACTAACTGTCAGGGTGACAACCTCG GGTAGACATGTCTGTGCCCTGCCCCGTGCCCTCAGCCTTCCTGTTAAGAGCACACCAGCT GGATTTGCAACTCCCAGCGCCTGCACCCAATGGGCTTTCTCTGGCCTCTGGAGCCCACAT TGCCCCTGCATGTGGCAGGCTGCAAGTGTCACAGCCACCAGCTCTTCCATTCCTCAACAA TGACTGTGGGTAAATAGCCCAGGAGCGTCCCCCTCCTGGGATGGTTCTGAGGTGCGTGTG
CCCAGTGGCTCCCTGAGTTGCCAGCAGGATTAAGTGCCAGTAGCCCTAGTGGTCAGCTGC TTGATAACACCCTGCTTCCTGGCTGCTCCCCCAGTCCCATCTGGTGTGTTCTGGGATCAT CTCCCAAAGAAACTGCTTACACTTGAAGCCTTGTCTGAGGTCTGTTTCTAGGGGAATTCA GATGACGATAATTATGCTTCAGGAAAGCCTAAATTTTCTGCTTTTCTCTCCCCTACCCAA ATCAGGACTTTTCTGGACACACACACCCTGTGGCAACCTTTCAGCCCAGCAGACCAGAGT
CCGTGAATGACTTGTTCCTCTGTCCCCAAAAGGAACTGACCAGAGGGGTCAGGCCGACGC CTCGAGTCAGGGCCCCAGCCACCCAACAGACAAGATGGAAGAAGGACCTTGCAGAGGACG AAGAGGAGGAGGATGAGGAGGACACAGAAGATGGCGTCAGCTTCCAGCCCTACATTGAAC CACCTTCTTTCCTGGGGCAAGAGCACCAGGCTCCAGGGCACTCGGAGGCTGGTGGGGTGG ACTCAGGGAGGCCCAGGGCTCCTCTGGTCCCAAGCGAAGGCTCCTCTGCTTGGGATTCTT
CAGACAGAAGCTGGGCCAGCACTGTGGACTCCTCCTGGGACAGGGCTGGGTCCTCTGGCT ATTTGGCTGAGAAGGGGCCAGGCCAAGGGCCGGGTGGGGATGGGCACCAAGAATCTCTCC CACCACCTGAATTCTCCAAGGACTCGGGTTTCCTGGAAGAGCTCCCAGAAGATAACCTCT CCTCCTGGGCCACCTGGGGCACCTTACCACCGGAGCCGAATCTGGTCCCTGGGGGACCCC CAGTTTCTCTTCAGACACTGACCTTCTGCTGGGAAAGCAGCCCTGAGGAGGAAGAGGAGG
CGAGGGAATCAGAAATTGAGGACAGCGATGCGGGCAGCTGGGGGGCTGAGAGCACCCAGA GGACCGAGGACAGGGGCCGGACATTGGGGCATTACATGGCCAGGTGAGCTGTCCCCCGAC ATCCCACCGAATCTGATGCTGCTGCTGCCTTTGCAAGGACTACTGGGCTTCCCAAGAAAC TCAAGAGCCTCCGTACCTCCCCTGGGCGGCGGAGGGGCATTGCACTTCCGGGAAGCCCAC CTAGCGGCTGTTTGCCTGTCGGGCTGAGCAATAAGATGCCCCTCCCTCCTGTGACCCGCC
CTCTTTAGGCTGAGCTATAAGAGGGGTGGACACAGGGTGGGCTGAGGTCAGAGGTTGGTG GGGTGTCATCACCCCCATTGTCCCTAGGGTGACAGGCCAGGGGGAAAAATTATCCCCGGA CAACATGAAACAGGTGAGGTCAGGTCACTGCGGACATCAAGGGCGGACACCACCAAGGGG CCCTCTGGAACTTGAGACCACTGGAGGCACACCTGCTATACCTCATGCCTTTCCCAGCAG CCACTGAACTCCCCCATCCCAGGGCTCAGCCTCCTGATTCATGGGTCCCCTAGTTAGGCC CAGATAAAAATCCAGTTGGCTGAGGGTTTTGGATGGGAAGGGAAGGGTGGCTGTCCTCAA ATCCTGGTCTTTGGAGTCATGGCACTGTACGGTTTTAGTGTCAGACAGACCGGGGTTCAA ATCCCAGCTCTGCTCTTCACTGGTTGTATGATCTTGGGGAAGACATCTTCCTTCTCTGCC TCGGCTTCCTCATCTGCAGCTACGCCTGGGTGTGGTGAGGGTTCTAGGGGATCTCAGATG TGTGTAGCACGGAGCCTGCTGTGTCCTGGGTGCTCTCTACGTGGTGGCCGGTAGAATTCT
CCATCTATCCAGGCTCCAGGAGACCCCTGGGCATCTCCCACCTGTGGCCCCTAAACCCAG AGTGACTGAGAGCACTTACCATTCAGCTTGTCTCATCCCCAGTCTACCTCCTTCCTTCTA CCCTCACTGCCTCCCAGTCAGGAGAGTGAGCTCTCAGAAGCCAGAGCCCCACCCAAGGGG ACCCTGGTCTCTCCGCCTTCACCTAGCAATGGGAACCCTGCTTCCCAGGGGAGGAACCAA CTGCTCCACCTTCTAGGGACCCAGTTTGTTGGAGTAGGACAGTAACATGGCAGGAATCGG
ACTTCTGGGCCTGTAATCCCAGTTTGGATGGCACGTTAGACTCTTGGTTGACCGTTGTGG TCCTTAGAAGTCCCATTCTCCCTTCCAGTTATGAGAAACCAATGCCTTCTAGATTCAGGT GACTATCCTTACCTGGGGGTGCTGATGCATCCTCAGTTAACCTACACCCACCTGAATATA GATGAGCGTAGCTGAGTTTTCACCCGTAGGACCGAAGTGTTTTGTGGTGGAGTATCTGAA CAACCTTGGCTCTGTGGCCATTCAACCTGCCAGGACTAACATTTCTGGATTTGTGAAGAA
GGGATCTTCAAAGCCATTGAACCCACAGAGCTGTGTTGCTTTAAAGCCACCACAAGGGTA CAGCATTAAATGGCAGAACTGGAAAAGCTTCTTAGGGCATCTCATCCAGGGATTCTCAAA CCATGTCCCCCAGAGGCCTTGGGCTGCAGTTGCAGGGGGCGCCATGGGGCTATAGGAGCC TCCCACTTTCACCAGAGCAGCCTCACTGTGCCCTGATTCACACACTGTGGCTTTCCACGT GAGGTTTTGTTTAGAGGGATCCACTACTCAAGAAAAAGTTAGCAAACCACTCCTTTTGTT
GCAAAGGAGCTGAGGTCAAGGGTGGCAAAGGCACTTGTCCAAGGTCGCCCAGCAGTGCTG CTCTGATGACTTGTGCACATCCCCAAGGGTAAGAGCTTCGATCTCTGCACAGCCGGGCCA ACCTCTGACCCCTTGTCCATGTCAGTAAAATATGAAGGTCACAGCCAGGATTTCTAAGGG TCAGGAGGCCTTCACCGCTGCTGGGGCACACACACACACATGCATACACACATACGACAC ACACCTGTGTCTCCCCAGGGGTTTTCCCTGCAGTGAGGCTTGTCCAGATGATTGAGCCCA
GGAGAGGAAGAACAAACAAACTACGGAGCTGGGGAGGGCTGTGGCTTGGGGCCAGCTCCC AGGGAAATTCCCAGACCTGTACCGATGTTCTCTCTGGCACCAGCCGAGCTGCTTCGTGGA GGTAACTTCAAAAAAGTAAAAGCTATCATCAGCATCATCTTAGACTTGTATGAAATAACC ACTCCGTTTCTATTCTTAAACCTTACCATTTTTGTTTTGTTTTGTTTTTTTGAGTCGGAG TTTTGTTCTTGTTGCCTAGGCTGGAGTGCAGTGGTGCGATCTCGGCTCACTGCAACCTCC ACCTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCA CCCGCCACCACACCTGGCTAATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGT TGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCGCCCGCCTCGGCCTCCCAAAG TGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCAAACCTTACTATTTTTTTAAAGAA TTTTTTCCAGAGTTTAATTTCTGACATAGCTTAAGTTTTCCAGTAACTCTAAACTCCATC
TCCTTTATCGTCATTAAGTCATTCACAAAAAGCCAGGAGAAGCATTTGGAAAGGGCATGA TAATCAGTATAATAATT (SEQ ID NO: 22)
Table 8 presents a correlation between the genomic sequence shown in Table 7 and the locations of the corresponding regions of the cDNA sequence shown in Table 1.
Table 8
Figure imgf000047_0001
Several sequence polymorphisms have been identified in the sequence shown in Table 7. These are summarized in the Table 9:
Table 9
Figure imgf000047_0002
Figure imgf000048_0001
CRF2-like nucleic acids and polypeptides ofthe invention (including those shown in Table 1) are referred to herein as "CRF2-13 " nucleic acids and polypeptides.
A CRF2-13 nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts. For example, sequence comparison reveals that the disclosed CRF2-13 nucleic acid (Table 1) encodes a Type II cytokine receptor. One or more secreted receptor chains may be associated with, and/or modulate the activity of, another membrane bound member of CRF2, or a membrane bound receptor of another family. Alternatively, or in addition, the receptor chains disclosed herein may act alone or in combination with another soluble receptor. In effect, the receptor can also be a ligand.
A soluble form ofthe CRF2-13 polypeptide ofthe invention (e.g., a polypeptide that includes amino acids 21-230, amino acids of SEQ ID NO:2) may additionally be used as a soluble receptor antagonist. Soluble receptor antagonists that block the activity of specific cytokines, e.g., TNF, are known in the art. A soluble CRF2-13 polypeptide of the invention can similarly block the activity of a cytokine that acts through a CRF2 member. Examples of such polypeptides include IL-10, IL-19, IL-20, IL-22, AK155, mda-7 or an interferon, such as interferon alpha, interferon beta, or interferon gamma. In one embodiment, a soluble CRF2-13 polypeptide of the invention is used to antagonize the function of IL-22. IL-22 is distantly related in sequence to IL-10 and is produced by activated T cells. IL-22 signaling into a cell is mediated by its receptor chains, IL-22R and CRF2-4, both members of the
CRF2 family. The CRF2-4 receptor was originally reported to serve as a second component in IL-10 signaling. IL-22 has been reported to inhibit IL-4 production from human Th2 T cells and to induce acute phase proteins in the liver of mice.
CRF2-13 nucleic acids and polypeptides according to the invention may additionally be used to identify cell types that make the invention or bind to the invention in a population of cells. The CRF2-13 nucleic acids and polypeptides can also be used for immunomodulation, inflammation, immunosuppression, allergy, asthma, autoimmunity (including rheumatoid arthritis and multiple sclerosis), repair of vascular smooth muscle cell after vascular injury or disease, transplantation and cancer based on the ligand that associates with this soluble receptor, alone or in conjunction with another receptor, and the impact that this ligand has on the above mechanisms and/or pathologies.
For example, a CRF2-13 polypeptide and/or soluble form of a CRF2-13 polypeptide ofthe invention may exhibit one or more of the following activities: (1) modulation, e.g., it may antagonize a signal transduction pathway mediated by a cytokine (such as EL- 10 or IL- 22); (2) modulation of cytokine production and/or secretion (e.g., production and/or secretion of a proinflammatory cytokine); (3) modulation of lymphokine production and/or secretion; (4) modulation of expression or activity of nuclear transcription factors (5) competition with cytokine receptors for cytokine ligands; (6) modulation of cell proliferation, development or differentiation, e.g., cytokine-stimulated (such as IL-10 or IL-22) production, development, or differentiation; (7) modulation of cellular immune responses; modulation of cytokine- meditated proinflammatory actions; and/or promotion and/or potentiation of immune reactions.
A CRF2-13 polypeptide of the invention may directly, by association with a membrane bound receptor, or indirectly, by its association with a soluble ligand affect or effect one or more ofthe following cell types: circulating or tissue-associated cells: T cells, B cells, NK cells, NK T cells, dendritic cells, macrophages, monocytes, neutrophils, mast cells, basophils, eosinophils, as well as cells in the respiratory tract, pancreas, kidney, liver, small and large intestine. A CRF2-13 polypeptide ofthe invention may additionally modulate upregulation of humoral immune responses and cell-mediated immune reactions; modulate the synthesis of proinflammatory cytokines and chemokines; and modulate inflammatory responses associated with injury, sepsis, gastrointestinal and cardiovascular disease, or inflammation following surgery. For efficient production ofthe protein, it is preferable to place the CRF2-13 sequences under the control of expression control sequences optimized for expression in a desired host. For example, the sequences may include optimized transcriptional and/or translational regulatory sequences (such as altered Kozak sequences). In addition, the mature amino terminus of a CRF2-13 protein may be operably linked to a non-CRF2-13 signal sequence based on a hypothetical or empirically determined of the mature amino terminal end of the protein.
A CRF2-13 fusion protein can be used to identify and determine binding partners using assays known in the art. These assays include, e.g., either histological, immunochemical, BIACORE or cell biology based assays.
Assays can also be performed in order to determine whether a CRF2-13 protein ofthe invention associates with cell types that already express other members ofthe CRF2 family. A CRF2-13 of the invention can also be examined for its ability to modulate the activity of known or novel cytokines (e.g., by inhibiting or otherwise antagonizing the functions of a cytokine).
For example, several novel IL-10 like molecules have been cloned. DL-22 is one of these molecules. It has been reported that this molecule blocks the production of EL-4 by Th2 cells (human) and initiates an acute phase response (mice). A finding that CRF2-13 binds to and inhibits IL-22 (or other IL-10 like molecules) indicates a CRF2-13 invention can be used to treat or prevent diseases associated with high levels of the IL-22 polypeptide.
It is also contemplated that a CRF2-13 polypeptide of the invention associates with other receptors and/or their associated cytokines within the CRF2 family. For example, a CRF2-13 ofthe invention may associate with either chain of the IL-22R and affect the function of the receptor or the IL-22 ligand.
CRF2-1 Nucleic Acids
The nucleic acids of the invention include those that encode a CRF2-13 polypeptide or protein. As used herein, the terms polypeptide and protein are interchangeable. In some embodiments, a CRF2-13 nucleic acid encodes a mature CRF2-13 polypeptide. As used herein, a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein. An example of a CRF2-13 nucleic acid encoding a mature form of a CRF2-13 polypeptide is a nucleotide sequence encoding amino acids 21-520 of SEQ ID NO:2 (e.g., nucleotides 61- 1560 of SEQ ID NO: 1). The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them. Among the CRF2-13 nucleic acids ofthe invenation are the nucleic acid whose sequence is provided in nucleotides 1-1560 of SEQ ID NO: 1, SEQ ID NO: 1 itself, or a fragment of one of these sequences. Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO: 1 , or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO:l, while still encoding a protein that maintains at least one of its CRF2-13 -like activities and physiological functions (i.e., modulating angiogenesis, neuronal development). The invention further includes the complement of the nucleic acid sequence of SEQ ID NO: 1 , including fragments, derivatives, analogs and homologs thereof. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. One aspect of the invention pertains to isolated nucleic acid molecules that encode CRF2-13 proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify CRF2-13 -encoding nucleic acids (e.g., CRF2-13 mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of CRF2-13 nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated CRF2-13 nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule ofthe present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe, CRF2- 13 nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et αl., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et αl., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to CRF2-13 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "ohgonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO:l, or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as probes.
In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NO: 1 , or a portion of this nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:l is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, thereby forming a stable duplex. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates. Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of CRF2-13 . Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by reference in its entirety).
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a CRF2-13 polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include nucleotide sequences encoding for a CRF2-13 polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human CRF2-13 protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:2, as well as a polypeptide having CRF2- 13 activity. Biological activities of the CRF2- 13 proteins are described below. A homologous amino acid sequence does not encode the amino acid sequence of a human CRF2-13 polypeptide. The nucleotide sequence determined from the cloning of the human CRF2-13 gene allows for the generation of probes and primers designed for use in identifying and/or cloning CRF2-13 homologues in other cell types, e.g., from other tissues, as well as CRF2-13 homologues from other mammals. The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1 ; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1 ; or of a naturally occurring mutant of SEQ ID NO: 1.
Probes based on the human CRF2-13 nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. , the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CRF2-13 protein, such as by measuring a level of a CRF2-13-encoding nucleic acid in a sample of cells from a subject e.g., detecting CRF2-13 mRNA levels or determining whether a genomic CRF2- 13 gene has been mutated or deleted.
A "polypeptide having a biologically active portion of CRF2-13 " refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically active portion of CRF2-13 " can be prepared by isolating a portion of SEQ ID NO:l that encodes a polypeptide having a CRF2-13 biological activity (biological activities of the CRF2-13 proteins are described below), expressing the encoded portion of CRF2-13 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of CRF2-13 . For example, a nucleic acid fragment encoding a biologically active portion of CRF2-13 can optionally include a cytokine-binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of CRF2-13 includes one or more regions.
Polymorphisms in CRF2-13 associated sequences The invention also provides polymorphic forms of CRF2-13 nucleic acid sequences as well as methods of detecting polymorphic sequences in CRF2-13 sequences The polymorphic forms include genomic sequences corresponding to exons and/or introns associated with CRF2-13. The polymorphisms can be provided on various isolated CRF2-13 nucleic acids. For example, the polymorphism can be provided on an isolated polynucleotide comprising at least 10 contiguous nucleotides of SEQ ID NO:3 that include the polymorphic sequences shown in Table 6. Alternatively, the polymorphism can be provided on an isolated polynucleotide comprising at least 10 nucleotides of SEQ ID NO:2 that include alternative forms of the polymorphic sequences shown in Table 9.
For example, an isolated CRF2-13 polymorphic sequence can include from nucleotide 30957 to nucleotide 30967 of SEQ ID NO:3, provided that position 30962 is "A or "G". In a second example, the isolated CRF2-13 polymorphic sequence can include at least 10 contiguous nucleotides from nucleotide 30650 to nucleotide 30660 of SEQ ID NO:3, provided that position 30655 is "A" or "G". In additional examples, the isolated CRF2-13 nucleic acid sequence includes at least 10 contiguous nucleotides from nucleotide 28739 to nucleotide 28749 of SEQ ID NO:3, wherein position 28744 is "A" or "G"; at least 10 contiguous nucleotides from nucleotide 28442 to 28452 of SEQ ID NO:3, wherein position 28448 is "C" or "T"; additional examples include an isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 9421 to 9431 of SEQ ID NO:3, wherein position 9426 of the polynucleotide is "A" or "G", or an isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 8806 to 8816 of SEQ ID NO:3, wherein position 8811 of the polynucleotide is "C or "T".
Alternatively, an isolated CRF2-13 polymorphic sequence can include from nucleotide 32954 to nucleotide 32964 of SEQ ID NO:22, provided that position 30962 is "C" or "A". Alternatively, the polymorphic sequence can include from nucleotide 31262 to 31272 of SEQ ID NO:22, provided that position 31266 is "C" or "T"; or nucleotides 30955 to 20965 of SEQ ID NO:22, provided that nucleotide 30960 is "T" or "C"; or nucleotides 29043 to 29053 of SEQ ID NO:22, provided that nucleotide 29048 is "C" or "T"; or nucleotides 28748 to 28758 of SEQ ID NO:22, provided that nucleotide 28753 is "G" or "A"; or nucleotides 23825 to 23835 of SEQ ID NO:22, provided that nucleotide 23830 is "G" or "A". In additional embodiments, the polymorphic nucleic acid includes at least 15, 20, 25, 50, 75, 100, 150, 250, 500, 750, or 1000 or more contiguous nucleotides from SEQ ID NO:3. In some embodiments, the polymorphic nucleotide sequence is 10-1000 nucleotides in length. For example, the polymorphic nucleotide sequence can be 20-750 nucleotides, 50-625 nucleotides, 75-500 nucleotides, 100-250 nucleotides in length.
Individuals carrying polymorphic alleles of the invention may be detected at either the DNA, the RNA, or the protein level using a variety of techniques that are well known in the art. Strategies for identification and detection are described in e.g., EP 730,663, EP 717,113, and PCT US97/02102. The present methods usually employ pre-characterized polymorphisms. That is, the genotyping location and nature of polymorphic forms present at a site have already been determined. The availability of this information allows sets of probes to be designed for specific identification of the known polymorphic forms.
Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. (1989), B. for detecting polymorphisms. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Apphcations 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202.
The genomic DNA used for the diagnosis may be obtained from any nucleated cells of the body, such as those present in peripheral blood, urine, saliva, buccal samples, surgical specimen, and autopsy specimens. The DNA may be used directly or may be amplified enzymatically in vitro through use of PCR (Saiki et al. Science 239:487-491 (1988)) or other in vitro amplification methods such as the ligase chain reaction (LCR) (Wu and Wallace Genomics 4:560-569 (1989)), strand displacement amplification (SDA) (Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992)), self-sustained sequence replication (3SR) (Fahy et al. PCR Methods P&J& 1:25-33 (1992)), prior to mutation analysis.
The detection of polymorphisms in specific DNA sequences, can be accomplished by a variety of methods including, but not limited to, restriction-fragment-length-polymo hism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy Lancet ii:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl. Acids Res. 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al. Proc. Natl. Acad. SCI. USA, 86:6230-6234 (1969)) or oligonucleotide arrays (Maskos and Southern Nucl. Acids Res 21:2269-2270 (1993)), allele-specific PCR (Newton et al. Nucl Acids Res 17:2503-_2516 (1989)), mismatch-repair detection (MRD) (Faham and Cox Genome Res 5:474-482 (1995)), binding of MutS protein (Wagner et al. Nucl Acids Res 23:3944-3948 (1995), denaturing-gradient gel electrophoresis (DGGE) (Fisher and Lerman et al. Proc. Natl. Acad. Sci. U.S.A. 80:1579-1583 (1983)), single-strand-conformation- polymorphism detection (Orita et al. Genomics 5:874-879 (1983)), RNAase cleavage at mismatched base-pairs (Myers et al. Science 230: 1242 (1985)), chemical (Cotton et al. Proc. Natl. w Sci. U.S.A, 8Z4397-4401 (1988)) or enzymatic (Youil et al. Proc. Natl. Acad. Sci. U.S.A. 92:87-91 (1995)) cleavage of heteroduplex DNA, methods based on allele specific primer extension (Syvanen et al. Genomics 8:684-692 (1990)), genetic bit analysis (GBA) (Nikiforov et al. &&I Acids 22:4167-4175 (1994)), the oligonucleotide-ligation assay (OLA) (Landegren et al. Science_241:1077 (1988)), the allele-specific ligation chain reaction (LCR) (Barrany Proc. Natl. Acad. Sci. U.S.A. 88:189-193 (1991)). gap-LCR (Abravaya et al. Nucl Acids Res 23:675-682 (1995)), radioactive and/or fluorescent DNA sequencing using standard procedures well known in the art, and peptide nucleic acid (PNA) assays (Orum et al., Nucl. Acids Res, 21:5332-5356 (1993); Thiede et al., Nucl. Acids Res. 24:983-984 (1996)).
CRF2-13 Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO:l due to the degeneracy ofthe genetic code. These nucleic acids thus encode the same CRF2-13 protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, e.g., the polypeptide of SEQ ID NO:2. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.
In addition to the human CRF2-13 nucleotide sequence shown in SEQ ID NO:l, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of CRF2-13 may exist within a population (e.g., the human population). Such genetic polymorphism in the CRF2-13 gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a CRF2- 13 protein, preferably a mammalian CRF2- 13 protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the CRF2-13 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in CRF2-13 that are the result of natural allelic variation and that do not alter the functional activity of CRF2-13 are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding CRF2-13 proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of SEQ JO NO: 1 are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe CRF2-13 cDNAs ofthe invention can be isolated based on their homology to the human CRF2-13 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a soluble human CRF2-13 cDNA can be isolated based on its homology to human membrane-bound CRF2-13 . Likewise, a membrane-bound human CRF2-13 cDNA can be isolated based on its homology to soluble human CRF2-13 .
Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Homologs (i.e., nucleic acids encoding CRF2-13 proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion ofthe particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm,
50% ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.
Conservative mutations In addition to naturally-occurring allelic variants of the CRF2-13 sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, thereby leading to changes in the amino acid sequence ofthe encoded CRF2-13 protein, without altering the functional ability ofthe CRF2-13 protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of CRF2-13 without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the CRF2-13 proteins ofthe present invention, are predicted to be particularly unamenable to alteration.
Another aspect ofthe invention pertains to nucleic acid molecules encoding CRF2-13 proteins that contain changes in amino acid residues that are not essential for activity. Such CRF2-13 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2. Preferably, the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO:2, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2. An isolated nucleic acid molecule encoding a CRF2-13 protein homologous to the protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the nucleotide sequence of SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in CRF2-13 is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CRF2-13 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CRF2-13 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant CRF2-13 protein can be assayed for (1) the ability to form protei protein interactions with other CRF2-13 proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant CRF2-13 protein and a CRF2- 13 receptor; (3) the ability of a mutant CRF2- 13 protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind CRF2-13 protein; or (5) the ability to specifically bind an anti-CRF2-13 protein antibody.
Antisense CRF2-13 Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire CRF2-13 coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a CRF2-13 protein of SEQ ID NO:2, or antisense nucleic acids complementary to a CRF2-13 nucleic acid sequence of SEQ ID NO:l are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding CRF2-13 . The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human CRF2-13 corresponds to SEQ ID NO:2). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding CRF2-13 . The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding CRF2-13 disclosed herein (e.g., SEQ ID
NO:l), antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of CRF2-13 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of CRF2-13 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CRF2-13 mRNA: An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CRF2-13 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).
Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
CRF2-13 Ribozymes and PNA moieties
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CRF2-13 mRNA transcripts to thereby inhibit translation of CRF2- 13 mRNA. A ribozyme having specificity for a CRF2-13 -encoding nucleic acid can be designed based upon the nucleotide sequence of a CRF2- 13 DNA disclosed herein (i.e., SEQ ID NO:l). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CRF2-13 -encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CRF2-13 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g. , Bartel et al, (1993) Science 261:1411-1418. Alternatively, CRF2-13 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CRF2-13 (e.g., the CRF2-13 promoter and/or enhancers) to form triple helical structures that prevent transcription of the CRF2-13 gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of CRF2-13 can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of CRF2-13 can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of CRF2-13 can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
In another embodiment, PNAs of CRF2-13 can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of CRF2-13 can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
CR 2-13 Polypeptides
A CRF2-13 polypeptide ofthe invention includes the CRF2-13 -like protein whose sequence is provided in SEQ ID NO:2. In some embodiments, a CRF2-13 polypeptide includes amino acid sequences 21-520, amino acids 21-230 of SEQ ID NO:2, amino acids v 21-246 of SEQ ID NO:2, amino acids 231-520 of SEQ ID NO:2, amino acids 247-520 of SEQ ID NO:2. The invention also includes a mutant or variant form of the disclosed CRF2- 13 polypeptide, or of any of the fragments of the herein disclosed CRF2-13 polypeptide sequences. Thus, a CRF2-13 polypeptide includes one in which any residues may be changed from the corresponding residue shown in SEQ ID NO:2 while still encoding a protein that maintains its CRF2-13 -like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more of the residues may be so changed in the mutant or variant protein. In some embodiments, the CRF2-13 polypeptide according to the invention is a mature polypeptide.
In general, a CRF2-13 -like variant that preserves CRF2-13 -like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated CRF2-13 proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-CRF2-13 antibodies. In one embodiment, native CRF2-13 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, CRF2-13 proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a CRF2-13 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
A "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the CRF2-13 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of CRF2-13 protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of CRF2-13 protein having less than about 30% (by dry weight) of non-CRF2-13 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-CRF2-13 protein, still more preferably less than about 10% of non-CRF2-13 protein, and most preferably less than about 5 % non-CRF2- 13 protein. When the CRF2-13 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of CRF2-13 protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of CRF2-13 protein having less than about 30% (by dry weight) of chemical precursors or non-CRF2-13 chemicals, more preferably less than about 20% chemical precursors or non-CRF2-13 chemicals, still more preferably less than about 10% chemical precursors or non-CRF2-13 chemicals, and most preferably less than about 5% chemical precursors or non-CRF2-13 chemicals.
Biologically active portions of a CRF2-13 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the CRF2-13 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 that include fewer amino acids than the full length CRF2-13 proteins, and exhibit at least one activity of a CRF2-13 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the CRF2-13 protein. A biologically active portion of a CRF2-13 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a CRF2-13 protein of the present invention may contain at least one ofthe above-identified domains conserved between the CRF2-13 proteins. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native CRF2-13 protein.
In an embodiment, the CRF2-13 protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the CRF2-13 protein is substantially homologous to SEQ ID NO:2 and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. Accordingly, in another embodiment, the CRF2- 13 protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the CRF2-13 proteins of SEQ ID NO:2.
Determining homology between two or more sequence
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either ofthe sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 910 J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. The term "percentage of positive residues" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues. Chimeric and fusion proteins
The invention also provides CRF2-13 chimeric or fusion proteins. As used herein, a CRF2-13 "chimeric protein" or "fusion protein" comprises a CRF2-13 polypeptide operatively linked to a non-CRF2- 13 polypeptide. An "CRF2-13 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to CRF2-13 , whereas a "non-CRF2-13 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the CRF2-13 protein, e.g., a protein that is different from the CRF2-13 protein and that is derived from the same or a different organism. Within a CRF2-13 fusion protein the CRF2- 13 polypeptide can correspond to all or a portion of a CRF2- 13 protein. An example of a CRF2- 13 fusion polypeptide is one that includes amino acids 21-230 of SEQ ID NO:2 (e.g., a polypeptide that includes amino acids 1-246 or amino acids 21-246 of SEQ ID NO:2). In one embodiment, a CRF2-13 fusion protein comprises at least one biologically active portion of a CRF2-13 protein. In another embodiment, a CRF2-13 fusion protein comprises at least two biologically active portions of a CRF2-13 protein. Within the fusion protein, the term
"operatively linked" is intended to indicate that the CRF2-13 polypeptide and the non-CRF2- 13 polypeptide are fused in-frame to each other. The non-CRF2-13 polypeptide can be fused to the N-terminus or C-terminus ofthe CRF2-13 polypeptide.
For example, in one embodiment a CRF2-13 fusion protein comprises a CRF2-13 polypeptide operably linked to either an extracellular domain of a second protein, i.e., non- CRF2-13 protein, or to the transmembrane and intracellular domain of a second protein, ie., non-CRF2-13 protein. Such fusion proteins can be further utilized in screening assays for compounds that modulate CRF2-13 activity (such assays are described in detail below).
In another embodiment, the fusion protein is a GST-CRF2-13 fusion protein in which the CRF2-13 sequences are fused to the C-terminus ofthe GST (i.e., glutathione
S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant CRF2-13 .
In another embodiment, the fusion protein is a CRF2-13 -immunoglobulin fusion protein in which the CRF2-13 sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.
The CFR2-13 fusion proteins (e.g., CRF2-13 -immunoglobulin fusion proteins) ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit or augment an interaction between a cell surface receptor and its ligand. This could occur either by 1) binding to and removing available ligand for the receptor (Fc mediated scavenging ofthe ligand affecting bioavailability); 2) binding to the ligand and blocking its ability to bind to the cell receptor (antagonizing or neutralizing); 3) associating with another CRF member and thereby modulating (e.g., inhibiting) a downstream signal transduction cascade; 4) associating with either a ligand or another CRF and facilitating the activity ofthe ligand. By all of these mechanisms, a CRF2-13 protein may be used to modulate the interaction between a CRF2 receptor and its cognate ligand (e.g., an interaction between IL-10 and an IL-10 receptor and interaction between JL-22 and an IL-22 receptor). Inhibition of the CRF2-13 ligand/CRF2-13 interaction can be used therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer, modulating (e.g., promoting or inhibiting) cell survival as well as immunomodulatory disorders, autoimmunity, transplantation, and inflammation by alteration of cyotokine and chemokine cascade mechanisms. Moreover, the CRF2-13 -immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-CRF2-13 antibodies in a subject, to purify CRF2-13 ligands, and in screening assays to identify molecules that inhibit the interaction of CRF2- 13 with a CRF2-13 ligand.
A CRF2-13 chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A CRF2-13 -encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CRF2-13 protein. Polypeptide libraries
In addition, libraries of fragments of the CRF2-13 protein coding sequence can be used to generate a variegated population of CRF2-13 fragments for screening and subsequent selection of variants of a CRF2-13 protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CRF2-13 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the CRF2- 13 protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CRF2-13 proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CRF2-13 variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
CRF2-13 Antibodies
Also included in the invention are antibodies to CRF2-13 proteins, or fragments of CRF2-13 proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab 1 and F(ab-)2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated CRF2-13 -related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecificaUy bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO:2, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of CRF2-13 -related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe human CRF2-13 -related protein sequence will indicate which regions of a CRF2-13 -related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, /. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecificaUy bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368. 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised ofthe sequence of a human immunoglobulin, and contain minimal sequence derived from a non- human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature. 332:323- 327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)).
Human Antibodies
Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779- 783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules. An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker. A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecificaUy to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fa fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature. 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBO J., 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted fromE. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radiocoηjugated antibodies. Examples include 212Bi, 1311, 131In, 90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
CRF2-13 Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a CRF2-13 protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous rephcation in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CRF2-13 proteins, mutant forms of CRF2-13 proteins, fusion proteins, etc.).
The recombinant expression vectors ofthe invention can be designed for expression of CRF2-13 proteins in prokaryotic or eukaryotic cells. For example, CRF2- 13 proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the CRF2-13 expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO I. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invirrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, CRF2-13 can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO I. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to CRF2-13 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, CRF2- 13 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding CRF2-13 or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) CRF2-13 protein. Accordingly, the invention further provides methods for producing CRF2-13 protein using the host cells ofthe invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding CRF2-13 protein has been introduced) in a suitable medium such that CRF2-13 protein is produced. In another embodiment, the method further comprises isolating CRF2-13 protein from the medium or the host cell.
Transgenic CRF2-13 Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which CRF2-13 protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous CRF2-13 sequences have been introduced into their genome or homologous recombinant animals in which endogenous CRF2-13 sequences have been altered. Such animals are useful for studying the function and/or activity of CRF2-13 protein and for identifying and/or evaluating modulators of CRF2-13 protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CRF2-13 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development of the animal.
A transgenic animal ofthe invention can be created by introducing CRF2-13 -encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retro viral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. Sequences including SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human CRF2-13 gene, such as a mouse CRF2-13 gene, can be isolated based on hybridization to the human CRF2-13 cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the CRF2-13 transgene to direct expression of CRF2-13 protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe CRF2-13 transgene in its genome and/or expression of CRF2-13 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding CRF2-13 protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a CRF2-13 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the CRF2-13 gene. The CRF2-13 gene can be a human gene (e.g., the DNA of SEQ ID NO: 1), but more preferably, is a non-human homologue of a human CRF2-13 gene. For example, a mouse homologue of human CRF2-13 gene of SEQ ID NO: 1 can be used to construct a homologous recombination vector suitable for altering an endogenous CRF2-13 gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous CRF2-13 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous CRF2-13 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous CRF2-13 protein). In the homologous recombination vector, the altered portion ofthe CRF2-13 gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the CRF2-13 gene to allow for homologous recombination to occur between the exogenous CRF2-13 gene carried by the vector and an endogenous CRF2- 13 gene in an embryonic stem cell. The additional flanking CRF2-13 nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced CRF2-13 gene has homologously-recombined with the endogenous CRF2-13 gene are selected. See, e.g., Li, etal, 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously- recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2:
823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The CRF2-13 nucleic acid molecules, CRF2-13 proteins, and anti-CRF2-13 antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments ofthe antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst, 81(19): 1484 (1989).
A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound
(e.g., a CRF2-13 protein or anti-CRF2- 13 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drug Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drag Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are prefeπed. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and or produced by recombinant DNA technology. See, e.g., Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893. The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules ofthe invention can be used to express CRF2-13 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect CRF2-13 mRNA (e.g., in a biological sample) or a genetic lesion in a CRF2-13 gene, and to modulate CRF2-13 activity, as described further, below. In addition, the CRF2- 13 proteins can be used to screen drugs or compounds that modulate the CRF2-13 protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of CRF2-13 protein or production of CRF2-13 protein forms that have decreased or abeπant activity compared to CRF2-13 wild-type protein . In addition, the anti-CRF2-13 antibodies of the invention can be used to detect and isolate CRF2-13 proteins and modulate CRF2-13 activity. For example, CRF2- 13 activity includes T-cell or NK cell growth and differentiation, antibody production, and tumor growth.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to CRF2-13 proteins or have a stimulatory or inhibitory effect on, e.g., CRF2-13 protein expression or CRF2-13 protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a CRF2- 13 protein or polypeptide or biologically-active portion thereof. The test compounds ofthe invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J. Med. Chem. 37: 2678; Cho, etal, 1993. Science 261: 1303; Carrell, etal, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, etal, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, etal, 1994. /. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. /. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to a CRF2-13 protein determined. The cell, for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the CRF2-13 protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the CRF2-13 protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds CRF2-13 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CRF2-13 protein, wherein determining the ability ofthe test compound to interact with a CRF2-13 protein comprises determining the ability of the test compound to preferentially bind to CRF2-13 protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of CRF2-13 protein, or a biologically-active portion thereof, on the cell surface with a test compound and determimng the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe CRF2-13 protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of CRF2-13 or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the CRF2-13 protein to bind to or interact with a CRF2-13 target molecule. As used herein, a "target molecule" is a molecule with which a CRF2-13 protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a CRF2-13 interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A CRF2-13 target molecule can be a non-CRF2-13 molecule or a CRF2-13 protein or polypeptide of the invention In one embodiment, a CRF2- 13 target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound CRF2-13 molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with CRF2-13 .
Determining the ability of the CRF2-13 protein to bind to or interact with a CRF2-13 target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability ofthe CRF2-13 protein to bind to or interact with a CRF2-13 target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a CRF2-13 -responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a CRF2-13 protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the CRF2-13 protein or biologically-active portion thereof. Binding of the test compound to the CRF2-13 protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the CRF2-13 protein or biologically-active portion thereof with a known compound which binds CRF2-13 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a CRF2-13 protein, wherein determining the ability ofthe test compound to interact with a CRF2-13 protein comprises determining the ability of the test compound to preferentially bind to CRF2-13 or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting CRF2-13 protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the CRF2-13 protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of CRF2-13 can be accomplished, for example, by determining the ability ofthe CRF2-13 protein to bind to a CRF2-13 target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of CRF2-13 protein can be accomplished by determimng the ability of the CRF2-13 protein further modulate a CRF2- 13 target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described above.
In yet another embodiment, the cell-free assay comprises contacting the CRF2-13 protein or biologically-active portion thereof with a known compound which binds CRF2-13 protein to form an assay mixture, contacting the assay mixture with a test compound, and deteπmning the ability of the test compound to interact with a CRF2-13 protein, wherein determining the ability of the test compound to interact with a CRF2-13 protein comprises determining the ability of the CRF2-13 protein to preferentially bind to or modulate the activity of a CRF2-13 target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of CRF2-13 protein. In the case of cell-free assays comprising the membrane-bound form of CRF2-13 protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of CRF2-13 protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl~N,N-dimethyl-3-ammonio-l-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholarmdopropyl)dimemylanuniniol-2-hydroxy-l-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either CRF2-13 protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to CRF2-13 protein, or interaction of CRF2-13 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-CRF2-13 fusion proteins or GST- target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or CRF2-13 protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of CRF2-13 protein binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the CRF2-13 protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated CRF2-13 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with CRF2-13 protein or target molecules, but which do not interfere with binding of the CRF2-13 protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or CRF2-13 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CRF2-13 protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the CRF2-13 protein or target molecule. In another embodiment, modulators of CRF2-13 protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of CRF2- 13 mRNA or protein in the cell is determined. The level of expression of CRF2-13 mRNA or protein in the presence of the candidate compound is compared to the level of expression of CRF2-13 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of CRF2-13 mRNA or protein expression based upon this comparison. For example, when expression of CRF2-13 mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of CRF2-13 mRNA or protein expression. Alternatively, when expression of CRF2-13 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CRF2-13 mRNA or protein expression. The level of CRF2-13 mRNA or protein expression in the cells can be determined by methods described herein for detecting CRF2-13 mRNA or protein.
In yet another aspect ofthe invention, the CRF2-13 proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, etal, 1993. Cell 72: 223-232; Madura, etal, 1993. /. Biol Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, etal, 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with CRF2-13 ("CRF2-13 -binding proteins" or "CRF2-13 -bp") and modulate CRF2-13 activity. Such CRF2-13 -binding proteins are also likely to be involved in the propagation of signals by the CRF2-13 proteins as, for example, upstream or downstream elements ofthe CRF2-13 pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for CRF2-13 is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a CRF2-13 -dependenht complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with CRF2-13 . The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
The invention will be further illustrated in the following non-limiting examples.
Example 1. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:4. The variant amino acid sequence is shown in bold-font. A valine at position 30 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an alanine in SEQ ID NO:4. MAGPER GPLLLCL QAAPGRPRLAPPQNATLLSQNFSVYLTW PG GNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMC KKQDLYNKF GRVRTVSPSSKSP VESEYLDY FEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAF KEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA IrøAFLVLPSL IIi LVIAAGGVI TLMGNPWFQRAK PRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEΞEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNVPGGPPVS QTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR(SEQ ID NO: 4)
Example 2. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:5. The variant amino acid sequence is shown in bold-font. A leucine at position 39 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an isoleucine in SEQ ID NO:5.
MAGPERWGPLLLC LQAAPGRPR APPQNVTLLSQNFSVYITWLPGLGNPQDVTYFVAYQSSPTRRRWREVEΞCA GTKELLCS MC KKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAF KEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPΞA IWAFLVLPSLLI L VIAAGGVI KTLMGNPWFQRAKMPRA DFSGHTHPVATFQPSRPESVND F CPQKELTR • GVRPTPRVRAPATQQTR KDLAEDEΞEΞDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYDAEKGPGQGPGGDGHQESLPPPΞFSKDSGFLEELPEDN SSWAT WGTLPPEPNLVPGGPPVS QT TFCWESSPΞEEEEARESEIEDSDAGSWGAESTQRTEDRGRT GHY AR (SEQ ID NO: 5)
Example 3. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:6. The variant amino acid sequence is shown in bold-font. An asparagine at position 49 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a threonine in SEQ ID NO:6. MAGPΞRWGP LLCL QAAPGRPRLAPPQNVTLIiSQNFSVYLTWLPGLGTPQDVTYFVAYQSSPTRRR REVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESΞY DYL.FEVEPAPPVLVLTQTEEILSANATYQ PPC MPPLDLKYEVAFW EGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAF VLPS LILIiLVIAAGGVIWKTLMGNPWFQRAK PRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRW KD AΞDEEEEDEEDTEDGVSFQPYIEPPSF GQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEE PEDNLSS AT WGTLPPEPN VPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 5)
Example 4. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:7. The variant amino acid sequence is shown in bold-font. An arginine at position 65 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO:7.
MAGPER GPLL CL QAAPGRPRLAPPQNVTLLSQNFSVYLTW PGLGNPQDVTYFVAYQSSPTKRR REVEECA GTKELLCSMMCLKKQDLYN FKGRVRTVSPSSKSPWVESΞYLDYLFEVEPAPPVLVLTQTEEILSANATYQ PPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFL EVPEA NWAFLVLPSL I L VIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSADSSDRSWASTVDSSWDRAGSSGYAEKGPGQGPGGDGHQES PPPEFSKDSGFLEELPEDNLSSWAT WGT PPEPN VPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHY AR (SEQ ID NO: 7)
Example 5. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 8. The variant amino acid sequence is shown in bold-font. A lysine at position 78 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an arginine in SEQ ID NO:8. MAGPERWGPL C LQAAPGRPRLAPPQNVTLLSQNFSVYLT LPG GNPQDVTYFVAYQSSPTRRRWREVEECA GTRELLCSMMCLKKQD YNKFKGRVRTVSPSSKSPWVESEY DYLFEVEPAPPVVLTQTEEILSANATYQLPPC MPP DLKYΞVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHC SARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNP FQRAKMPRA DFSGHTHPVATFQPSRPESVNDLF CPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAP V PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYAEKGPGQGPGGDGHQESLPPPEFSKDSGF EELPEDNLSSWAT WGT PPEPNLVPGGPPVSLQT TFCWESSPEEΞEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 8)
Example 6. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:9. The variant amino acid sequence is shown in bold-font. A Q {glutamine?} at position 90 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an asparagine in SEQ ID NO:9.
MAGPΞR GP CLLQAAPGRPRLAPPQNVTLLSQNFSVYLTW PGLGNPQDVTYFVAYQSSPTRRRWRΞVEECA GTKELLCSMMC KKND YNKF GRVRTVSPSSKSPWVESEYLDY FEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDL YEVAF KΞGAGNKTLFPVTPHGQPVQIT QPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLIL LVIAAGGVIWKTLMGNP FQRA MPRAIiDFSGHTHPVATFQPSRPESVNDLFLCPQKE TR GVRPTPRVRAPATQQTRKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAP V PSEGSSA DSSDRS ASTVDSSWDRAGSSGY AE GPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSS AT GTLPPEPNIiVPGGPPVS QTLTFC ESSPEEEEEARESEIEDSDAGS GAΞSTQRTEDRGRTLGHYMAR (SEQ ID NO: 9)
Example 7. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 10. The variant amino acid sequence is shown in bold-font. A arginine at position 99 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an lysine in SEQ ID NO: 10.
Ill MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWRΞVEECA GTKELLCSMMCLKKQDLYNKFKGKVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKEI.TR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIΞPPSFLGQEHQAPGHSΞAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPΞΞEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ
ID NO: 10)
Example 8. A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence ofSEQ ID NO:2 is shown in SEQ ID NO:11. The variant amino acid sequence is shown in bold-font. A valine atposition 112 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an leucine in SEQ ID NO:.11.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRKRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWLESEYLDYLFEVEPAPPVLVLTQTΞEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIΞPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEΞEEEARESΞIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ
ID NO: 11)
Example 9. A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 12. The variant amino acid sequence is shown in bold-font. A tyrosine at position 119 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a phenylalanine in SEQ ID NO: 12. MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEFLDYLFΞVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEΞEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTΞDRGRTLGHYMAR (SEQ
ID NO: 12)
Example 10. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 13. The variant amino acid sequence is shown in bold-font. A valine at position 129 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an isoleucine in SEQ ID NO: 13.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPILVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPΞSVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEΞDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEΞARESEIEDSDAGSWGAESTQRTΞDRGRTLGHYMAR (SEQ ID NO: 13)
Example 11. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 14. The variant amino acid sequence is shown in bold-font. A threonine at position 144 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an asparagine in SEQ ED NO: 14.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEΞCA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANANYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 14)
Example 12. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 15. The variant amino acid sequence is shown in bold-font. A leucine at position 154 in the polypeptide sequence shown in SEQ ED NO:2 is replaced with an alanine in SEQ ID NO: 15.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTΞEILSANATYQLPPC MPPADLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLΞVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPΞSVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDΞΞEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 15)
Example 13. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2) A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 16. The variant amino acid sequence is shown in bold-font. A lysine at position 170 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an arginine in SEQ ID NO: 16.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKΞLLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEΞILSANATYQLPPC MPPLDLKYEVAFWKEGAGNRTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDΞEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 16)
Example 14. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 17. The variant amino acid sequence is shown in bold-font. A valine at position 175 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a leucine in SEQ ID NO: 17.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPLTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPΞA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEΞDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEΞEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 17) Example 15. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 18. The variant amino acid sequence is shown in bold-font. An alanine at position 189 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a valine in SEQ ID NO: 18.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPVASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPΞSVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEΞEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPΞDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEΞARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAR (SEQ ID NO: 18)
Example 16. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 19 The variant amino acid sequence is shown in bold-font. An arginine at position 199 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO:.19
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSAKTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPΞSVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDΞEEEDEEDTΞDGVSFQPYIEPPSFLGQEHQAPGHSΞAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEΞEEEARESEIEDSDAGSWGAΞSTQRTEDRGRTLGHYMAR (SEQ ID NO: 19) Example 17. A sequence variant of the disclosed CRF2-13 polypeptide amino acid sequence (SEQ ED NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:20. The variant amino acid sequence is shown in bold-font. A phenylalanine at position 212 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with an a tryptophan in SEQ ID NO:20.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKΞGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSK SKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWGAESTQRTΞDRGRTLGHYMAR (SEQ ID NO: 20)
Example 18. A sequence variant ofthe disclosed CRF2-13 polypeptide amino acid sequence (SEQ ID NO:2)
A polypeptide sequence differing by one amino acid sequence from the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO:21. The variant amino acid sequence is shown in bold-font. An arginine at position 230 in the polypeptide sequence shown in SEQ ID NO:2 is replaced with a lysine in SEQ ID NO21:.
MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGNPQDVTYFVAYQSSPTRRRWREVEECA GTKELLCSMMCLKKQDLYNKFKGRVRTVSPSSKSPWVESEYLDYLFΞVEPAPPVLVLTQTEEILSANATYQLPPC MPPLDLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVPKYSKFSKPTCFLLEVPEA NWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQRAKMPRALDFSGHTHPVATFQPSRPESVNDLFLCPQKELTR GVRPTPRVRAPATQQTRWKKDLAEDEEΞEDEEDTΞDGVSFQPYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLV PSEGSSAWDSSDRSWASTVDSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPΞFSKDSGFLEELPEDNLSSWAT WGTLPPEPNLVPGGPPVSLQTLTFCWΞSSPEEEEEARESEIEDSDAGSWGAESTQRTEDRGRTLGHYMAK (SEQ ID NO: 21) Example 19. Identification of a CRF2-13 Sequence in a Human Placental cDNA Library
A 310 nucleotide fragment corresponding to nucleotides XX to XX [41-352 of SEQ ID No.l] in Table 1 was identified in a human placental cDNA library (BD Biosciences Clontech, Palo Alto, CA, USA) by PCR using an Advantage II PCR kit (BD Biosciences Clontech, Palo Alto, CA, USA) and primers specific for the 5' region of the human CRF2-13. The primers included Ax5-1 (GCTGCAGGCCGCTCCAGGGAGGCCCCG; SEQ ID:23) and Ax3-1 (CCAGGTATTCGGACTCCACCCAGGGGGAC; SEQ ID NO:24). The primers were used for thirty eight thermal cycles of PCR. The CRF2-13 nucleic acid product was gel purified and sequenced. The sequence corresponds to the corresponding sequences in the CRF2-13 sequence disclosed in Table 1.
Based on these findings a Rapid-Screen™ Arrayed cDNA Library Panel of Human Placenta Sub-Plate 2H (Origene Technologies, Inc., Rockville, MD, USA) was selected for screening and isolation of the CFR2-13 clone coding for the mature protein. [11-1563 of SEQ ID No.l]. The existence of the first 10 bases of SEQ ID No.1 was verified by PCR. The library quality was improved by first isolating double-stranded cDNAs of different size- fractions and then ligating them separately into the vector. The cDNA library is arrayed in a 96-well plates.
Since the cDNAs ofthe Human Placenta Sub-Plate 2H human placental library were directionally-cloned into the CMV expression vector pCMV6-XL4, a vector-derived 5' PCR primer was used in conjunction with a gene-specific 3' reverse primer to identify the CRF2- 13 clone. In this study, the cDNA library was screened by a PCR-based procedure using the Advantage II PCR kit (BD Biosciences Clontech, Palo Alto, CA, USA) and Ax5-1 (SEQ ID:25 and Ax3-2 (TTGGTTCCCGCACACTCTTCCACTTCG; SEQ ID NO:26) as PCR primers. PCR analysis was carried out in a 96-well arrayed at 50 clones per well. The PCR positive well (E2) was identified and the E. coli cells from that well were subsequently diluted, plated out and analyzed to yield the clone full-length CRF2-13 clone. The identity of the CRF2-13 clone was then verified by sequence analysis.
OTHER EMBODIMENTS While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is defined by the scope ofthe appended claims. Other aspects, advantages, and modifications are within the scope ofthe following claims.

Claims

What is claimed is:
1. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 85% identical to amino acids 21-520 of SEQ ID NO:2.
2. A vector comprising the nucleic acid molecule of claim 1.
3. A cell including the vector of claim 2.
4. A pharmaceutical composition comprising the nucleic acid molecule of claim 1 and a pharmaceutically acceptable carrier.
5. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 21-520 of SEQ ED NO:2.
6. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 95% identical to amino acids 21-520 of SEQ ID NO:2.
7. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 98% identical to amino acids 21-520 of SEQ ED NO:2.
8. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 99% identical to amino acids 21-520 of SEQ ID NO:2.
9. The nucleic acid molecule of claim 8, wherein said nucleic acid molecule encodes a polypeptide with an amino acid sequence having one or more substitutions relative to the amino acid sequence of amino acids 21-520 of SEQ ID NO:2.
10. The nucleic acid molecule of claim 9, wherein said molecule hybridizes under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule comprising SEQ ID NO:l
11. The nucleic acid molecule of claim 9, wherein said encoded polypeptide binds specifically to a polypeptide ligand.
12. An isolated nucleic acid molecule encoding a polypeptide that includes amino acids 21-520 of SEQ ED NO:2.
13. The nucleic acid molecule of claim 12, wherein said encoded polypeptide consists of amino acids 1-520 of SEQ ID NO:2.
14. The nucleic acid molecule of claim 12, wherein said isolated nucleic acid molecule comprises nucleotides 1-1563 of SEQ ED NO:l.
15. The nucleic acid molecule of claim 12, wherein said encoded polypeptide includes amino acids 1-520 of SEQ ID NO:2.
16. A vector comprising the nucleic acid molecule of claim 12 and a pharmaceutically acceptable carrier.
17. A cell containing the vector of claim 16.
18. An isolated nucleic acid encoding a polypeptide of at least 499 amino acids, wherein said nucleic acid hybridizes under high stringency conditions to SEQ ID NO:l.
19. An isolated nucleic acid encoding a polypeptide of at least 499 amino acids, wherein said nucleic acid hybridizes under moderate stringency conditions to SEQ ED NO: 1.
20. An isolated nucleic acid encoding a polypeptide of at least 499 amino acids, wherein said nucleic acid hybridizes under low stringency conditions to SEQ ID NO:l.
21. A substantially purified polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence of amino acids 21-520 of
SEQ ED NO:2.
22. A pharmaceutical composition comprising the polypeptide of claim 20 and a pharmaceutically acceptable carrier.
23. A substantially purified polypeptide comprising an amino acid sequence at least 95% identical to a polypeptide comprising the amino acid sequence of amino acids 21-520 of SEQ ID NO:2.
24. A substantially purified polypeptide comprising an amino acid sequence at least 99% identical to a polypeptide comprising the amino acid sequence of amino acids 21-520 of SEQ ID NO:2.
25. The substantially purified polypeptide of claim 24, wherein said polypeptide differs by one or more substitutions from amino acids 21-520 of SEQ ID
NO:2.
26. A substantially purified polypeptide comprising amino acids 21-520 of SEQ ID NO:2.
27. The substantially purified polypeptide of claim 26, wherein said polypeptide comprises the amino acid sequence of SEQ ED NO:2.
28. The substantially purified polypeptide of claim 26, wherein said polypeptide consists of amino acids 21-520 of SEQ ID NO:2.
29. The substantially purified polypeptide of claim 26, wherein said polypeptide consists of the amino acid sequence of SEQ ID NO:2.
30. A polypeptide at least 85% homologous to amino acids 21-230 of SEQ ED NO:2.
31. The polypeptide of claim 30, wherein said polypeptide binds specifically to a polypeptide ligand.
32. A polypeptide at least 95% homologous to amino acids 21-230 of SEQ ED NO:2.
33. A polypeptide at least 98% homologous to amino acids 21-230 of SEQ ED NO:2.
34. A polypeptide at least 99% homologous to amino acids 21-230 of SEQ
ID NO:2.
35. The polypeptide of claim 34, wherein said polypeptide differs by one or more substitutions from amino acids 21-230 of SEQ ED NO:2.
36. A substantially purified polypeptide comprising amino acids 21-230 of SEQ ID NO:2.
37. The polypeptide of claim 26, wherein said polypeptide consists of amino acids 21-230 of SEQ ED NO:2.
38. A fusion polypeptide comprising the polypeptide of claim 18 operably linked to a non-CRF2-13 polypeptide.
39. The fusion polypeptide of claim 38, wherein said non-CRF2- 13 polypeptide comprises at least one member selected from the group consisting of an
Fc region of an immunoglobulin molecules or a FLAG epitope, a HIS tag, and a MYC tag.
40. A pharmaceutical composition comprising the fusion polypeptide of claim 26 and a pharmaceutically acceptable carrier.
41. A fusion polypeptide comprising the polypeptide of claim 26 operably linked to a non-CRF2-13 polypeptide.
42. The fusion polypeptide of claim 41 , wherein said non-CRF2- 13 polypeptide comprises at least one member selected from the group consisting of an Fc region of an immunoglobulin molecules or a FLAG epitope, a HES tag, and a MYC tag.
43. A pharmaceutical composition comprising the fusion polypeptide of claim 41 and a pharmaceutically acceptable carrier.
44. A fusion polypeptide comprising the polypeptide of claim 30 operably linked to a non CRF2-13 polypeptide.
45. The fusion polypeptide of claim 44, wherein said non-CRF2- 13 polypeptide comprises at least one member selected from the group consisting of an Fc region of an immunoglobulin molecules or a FLAG epitope, a HIS tag, and a MYC tag.
46. A pharmaceutical composition comprising the fusion polypeptide of claim 44 and a pharmaceutically acceptable carrier.
47. A fusion polypeptide comprising the polypeptide of claim 36 operably linked to a non CRF2-13 polypeptide.
48. The fusion polypeptide of claim 47, wherein said non-CRF2- 13 polypeptide comprises at least one member selected from the group consisting of an Fc region of an immunoglobulin molecules or a FLAG epitope, a HIS tag, and a MYC tag.
49. A pharmaceutical composition comprising the fusion polypeptide of claim 47 and a pharmaceutically acceptable carrier.
50. An antibody that binds selectively to the polypeptide of claim 1, the polypeptide of claim 26, the polypeptide of claim 26, the polypeptide of claim 30, the polypeptide of claim 36, the fusion polypeptide of claim 38, the fusion polypeptide of claim 41, the fusion polypeptide of claim 44, or the fusion polypeptide of claim 47.
51. The antibody of claim 50, wherein said antibody neutralizes binding of a CRF2-13 polypeptide to a CRF2-13 ligand.
52. The antibody of claim 50, wherein said antibody is a polyclonal antibody.
53. The antibody of claim 50, wherein said antibody is a monoclonal antibody.
54. The monoclonal antibody of claim 53, wherein said monoclonal antibody is selected from the group consisting of a murine monoclonal antibody, and a humanized monoclonal antibody.
55. The monoclonal antibody of claim 54, wherein said monoclonal antibody is a humanized monoclonal antibody.
56. A kit comprising in one or more containers a compound selected from the group consisting of an CRF2-13 nucleic acid, an CRF2-13 polypeptide and an
- antibody to an CRF2- 13 polypeptide.
57. The kit of claim 56, wherein said compound is present with a pharmaceutically acceptable carrier.
58. A method of producing a CRF2-13 polypeptide, said method comprising culturing a cell including the nucleic acid molecule of claim 1 under conditions allowing for expression of a polypeptide encoded by said nucleic acid molecule.
59. A method of producing a CRF2-13 polypeptide, said method comprising culturing a eel including the nucleic acid molecule of claim 12 under conditions allowing for expression of a polypeptide encoded by said nucleic acid molecule.
60. A method of detecting the presence of a CRF2-13 nucleic acid molecule in a biological sample, the method comprising: contacting the sample with a nucleic acid probe; and identifying the bound probe, if present, thereby detecting the presence of CRF2-13 nucleic acid molecule in said sample.
61. The method of claim 60, wherein said CRF2-13 nucleic acid molecule is detected in a PCR reaction using primers (GCTGCAGGCCGCTCCAGGGAGGCCCCG; (SEQ ID:23) and
(CCAGGTATTCGGACTCCACCCAGGGGGAC (SEQ ID NO:24).
62. A method of detecting the presence of a CRF2- 13 polypeptide in a sample, the method comprising: contacting the sample with a compound that selectively binds to said polypeptide under conditions allowing for formation of a complex between said polypeptide and said compound; and detecting said complex, if present, thereby identifying said polypeptide in said sample.
63. A method of modulating the activity of a CRF2- 13 polypeptide, the method comprising contacting a cell sample comprising said polypeptide with a compound that binds to said polypeptide in an amount sufficient to modulate the activity of the polypeptide.
64. The method of claim 63, wherein said compound is a soluble CRF2-13 polypeptide inhibitor.
65. The method of claim 64, wherein said soluble CRF2-13 inhibitor includes a polypeptide at least 85% homologous to amino acids 21-260 of SEQ ID NO:2.
66. A method for screening for a modulator of activity or of latency or predisposition to a cytokine-mediated immune disorder, the method comprising: contacting a test compound with a CRF2-13 polypeptide; and determining if said test compound binds to said CRF2-13 polypeptide, wherein binding of said test compound to said polypeptide indicates the test compound is a modulator of activity or of latency or predisposition to a cytokine- mediated immune disorder.
67. A method for screening for a modulator of activity or of latency or predisposition to a cytokine-mediated immune disorder, the method comprising: administering a test compound to a test animal suffering from or at increased risk for said immune disorder, wherein said test animal recombinandy expresses a CRF2-13; measuring expression ofthe activity of said polypeptide in said test animal; measuring the activity of said polypeptide in a control animal that recombinantly expresses said polypeptide and is not at increased risk for said immune disorder; and comparing expression of said polypeptide in said test animal and said control animal, wherein a change in the activity of said polypeptide in said test animal relative to said control animal indicates the test compound is a modulator of latency of said immune disorder, and wherein said cytokine-mediated immune disorder is selected from the group consisting of an autoimmune disorder, a T-lymphocyte-associated disorder, a cell-prohferation disorder, a cell differentiation disorder, and an immune deficiency order.
68. A method for determining the presence of or predisposition to a disease associated with altered levels of a CRF2-13 polypeptide, the method comprising: a) measuring the amount of said polypeptide in a sample from said subject; and b) comparing the amount of the polypeptide in step (a) to the amount of the polypeptide present in a control sample, wherein an alteration in the level of said polypeptide in step (a) as compared to the level of the polypeptide in said control sample indicates the presence of or predisposition to a disease in said subject.
69. The method of claim 68, wherein said subject is a human.
70. A method for determining the presence of or predisposition to a disease associated with altered levels of a CRF2-13 nucleic acid molecule, the method comprising: a) measuring the amount ofthe nucleic acid in a sample from said subject; and b) comparing the amount of said nucleic acid in step (a) to the amount of the nucleic acid present in a control sample, wherein an alteration in the level of said nucleic acid in step (a) as compared to the level of the nucleic acid in the control sample indicates the presence of or predisposition to said disease in said subject.
71. The method of claim 70, wherein said subject is a human.
72. A method of treating or preventing a pathological condition associated with a cytokine-mediated disorder, the method comprising administering to a subject an agent that increases levels of a polypeptide comprising the extracellular amino acid sequence of a CRF2-13 polypeptide in an amount sufficient to alleviate or prevent the pathological condition in said subject
73. The method of claim 72, wherein said subject is a human.
74. The method of claim 72, wherein said agent is a polypeptide that includes the extracellular amino acid sequence of a CRF2-13 polypeptide.
75. The method of claim 74, wherein said polypeptide is a fusion polypeptide comprising the extracellular amino acid sequence of a CRF2-13 polypeptide fused to a non-CRF2-13 polypeptide.
76. The method of claim 72, wherein said agent is a nucleic acid encodes a polypeptide that includes the extracellular amino acid sequence of a CRF2-13 polypeptide.
77. A method of treating or preventing a pathological condition, the method comprising administering an antibody that binds specifically to a CRF2-13 polypeptide in an amount sufficient to alleviate or prevent the pathological condition.
78. The method of claim 77, wherein said subject is a human.
79. A method of treating rheumatoid arthritis in a subject, the method comprising administering to the subject an agent that modulates the amount of a CRF2-13 polypeptide in said subject.
80. The method of claim 79, wherein said subject is a human.
81. The method of claim 79, wherein said agent is a CRF2- 13 nucleic acid or polypeptide.
82. The method of claim 79, wherein said agent increases the amount of said CRF2-13 polypeptide in said subject.
83. The method of claim 79, wherein said agent decreases the amount of said CRF2-13 polypeptide in said subject.
84. The method of claim 79, wherein said agent is an anti-CRF2-13 antibody. '
85. A method of treating multiple sclerosis in a subject, the method comprising administering to the subject an agent that modulates the amount of a CRF2-13 polypeptide in said subject.
86. A method of modulating vascular smooth muscle cell proliferation, the method comprising contacting a vascular smooth muscle cell with an agent that modulates the amount of CRF2-13 polypeptide in said cell.
87. A method of treating or preventing inflammation in a subject, the method comprising administering to said subject an agent that modulates the amount of a CRF2-13 polypeptide in said subject.
88. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 30957 to nucleotide 30967 of SEQ ID NO:3, provided that position 30962 of said polynucleotide is "A or "G".
89. The polynucleotide of claim 88, wherein position 30962 of said sequence is "A".
90. The polynucleotide of claim 88, wherein position 30962 of said sequence is "G".
91. The polynucleotide of claim 88, wherein said polynucleotide includes at least 15 contiguous nucleotides of SEQ ID NO:3.
92. The polynucleotide of claim 88, wherein said polynucleotide includes at least 20 contiguous nucleotides of SEQ ID NO:3.
93. The polynucleotide of claim 88, wherein said polynucleotide is between about 10 and about 100 nucleotides in length.
94. The polynucleotide of claim 88, wherein said polynucleotide sequence is between about 10 and about 90 nucleotides in length.
' 95. The polynucleotide of claim 88, wherein said polynucleotide sequence is between about 10 and about 75 nucleotides in length.
96. The polynucleotide of claim 88, wherein said polynucleotide is between about 10 and about 50 bases in length.
97. The polynucleotide of claim 88, wherein said polynucleotide is between about 10 and about 40 bases in length.
98. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 30650 to nucleotide 30660 of SEQ ID NO:3, provided that position 30655 of said polynucleotide is "A" or "G".
99. The polynucleotide of claim 98, wherein position 30655 of said sequence is "A".
100. The polynucleotide of claim 98, wherein position 30655 of said sequence is "G".
101. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 28739 to nucleotide 28749 of SEQ ID NO:3, wherein position 28744 of said polynucleotide is "A" or "G".
102. The polynucleotide of claim 101 , wherein position 28744 of said sequence is "A".
103. The polynucleotide of claim 101, wherein position 28744 of said sequence is "G".
104. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 28442 to 28452 of SEQ ID NO:3, wherein position 28448 of said polynucleotide is "C" or "T".
105. The polynucleotide of claim 104, wherein position 28448 of said polynucleotide is "C".
106. The polynucleotide of claim 104, wherein position 28448 of said polynucleotide is "T".
107. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 9421 to 9431 of SEQ ED NO:3, wherein position 9426 of said polynucleotide is "A" or "G".
108. The polynucleotide of claim 107, wherein position 9426 of said polynucleotide is "A".
109. The polynucleotide of claim 107, wherein position 9426 of said polynucleotide is "G".
110. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 9157 to 9167 of SEQ ED NO:3, wherein position 9162 of said polynucleotide is "A" or "G".
111. The polynucleotide of claim 110, wherein position 9162 of said polynucleotide is "A".
112. The polynucleotide of claim 110, wherein position 9162 of said polynucleotide is "T".
113. An isolated polynucleotide comprising at least 10 contiguous nucleotides from nucleotide 8806 to 8816 of SEQ ED NO:3, wherein position 8811 of said polynucleotide is "C or "T".
114. The polynucleotide of claim 113, wherein position 8811 of said polynucleotide is "C".
115. The polynucleotide of claim 113, wherein position 8811 of said polynucleotide is "T".
PCT/US2002/036316 2001-11-09 2002-11-12 Type 2 cytokine receptor and nucleic acids encoding same WO2003040345A2 (en)

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EP2251353A1 (en) 2003-08-07 2010-11-17 ZymoGenetics, L.L.C. Homogeneous preparations of IL-28 and IL-29
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ZA200403284B (en) 2005-05-27
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