WO2002066600A2 - Procedes et materiaux concernant des polypeptides et polynucleotides semblables a des recepteurs de leucocytes de type immunoglobuline (lir) - Google Patents

Procedes et materiaux concernant des polypeptides et polynucleotides semblables a des recepteurs de leucocytes de type immunoglobuline (lir) Download PDF

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Publication number
WO2002066600A2
WO2002066600A2 PCT/US2001/049435 US0149435W WO02066600A2 WO 2002066600 A2 WO2002066600 A2 WO 2002066600A2 US 0149435 W US0149435 W US 0149435W WO 02066600 A2 WO02066600 A2 WO 02066600A2
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Prior art keywords
polypeptide
polynucleotide
seq
protein
cell
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PCT/US2001/049435
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English (en)
Inventor
Bryan J. Boyle
Chiaoyun Kuo
Nancy K. Mize
Dana A. Haley-Vicente
Matthew C. Arterburn
Y. Tom Tang
Ping Zhou
Chenghua Liu
Vinod Asundi
Radoje T. Drmanac
George Yeung
Servando Palencia
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Hyseq, Inc.
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Publication of WO2002066600A2 publication Critical patent/WO2002066600A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6432—Coagulation factor Xa (3.4.21.6)
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21006—Coagulation factor Xa (3.4.21.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.
  • the invention relates to a novel leukocyte immunoglobuiin receptor-like polypeptide (LIR-like). 2.2 BACKGROUND ART
  • Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other t pes of data and products dependent on DNA and arru o acid se uences. — " p-c-e' "_:_ . .” '”e c se A- -- - ⁇ - -e ⁇ -.ercs cr. rg. ?_» vr..r -- * -: er _.q r-r-.iss-.e so- e in tne case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed. In particular, this invention is directed to a novel soluble LIR-like polypeptides and polynucleotides.
  • Immune system functions are governed by a complex network of cell surface interactions and associated signaling processes.
  • a cell surface receptor When a cell surface receptor is activated by its ligand a signal is sent into the cell; depending upon the signal transduction pathway that is engaged, the signal can be inhibitory or activatory.
  • cellular activity is regulated by a balance between activatory signajs and inhibitory signals.
  • ACM immunoreceptor tyrosine-based activation motifs
  • ITIM immunoreceptor tyrosine based inhibitory motifs
  • Receptors having these motifs are involved in inhibitory signaling because these motifs provided binding sites for tyrosine phosphatases which block signaling by removing phosphate from activated receptors or signal transduction molecules.
  • NK cells Natural Killer cells
  • the cytolytic activity of Natural Killer (NK) cells is an example of immune system activity which is regulated by a balance between positive signals that initiate cell function and inhibitory signals which prevent the activity.
  • the receptors that activate NK cytotoxicity are not fully understood. If the target cell expresses cell surface MHC class I antigens for which the NK cell has a specific receptor, the NK cell is inhibited from killing the target cell.
  • KIRs Killer Inhibitory Receptors
  • Immunoglobuiin (lg) superfamiiy genes encode for diverse proteins characterized by a common lg fold.
  • the lg superfamiiy proteins include antibodies, T cell receptors, B cell receptors, NK, myeloid and leukocyte immunoglobuiin receptors like killer inhibitory receptors (KIRs) and activating receptors (ARs).
  • KIRs killer inhibitory receptors
  • ARs activating receptors
  • Ig Variable domains are utilized to create a specific binding site while Ig Constant domains may serve as more conserved counter receptor binding module.
  • CMRF-35 and PIGR-1 immunoglobuiin members have been cloned that have only one Ig-variable domain (Shujian et al (1999). EP 0897981A1, incorporated herein by reference). It is becoming apparent that inhibitory receptors are present on most of the haemopoietic cells, including dendritic cells, monocytes, CD19+ B cells; and CD3+ T cells (Borges and Cosman (2000). Cytokine and Growth Factor Reviews, 11, 209-217; De Maria, AD et al, (1997) Proc. Natl. Acad.
  • TTAMs Transmembrane molecules with immunoreceptor tyrosine activation motifs (TTAMs) (YxxL where x is any amino acid, SEQ ID NO: 78) are activating receptors.
  • TTIMs immunoreceptor tyrosine inhibition motifs (17L/NxxYx ⁇ ]JV where x is any amino acid, SEQ ID NO: 77) are inhibitory in nature.
  • a third class of transmembrane receptors like LIR-4, lack a cytoplasmic tail with an activation or an inhibition motif. Rather, these proteins contain a positively charged argmine residue within their transmembrane domain, which allows them to interact with transducer molecules such as CD3 ⁇ , Fc ⁇ RI ⁇ , or KARAP/DAP12. These proteins contain a negatively charged aspartic acid residue in their transmembrane region, allowing them to interact with the imm r-og-obu.in-recep-or -. ⁇ >d ha'- ⁇ ar. IT AM. or possibly TT-LM. motif within their cy.cpl-. ⁇ .r.c merely' • :. »r ⁇ -:a-e s.znJi r-ns -rt or.
  • Ig receptor monoclonal antibody to T cells induced their cytolytic activity for HTV infected target cells. It is apparent that the down regulation of an inhibitory receptor could lead to generalized activation of NK/T cells, which may cause autoimmune disorders like rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), psoriasis, and inflammatory bowel disease (IBD) among others.
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • IBD inflammatory bowel disease
  • the immune systems activatory and inhibitory signals mediated by opposing kinases and phosphatases are very important for maintaining balance in the immune systems.
  • Systems with a predominance of activatory signals will lead to autoimmunity and inflammation.
  • Immune systems with a predominance of inhibitory signals are less able to challenge infected cells or cancer cells. Isolating new activatory or inhibitory receptors is highly desirable for studying the biological signal(s) transduced via the receptor. Additionally, identifying such molecules provides a means of regulating and treating diseased states associated with autoimmunity, inflammation and infection.
  • engaging a newly discovered cell surface receptor having ITIM motifs with an agonistic antibody or ligand can be used to downregulate a cell function in disease states in which the immune system is overactive and excessive inflammation or immunopathology is present.
  • using an antagonistic antibody specific to the receptor or a soluble form of the receptor can be used to block the interaction of the cell surface receptor with the receptor's ligand to activate the specific immune function in disease states associated with suppressed immune function.
  • This invention is based on the discovery of novel LIR-like polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
  • polynucleotides of the present invention are based on an LIR-like polynucleotide isolated from a cDNA library prepared from human leukocyte mRNA (GIBCO Laboratories) (SEQ ID NO: 1 , SEQ ID NO: 16); from infant brain mRNA (Columbia University) (SEQ ID NO: 35); from human mammary gland mRNA (Invitrogen) (SEQ ID NO: 47); and from bone marrow mRNA (Clontech) (SEQ ID NO: 63).
  • a cDNA library prepared from human leukocyte mRNA (GIBCO Laboratories) (SEQ ID NO: 1 , SEQ ID NO: 16); from infant brain mRNA (Columbia University) (SEQ ID NO: 35); from human mammary gland mRNA (Invitrogen) (SEQ ID NO: 47); and from bone marrow mRNA (Clontech) (SEQ ID NO: 63).
  • compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
  • compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67; or a fragment of SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67; a polynucleotide comprising the full length protein coding sequence of the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 (for example, SEQ ID NO: 3, 6, 19, 22, 37, 40, 50, or 66); and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of any of S
  • the polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67: (b) a nucleotide sequence encoding any of SEQ ID NO: 3. 6, 8-13. 19. 22. 24-28. 33-34, ?". 40 42-45. 50, 52-? ⁇ . 66. or 6S-7- 1 .
  • a collection as used in this application can be a collection of only one polynucleotide.
  • the collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array.
  • segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment.
  • the array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.
  • the collection can also be provided in a computer-readable format.
  • This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors.
  • Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.
  • the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
  • the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
  • Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
  • compositions of the present invention include polypeptides comprising, but not limited to, an isolated polypeptide selected from the group comprising the amino acid sequence of SEQ ID NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58, 66, or 68-74; or the corresponding full length or mature protein.
  • Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: 1-2, 4-5, 7, 16- 18. 20-21. 23, 35-36, 38-39, 41. 47-49, 51. 63-65, or 67: or (b) polynucleotides that
  • polypeptides of the invention may be wholly or partially chemically
  • compositions comprising a polypeptide of the invention.
  • Pharmaceutical compositions of the invention may comprise a polypeptide of the invention and an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically
  • the invention also relates to methods for producing a polypeptide of the invention comprising culturing host cells comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and
  • Preferred embodiments include those in which the protein produced by such a process is a mature form of the protein.
  • Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These
  • 25 techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like.
  • hybridization probes use as oligomers, or primers, for PCR
  • use in an array use in computer-readable media
  • use for chromosome and gene mapping use in the recombinant production of protein
  • use in generation of antisense DNA or RNA their chemical analogs and the like.
  • polynucleotides of the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the
  • the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence c tags for physical mapping of the human genome.
  • Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier.
  • polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of .immunological diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus and inflammatory bowel disease.
  • .immunological diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus and inflammatory bowel disease.
  • the polynucleotides and polypeptides may also be beneficial in the treatment of viral infections and in the
  • the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering a composition comprising compounds and other substances that modulate the overall activity of the target gene products and a pharmaceutically acceptable carrier. Compounds and other substances can effect such modulation either on the level of target
  • methods for preventing, treating or ameliorating a medical condition, including viral diseases which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g.. antibody specifically reactive for) LIR-like polypeptides of the invention.
  • a mammalian subject including but not limited to humans
  • a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g.. antibody specifically reactive for) LIR-like polypeptides of the invention e.g. antibody specifically reactive for
  • polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of cellular function.
  • a polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies.
  • protein or other active ingredients of the present invention may be included in formulations of a particular agent to minimize side effects of such an agent.
  • the invention further provides methods for manufacturing medicaments useful in the above-described methods.
  • the present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.
  • the invention provides a method for detecting a polypeptide of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting formation of the complex, so that if a complex is formed, the polypeptide is detected.
  • kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.
  • the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
  • the invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g.. bind to) the polypeptides of the invention.
  • the invention .pro ides a method for identif ing acompo ' und 'hat binds to the
  • Also provided is a method for identifying a compound that binds to the polypeptide comprising contacting the compound with the polypeptide in a cell for a time sufficient to form a polypeptide/compound complex wherein the complex drives expression of a reporter gene sequence in the cell and detecting the complex by detecting reporter gene sequence expression so that if the polypeptide/compound complex is detected a compound that binds to the polypeptide is identified.
  • Figure 2 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID: 2 (i.e. SEQ ID NO: 3) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like”) and human PIGR-1 (Patent Application No.
  • .Gaps are presented as dashes. - Figure 3 shews the BLASTX amino acid sequence alignment beiween-the protein erc e - . 5I ⁇ Q ID: .5...i.e. S ⁇ Q ID NO: 6. .t'J ⁇ c . t ⁇ i-r.ur.ogicb lir. recectcr- e " .. . pc; pep-..-e.-__:so : er::::ied :.s ' " IR-i ⁇ ' -- and h iran IRCIa.prc e.r 5 ⁇ ID NO: 14.
  • Figure 4 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 5 (i.e. SEQ ID NO: 6) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like”) and human PIGR-1 (Patent Application No.
  • Figure 5 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 18 (i.e. SEQ ID NO: 19) leukocyte immunoglobuiin receptor- like polypeptide (also identified as "LIR-like”) and putative inhibitory receptor (Rojo et al, (1997) J.
  • FIG. 6 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 18 (i.e. SEQ ED NO: 19) leukocyte immunoglobuiin receptorlike polypeptide (also identified as "LIR-like") and human GP49 HM18 polypeptide (Patent Application No. WO9809638) SEQ ID NO: 30, indicating that the two sequences share 50% similarity over 123 amino acid residues and 34% identity over the -same 1-23 . -i ⁇ :r . ' _.c:d re_.x_.es. v. heror. _
  • Q GIutamine.
  • R Arginme.
  • FIG. 8 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 21 (i.e SEQ ID NO: 22) leukocyte immunoglobuiin receptorlike polypeptide (also identified as "LIR-like”) and human LIR-pbm36-2 protein (Patent Application No.
  • Figure 10 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e. SEQ ID NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human CMRF 35 protein (Jackson et al, (1992) Eur. J.
  • Figure 12 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e. SEQ ID NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human PIGR-2 protein (Patent Application No.
  • SEQ TD NO: 65 i.e. SEQ ID NO: 66
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like”
  • human platelet glycoprotein VI-2 protein SEQ ID NO: 75, indicating that the two sequences share 50% similarity over residues 1-219 of SEQ ID NO: 66, and 37% identity over the same residues 1-219 of SEQ ID NO: 66,
  • Figure 15 shows the BLASTP amino acid sequence alignment between the
  • SEQ ID NO: 65 i.e. SEQ ID NO: 66
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like”
  • human FcR-II protein Patent Application No. WO9831806
  • D Aspartic Acid
  • E Glutamic Acid
  • F Phenylalanine
  • G Glycine
  • H Histidine
  • I Isoleucine
  • K Lysine
  • L Leucine
  • M Methionine
  • N Asparagine
  • P Proline
  • Q Glutamine
  • R Arginine
  • S Serine
  • T Threonine
  • V Valine
  • Y Tyrosine. Gaps are presented as dashes.
  • Protein database searches with the BLASTX algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403- 10 (1990), herein incorporated by reference) indicate that SEQ ID NO: 3 is homologous to leukocyte immunoglobuiin receptors like IRCla and NK cell inhibitory receptors like PIGR-1.
  • Protein database search with eMATRIX software (Stanford University, Stanford CA) further show that a portion of SEQ ID NO: 3 (i.e. SEQ ID NO: 8) is homologous to poly Ig receptors.
  • Figure 1 shows the BLASTX amino acid sequence alignment between the protein
  • SEQ ID NO: 2 i.e. SEQ ID NO: 3
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like”
  • human IRCla protein SEQ RO NO: 14 indicating that the two sequences share 55% similarity over 297 amino acid residues and 37% identity over the same 297 amino acid residues.
  • Figure 2 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID: 2 (i.e. SEQ ID NO: 3) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human PIGR-1 (Patent Application No.
  • SEQ ID NO: 15 A predicted soluble, secreted splice variant of SEQ ID NO: 3 is SEQ ID NO: 6. It is an approximately 162 amino acid protein with a predicted molecular mass of approximately 18 kDa unglycosylated. Protein database searches with the BLASTX algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol.
  • SEQ ID NO: 6 is homologous to leukocyte immunoglobuiin receptors like IRCla and NK cell inhibitory receptor like PIGR-1.
  • Protein database search with eMATRIX software (Stanford University, Stanford CA) further show that a portion of SEQ ID NO: 7 (i.e.
  • SEQ ID NO: 9 is homologous to poly Ig receptors.
  • Figure 3 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID: 5 (i.e. SEQ ID NO: 6) leukocyte immunoglobuiin receptor-like ⁇ 5 ⁇ "" .palypeptideTalso identified as 'T_.IRr.ike") and human -IRCl protein SEQpTD NO:-14, . " indicating that the ' two sequences share "57 '-I. si_ni].
  • Figure 4 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 5 (i.e. SEQ ID NO: 6) leukocyte immunoglobulm receptor-like 10 polypeptide (also identified as "LIR-like”) and human PIGR-1 (Patent Application No.
  • EP897981 SEQ ID NO: 15, indicating that the two sequences share 67% similarity over
  • SEQ ID NO: 12 is the peptide resulting when the signal peptide is removed from SEQ ID NO: 3.
  • SEQ ID NO: 13 is the peptide resulting when the signal peptide is removed from SEQ ID NO: 6.
  • a predicted approximately fourteen residue transmembrane peptide is encoded from approximately residue 170 to residue 193 of SEQ ID NO: 3 (SEQ ID NO: 11). This 25 can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol,
  • both the membrane-bound and soluble ILR-like polypeptide is expected to have a poly Ig receptor domain at residues 82 - 129 of SEQ ID NO: 3 and residues 82 - 129 of SEQ ID NO: 6 (SEQ ID NO: 8 and SEQ ID NO: 9, respectively).
  • SEQ ID NO 8 has serine in the position 129 while SEQ ID NO: 9 contains proline in the same position.
  • SEQ ID NO: 19 is an approximately 236-amino acid protein with a predicted molecular mass of approximately 26 kDa unglycosylated.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol.21:403-10 (1990), herein incorporated by reference
  • SEQ ID NO: 19 is homologous to leukocyte immunoglobuiin receptors (IIRs) and immunoglobulin- like protein IGSF-1.
  • Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, CA) further show that SEQ ID NO: 19 is homologous to killer cell inhibitory receptors.
  • Figure 5 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 18 (i.e. SEQ ID NO: 19) leukocyte immunoglobuiin receptorlike polypeptide (also identified as "LIR-like") and putative inhibitory receptor (Rojo et al, (1997) J. Immunol., 158, 9-12) SEQ ID NO: 29, indicating that the two sequences share 51% similarity over 145 amino acid residues and 33% identity over the same 145 amino acid residues.
  • Figure 6 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 18 (i.e. SEQ ID NO: 19) leukocyte immunoglobuiin r ⁇ ceptor- e poivpeptie ⁇ al o -identified as "LJR-like” and human GP49 " HM18 polypeptide .Paten: Application No. " ⁇ :O9.S0 6 3") S ⁇ Q ID NO: X.
  • a predicted soluble, secreted splice variant of SEQ ID NO: 19 is SEQ ID NO: 22. It is an approximately 199 amino acid protein with a predicted molecular mass of approximately 22 kDa unglycosylated. Protein database searches with the BLASTP algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated by reference) indicate that SEQ ID NO: 22 is homologous to leukocyte immunoglobuiin receptors (LIRs) and immunoglobulin- like protein IGSF-1. Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, CA) further shows that SEQ ID NO: 22 is homologous to killer cell inhibitory receptors.
  • LIRs leukocyte immunoglobuiin receptors
  • Figure 7 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 21 (i.e SEQ ID NO: 22) leukocyte immunoglobulm receptor- like polypeptide (also identified as "LER-like") and immunoglobulin-like protein IGSFl (Mazzarella et al, (1998) Genomics 48, 157-162) SEQ ID NO: 31, indicating that the two sequences share 53% similarity over 209 amino acid residues and 38% identity over the same 209 amino acid residues.
  • SEQ ID NO: 21 i.e SEQ ID NO: 22
  • leukocyte immunoglobulm receptor- like polypeptide also identified as "LER-like
  • IGSFl immunoglobulin-like protein IGSFl
  • Figure 8 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 21 (i.e SEQ ID NO: 22) leukocyte immunoglobulm receptorlike polypeptide (also identified as "LIR-like") and human LIR-pbm36-2 protein (Patent Application No. WO9848017) SEQ ID NO: 32, indicating that the two sequences share 46% similarity over 236 amino acid residues and 32% identity over the same 236 amino acid residues.
  • a predicted approximately sixteen residue signal peptide is encoded from approximately residue 1 to residue 16 of both SEQ ID NO: 19 and SEQ ID NO: 22 (SEQ ID NO: 25). The extracellular portion is useful on its own.
  • the signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol. 157. pp. 105-31 (1982), incorporated herein by reference).
  • transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • One of skill in the art will recognize that the actual transmembrane region may be different
  • LIR-like polypeptides of SEQ ID NO: 19 and 22 were determined to have a region at residues 26 - 97 with characteristic motifs to the killer cell inhibitory receptor domain (SEQ ID NO: 24). The corresponding domain within SEQ ID NO: 19
  • the leukocyte immunoglobuiin receptor-like polypeptide of both SEQ ID NO: 37 and 40 is an approximately 230-amino acid protein with a predicted molecular mass of approximately 26 kDa unglycosylated. Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, CA) ⁇ -5 " further shows that- ' both --SEQ ID NO: and 40 and are homologous to P?8 killer ceil " " " - Inhibitory receptor. ' . • - - . : ⁇ •-. ' ⁇
  • ..'Fi sere 9 shows ' :he-Ger.e ' .-'.t " .as amino ecie sequence alignment betv.-ijter.-the - protein encoded by SEQ ID NO: 36 and 39 (i.e SEQ ID NO: 37 and 40, respectively) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like”) and
  • a predicted approximately fifteen residue signal peptide is encoded from approximately residue 1 to residue 15 of both SEQ ID NO: 37 and SEQ ID NO: 40 (SEQ ID NO: 40).
  • SEQ ID NO: 45 is the peptide resulting when the predicted signal peptide is removed from either SEQ ID NO: 37 or SEQ ID NO: 40.
  • a predicted approximately twenty-seven residue transmembrane peptide is encoded from approximately residue 116 to residue 143 of SEQ ID NO: 37 and SEQ ID NO: 40 (SEQ ID NO: 44).
  • the transmembrane portion may be useful on its own. This
  • transmembrane peptide region 25 can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • the actual transmembrane region may be different than that predicted by the computer program.
  • the leukocyte immunoglobuiin receptor-like polypeptide of SEQ ID NO: 50 is an approximately 201 -amino acid protein with a predicted molecular mass of approximately 22 kDa unglycosylated.
  • Protein database searches with the BLASTX algorithm Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403- 10 (1990), herein incorporated by reference
  • SEQ ED NO: 50 is homologous to leukocyte immunoglobuiin receptors like CMRF35.
  • F-i-g-re 10 S ⁇ .O A S tne BLASTX amino ⁇ cd sequence alignment bet • een - ⁇ e prct ⁇ > cncodee b ⁇ /SEQ NO: -1- . i.e. -SEQ ID NO: 50 ie. ccy.e ' r-ir'- ; ⁇ Ar . receptor-like polypeptide (also identified as ''LER-like " ) and human CMRF 35 protein
  • Figure 11 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e. SEQ ED NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LER-like") and human Natural Killer inhibitory receptor protein SEQ ID NO: 61 , indicating that the two sequences share 64% similarity over 145 amino acid residues and 51% identity over the same 145 amino acid residues.
  • SEQ ID NO: 49 i.e. SEQ ED NO: 50
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LER-like
  • SEQ ID NO: 61 human Natural Killer inhibitory receptor protein
  • Figure 12 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e. SEQ ID NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like”) and human PIGR-2 protein
  • Figure 13 shows the GeneAtlas amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e SEQ ID NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LER-like") and T cell receptor, pdb
  • a predicted approximately twenty-four residue transmembrane peptide is encoded from approximately residue 167 to residue 191 of SEQ ID NO: 50 (SEQ ID NO: 56).
  • the transmembrane portion may be useful on its own. This can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • One of skill in the art will recognize that the actual transmembrane region may be different than that predicted by the computer program.
  • LER-like polypeptide is expected to have two poly-Ig receptor domains.
  • Poly Immunoglobuiin receptor domain ⁇ TOEIPGDLTCTVTLENLTADDAGKYRCGIATILQEDGLSGFLPDPFFQ designated as (SEQ ID NO: 53) p-value of 4.504e-9, DM01688B 2 (identification number correlating to signature); located at residues 85-132 of SEQ ID NO: 50.
  • MGAVGESLSVQCRYEEKYKTFNKY CRQPCLPIWHEM designated as (SEQ ID NO: 54) p-value of 8.364e-9, DM01688J 2 (identification number correlating to signature); located at residues 32-68 of SEQ ID NO: 50.
  • the leukocyte immunoglobuiin receptor-like polypeptide of SEQ ID NO: 66 is an approximately 777-amino acid protein with a predicted molecular mass of approximately 87.8 kDa unglycosylated. Protein database searches with the BLASTP algorithm (Altschul S.F. et al., J. Mol. Evol.36:290-300 (1993) and Altschul S.F..et al., J. Mol.
  • SEQ ID NO: 65 i.e. SEQ ID NO: 66
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like”
  • human platelet glycoprotein VI-2 protein SEQ ED NO: 75, indicating that the two sequences share 50% similarity over residues 1-219 of SEQ ED NO: 66, and 37% identity over the same residues 1-219 of SEQ ED NO: 66.
  • Figure 15 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 65 (i.e. SEQ ID NO: 66) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LER-like”) and human FcR-II protein
  • a predicted approximately sixteen-residue signal peptide is encoded from approximately residue 1 through residue 16 of SEQ ID NO: 66 (SEQ ID NO: 71).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • SEQ ID NO: 72 is the peptide resulting when the predicted signal peptide is removed from SEQ ID NO: 66.
  • a predicted approximately twenty four-residue transmembrane region is encoded from approximately residue 231 through residue 254 of SEQ ID NO: 66 (SEQ 3D NO: 0 73).
  • the transmembrane portion may be useful on its own.
  • the transmembrane region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • Receptor cell NK glycoprotein immunoglobuiin GSLPKPSLSAWPSSVVPANSNVTLRCWTPARGVSFV designated as (SEQ ID NO: 68) p-value of 6.625e-10, PD01652A (identification 15 number correlating to signature); located at residues 24 - 59 of SEQ ED NO: 66.
  • Receptor cell NK glycoprotein immunoglobuiin GSLPKPSLSAWPSSVVPANSNVTLRCWTPARGVSFV designated as (SEQ ID NO: 68) p-value of 6.625e-10, PD01652A (identification 15 number correlating to signature); located at residues 24 - 59 of SEQ ED NO: 66.
  • RSDVUI VTGHLSKPFLRTYQRGTVTAGGRVTLQCQKRDQ FVPIMFAL LK designated as (SEQ ID NO: 70) p-value of 4.021e-9, PD01652B (identification number correlating to signature); located at residues 111 - 162. 25
  • the sequences of the present invention are expected to have both membrane bound and soluble LER-like activity.
  • polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for the prevention and/or treatment of disorders mediated by loss or overexpression of LIR-like polypeptide.
  • disorders include, psoriasis, multiple 30 sclerosis, rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease among others.
  • the polypeptides of the inventions could also be used to treat bone marrow transplant patients to inhibit graft versus host disease.
  • the polypeptides and polynucleotides may also be used to boost the killer cell and cytolytic activity of leukocytes of human immune deficiency disease patients.
  • active refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
  • biologically active or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.
  • LIR-like refers to biological activity that is similar to the biological activity of a leukocyte immunoglobuiin receptor.
  • biologically active or “biological activity” refers to the capability of the natural, recombinant or synthetic LER-like peptide, or any peptide thereof, to induce a specific biological response in appropriate animals or cells and to bind with specific antibodies.
  • activated cells are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.
  • complementarity refers to the natural binding of polynucleotides by base pairing.
  • sequence 5'-AGT-3' binds to the complementary sequence 3'-TCA-5'.
  • Complementarity between two single-stranded molecules may be "partial” such that only some of the nucleic acids bind or it may be "complete” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
  • Embryonic stem cells refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.
  • GSCs germ line stem cells
  • primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.
  • primordial germ cells PGCs
  • PGCs primordial germ cells
  • the GSCs and the ⁇ S cells are capab e.of self-renewal.
  • these cells nct-onlv popul te .the germ -line and zx e r.se to e pi.'.r-r-.t of termmai e ⁇ :e-ert:..te-/:elis t" ⁇ t- comprise the adii't specialized orge-s, ut ere able to regenerate themsel- es.
  • EMF expression modulating fragment
  • a sequence is said to "modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.
  • EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).
  • One class of EMFs is nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.
  • nucleotide sequence or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to _a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • A is adenine
  • C cytosine
  • G guanine
  • T thymine
  • N A, C, G, or T (U).
  • nucleic acid segments may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
  • oligonucleotide fragment or a "polynucleotide fragment", “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.
  • the fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 " nucleotides and-most less than 30 nucleotides.
  • the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
  • a fragment or segment may uniquely identify each polynucleotide sequence of the present invention.
  • the fragment comprises a sequence substantially similar to a portion of SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67.
  • Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P.S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, both of which are incorporated herein by reference in their entirety.
  • the nucleic acid sequences of the present invention also include the sequence information from any of the nucleic acid sequences of SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67.
  • the sequence information can be a segment of SEQ ED NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-2, 4-5, 1, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67.
  • One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 4 20 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosome. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segments can be used. The probability that the ⁇ ⁇ ' i fteen-mer.is- fu'J matcKec in the expressed sequences is. also approximately ne in/five -beeues ⁇ -e .presse .sequences eo .prise.'.ess than approximately 5 ⁇ or "the entire genome
  • a segment when using sequence information for detecting a single mismatch a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in
  • a human genome with a single mismatch is calculated by multiplying the probability for a full match (1 ⁇ 4 25 ) times the increased probability for mismatch at each nucleotide position (3 x 25).
  • the probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.
  • the probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.
  • ORF open reading frame
  • operably linked or “operably associated” refer to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably
  • nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.
  • pluripotent refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.
  • polypeptide or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to
  • segment is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.
  • the peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.
  • the peptide is from about 5 to about 200
  • any ⁇ p.elypeptide must have sufficient length -to-di s l y . - bioicgicai ⁇ nd'lo imm-inologicai. activity. _ "
  • translated protein coding portion means a sequence which encodes for the full length protein which may include any leader sequence or a processing sequence.
  • mature protein coding sequence refers to a sequence which encodes a
  • the "mature protein portion” refers to that portion of the protein without the leader/signal sequence.
  • the peptide may have the leader sequences removed during processing in the cell or the protein may have been produced synthetically or using a polynucleotide only encoding for the mature protein coding sequence. It is contemplated that the mature protein portion may or may
  • the initial methiomne is often removed during processing of the peptide.
  • derivative refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol)
  • variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques. Guidance in determining which amino acid
  • 30 residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.
  • recombinant variants encoding these same or similar polypeptides may be synthesized nr selected by -making use of the "redundancy" in the genetic "code.
  • m._y be intrc iuced to optimiee cloning nto u. plasmid c-r '. _ral /ector or expression - in a particular prokaryotic or eukaryotic system.
  • Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.
  • conservative amino acid replacements may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
  • insertions, deletions or non- conservative alterations can be engineered to produce altered polypeptides.
  • Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.
  • such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • the ;terr 5 is a nucleic acid residues, or substituted with another amino acid residue in order to eliminate disulfide bridges.
  • the polynucleotide or polypeptide is pu ⁇ fied such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).
  • isolated refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.
  • the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other components normally present in a solution of the same.
  • isolated and purified do not encompass nucleic acids or polypeptides present in their natural source.
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • recombinant microbial defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
  • recombinant expression vehicle or vector refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.
  • An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed -
  • recombinant expression system means host cells which have integrated a recombinant transcriptional unit into chromosomal DNA or carry the 0 recombinant transcriptional unit extrachromosomally.
  • Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters 5 or enhancers.
  • Recombinant expression systems as defined herein will express
  • the cells pan be prokaryotic or eukaryotic.
  • secreted includes a protein that is transported across or through a 0 membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.
  • “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.
  • “Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplas ic reticulum.
  • “Secreted” 5 proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P.A. and Young, P.R.
  • an expression vector may be designed to contain a "signal or 0 leader sequence" which will direct the polypeptide through the membrane of a cell.
  • a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
  • stringent is used to refer to conditions that are commonly understood in the art as stringent.
  • Stringent conditions can include highly stringent conditions (i.e., hybridization to " i.eV-bour.d DNAlrTO.-S M NaHPO., 7% sodium dodecyl sulfate”. SDS).
  • I ' - SDS at -i "Ci Ot'-er exemplary hybridization conditions are described herein in the examples.
  • additional exemplary stringent hybridization conditions include washing in 6X SSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
  • substantially equivalent can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an 'adverse functional dissimilarity between the reference and subject sequences.
  • a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).
  • Such a sequence is said to have 65% sequence identity to the listed sequence.
  • a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).
  • Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 90% sequence identity.
  • nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.
  • nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, and most preferably at least about 95 identity.
  • sequences having-substantially • equivalent bi uogieai ccti-.it> and substantially-equivalent expression characteristics are " ⁇ considered substantial ' .;- ecei'.ussi.-For the perpcses of de t ermining eceival&nce.
  • Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.
  • totipotent refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
  • transformation means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.
  • transfection refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.
  • infection refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.
  • an "uptake modulating fragment,” UMF means a series of nucleotides which mediate the uptake of a ' linked DNA fragment into a cell.
  • UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.
  • the invention is based on the discovery of a novel secreted LIR-like polypeptide, the polynucleotides encoding the LER-like polypeptide and the use of these compositions for the diagnosis, treatment or prevention of neurological conditions and disorders.
  • the isolated polynucleotides of the invention include, but are not limited to a pol>ne*ieC " tide comp-smg _.m. er " the n_-c!eo:;de sect ences oi ' SEQ ID NO 1-2. -i-5. 7. :6-Ic..2T-21 23. 35-5c.-3S-39. - :. -!- " -i ⁇ 51:63-55. or 6 " . c r ⁇ grnent :: ' SEQ ID-NO: 1- 2, 4-5, 7, 16-18, 20-21, 23. 35-36, 38-39, 41, 47-49, 51, 63-65, or 67; a polynucleotide comprising the full length protein coding sequence of SEQ ED NO: 2, 5, 18, 21, 36, 39,
  • polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67.
  • the polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a)
  • Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic
  • domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains
  • domains in enzyme-like polypeptides include catalytic and substrate binding domains
  • domains in ligand polypeptides include receptor-binding domains.
  • polynucleotides of the invention include naturally occurring or wholly or
  • the polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.
  • the present invention also provides genes corresponding to the cDNA sequences disclosed herein.
  • the corresponding genes can be isolated in accordance with known methods using the sequence in of-ma i on-clisc osed here ⁇ .7 Suc-fi -methods " include tFe ⁇ rep ⁇ rat ⁇ on-o ⁇ prooes-or p ⁇ mers " mcm seque-ce in erm ⁇ eon for . ⁇ i c-i.c" end cr ⁇ mcl.ficaLo ⁇ of geneb -.pprpp ⁇ -e ''bmres or o"- ⁇ e ⁇ sc ernes of gerem-c materials. Further 5' and 3 ' sequence can be obtained using methods known in the art.
  • full length cDNA or genomic DNA that corresponds to any of the polynucleotides of the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 or a portion thereof as a probe.
  • polynucleotides of the SEQ ID NO: SEQ ID NO: 1-2, 4-5, 7, 16-18, 20- 21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
  • the nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.
  • the EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.
  • polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.
  • Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, more typically at least about 98% or most typically at least about 99% sequence identity to a polynucleotide recited above.
  • nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to any one of the polynucleotides
  • Probes capable of specifically hybridizing to a pccHnucieetide can differenti ⁇ te_pei _. cle tide sequences ot " th -invention frsra g ⁇ her .. ' . poiynucieotide -sequences In the same famely.of -genes or can di-i:erenti ⁇ te human genes from genes of other species, and are preferably based on unique nucleotide sequences.
  • the sequences falling within the scope of the present invention are not limited to 0 these specific sequences, but also include allelic and species variations thereof.
  • Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-
  • the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated. 0 The nearest neighbor result for the nucleic acids of the present invention, including
  • SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 can be obtained by searching a database using an algorithm or a program.
  • a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S.F. J Mol. Evol. 36290-300 (1993) and Altschul S.F. et al. J. Mol.
  • Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.
  • nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may.be prepared vby methods ; known in the -art-by. ' introducing .appropriate
  • Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).
  • Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g.; hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.
  • Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
  • terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.
  • polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site being changed.
  • a further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.
  • Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
  • the polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
  • the polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.
  • polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58,66, or 68-74 or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.
  • a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see _ Sambrook j et al. (19.S9 M ⁇ lec ⁇ lar Cloning: -A Laboratory Mahual7 C ⁇ Id Sp ⁇ ring " 3fefbor ⁇ -L ⁇ b ⁇ rato ⁇ yNYi.
  • Useful nuc ' ieetide sequences for joining tC ⁇ polynuc'.et-tiee's include an '" - ⁇ ssortmer,tx.f vectors, e.g..-.plasmids.
  • the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
  • the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
  • Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
  • the present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of the SEQ ID NO: 1-2, 4-5, 7, 16- 18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 or a fragment thereof or any other polynucleotides of the invention.
  • the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of the SEQ ID NO: 1-2, 4-5, 7, 16- 18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 or a fragment thereof is inserted, in a forward or reverse orientation.
  • the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
  • Bacterial Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRlT5 (Pharmacia).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSNK3, pBPV, pMSG, pSNL (Pharmacia).
  • the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art.
  • pei n ⁇ cieotide ef the .invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • CAT chloramphenicol transferase
  • Two appropriate vectors are pKK232-8 and ⁇ CM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, and trc.
  • Eukaryotic promoters include CMV
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a
  • promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
  • Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-f actor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a
  • the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Stre toirr ces. and Staphylococcus. although ethers may also be employed as a matter of ercice. - ⁇ •
  • e-.pressiC ' n . 1 ecto-s for bacte ⁇ al use can comprise a selectable marker and bacte ⁇ al origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, USA).
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Polynucleotides of the invention can also be used to induce immune responses.
  • nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical admii-istration of naked plasmid D ⁇ A or following injection, and preferably intramuscular injection of the D ⁇ A.
  • the nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked D ⁇ A.
  • antisense nucleic acid molecules that can hybridize to, or are complementary to, the nucleic acid molecule comprising the LIR-like nucleotide sequence, 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 are provided that comprise a sequence complementary to at least about 10. 25, 50. 100. 250 or 500
  • an antisense nucleic acid molecule is antisense to a "coding
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "conceding region" of the coding strand of a nucleotide sequence encoding the LIR-like protein.
  • conceding region refers
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of LER-like mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
  • 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 of the 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, eihydrouracil.-peta- ⁇ -gai ⁇ ctosylqueos-ine.
  • N6 isopentenyl ⁇ denine. 1- met.V-l ⁇ u ⁇ nine. l-methv 1 .incsine. " 2.2-d'imeti'-.”. lee ⁇ nine. 2-rretb eiderine. 2- -ethy uanine. 3-methyl cyesire.-5-metr.-v icy.osine. N ⁇ -s erine. "" -me: y.g-. ⁇ rme.
  • 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 section).
  • 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 an LER-like 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 of the 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 IH promoter are preferred.
  • the antisense nucleic acid molecule of the invention is - an-a-ph-.-anorr.eric n ⁇ :c ;c aeid-rr.o -cule.
  • the antisense nucleic acid molecule can also comprise a 2 -o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987. FEBS Lett. 215, 327-330).
  • Nucleic acid modifications include, by way of non-limiting 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 of the modified nucleic acid, such that they can 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 an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334, 585-591
  • a ribozyme having specificity for an LIR -like-encoding nucleic acid can be designed based upon the nucleotide sequence of an LER-like cDNA disclosed herein.
  • 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 an UR-like-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al.
  • LTR-like mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel et al., (1993) Science 261, 1411-1418.
  • LER-like gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the LIR-like nucleic acid (e.g.. the L ⁇ R-h e promoter ⁇ n 'or enhancers; t ⁇ form triple hekcab structures that brevent "r- ⁇ nsc.-p-ion of "he LIR-like gene-"' target cell* ; . See. ⁇ g . ⁇ elene. iX>l A-nticanc ⁇ r Dreg I?es. c, 5c9-S4: Helcne. et ⁇ .. 1992 'Ann. N Y. Ac. ⁇ . Sc- 560. 2 ⁇ -3c. M rer. 15 2. Bioassays 14, 807-15.
  • nucleotide sequences complementary to the regulatory region of the LIR-like nucleic acid e.g. the L ⁇ R-h e promoter ⁇ n 'or enhancers; t
  • the LER-like nucleic acids 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, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4, 5-23.
  • peptide nucleic acids 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. supra; Perry-OKeefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93, 14670-14675.
  • PNAs of LER-like 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 LIR-like can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (see, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al., 1996, supra; Perry-OKeefe, et al., 1996. supra).
  • PNA directed PCR clamping as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (see, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al., 1996, supra; Perry-OKeefe, et al., 1996. supra).
  • PNAs of LIR-like 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 LIR-like 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 -.pec'f'C").
  • PNA-DNA chimeros can he Im ⁇ d u ⁇ mg linkers of appropriate lengths ⁇ electee .n terms of base «t ⁇ c " vir ⁇ g. -number :: norths be: % eer, : e r.rciec ase . ⁇ -d - or-ertation -see. Hy-.p e: al . 1996 s..p- ⁇ T e - : ' --.- ⁇ of PNA-DNA cnimer ⁇ s can re performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24, 3357-3363.
  • 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. See, e.g., Mag, et al., 1989. Nucl Acid Res 17, 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., 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. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • 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
  • 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 can be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • the present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.
  • host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.
  • the present invention still further provides host cells poKnucieotfdes -of che invention, '-neretn such «- ⁇ ' -..c' - t-ees- ⁇ -; in ⁇ ssec a:: or '.vr ⁇ -e ⁇ e' ⁇ te" seceeree "cerol ec ⁇ s to
  • LER-like DNA sequences allows for modification of cells to permit, or increase, expression of LER-like polypeptide.
  • Cells can be modified (e.g., by homologous recombination) to provide increased LIR-like polypeptide expression by replacing, in whole or in part, the naturally occurring LIR-like promoter with all or part of a heterologous promoter so that the cells LER-like polypeptide is expressed at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to LIR-like encoding sequences. See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO92/20808, and PCT
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the LIR-like coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the LER- like coding sequences in the cells.
  • the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
  • the host cells containing one of polynucleotides of the invention can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.
  • Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.
  • the most preferred cells are those which do not normally express the -c:trt ⁇ c , -f i .a--p' ⁇ ir p ⁇ e-'r't ⁇ de -or-pt-ctem or .which the polypeptide or protein -vHow ⁇ ""-..terJ ⁇ ' e " ⁇ tere protein-, "can be expressed in mammal. n celis. >e..sr, bacteria, or c' ⁇ er ce s ..r ⁇ er the control of appropriate prcmeters Ce ' i-frce tr-rslc-i-m ⁇ ytem-s cen also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.
  • Mammalian expression vectors wilLcomprise an origin of replication, a suitable promoter, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SN40 viral genome for example, SN40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting- out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • a number of types of cells may act as suitable host cells for expression of the protein.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells.
  • CN-1 cells other transformed primate cell lines, normal ⁇ .i : ⁇ 'oid-cel.s. eel! strains derived from-m vtro culture .of p ⁇ rnery 'issue, primary exnlants. -HeLa DC's. mouse L ce! EHK.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • yeast strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.
  • the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.
  • Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
  • gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
  • regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, and regulatory protein binding sites or combinations of said sequences.
  • sequences which affect the structure or stability of the R ⁇ A or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals, mR ⁇ A stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or R ⁇ A molecules.
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promotei or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative •5' • regulatory ' element.
  • y.-.t e targeting e ⁇ en may replace an existing.e-lemenf. f - r- ⁇ xcmple. ⁇ tissue-specific enhance:-;: ⁇ ntbe- -enl-.c--e by an-e ' nh ⁇ ncer -th ⁇ t-h ⁇ s breeder or-di.:er ⁇ n: cell-type ⁇ pesificitythan the ' n ⁇ tur ⁇ lly ccce ing elements.
  • the - naturally occurring sequences are deleted and new sequences are added.
  • the identification of the targeting event may be facilitated by the use of one or more 0 selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the 5 negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
  • TK Herpes Simplex Virus thymidine kinase
  • gpt bacterial xanthine-guanine phosphoribosyl-transferase
  • the gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is 5 inco ⁇ orated by reference herein
  • an LER-like "chimeric protein” or “fusion protein” comprises an LIR-like polypeptide 0 operatively linked to either a different LER-like polypeptide or a non-LTR-like polypeptide.
  • An "LER-like polypeptide” refers to a polypeptide having an amino acid sequence corresponding to an LER-like protein
  • a “non-LIR-like polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the LIR-like protein, e.g., a protein that is different from the LIR-like protein and that is derived from the same or a different organism.
  • LIR-like ol vpeptide can co" ⁇ ejspor.d to all -or a portion cf ⁇ VR-l'Ac r - ⁇ .en.
  • en LIR- ke fusion protein comprises at least two biologically active portions of an LIR-like protein.
  • an LER-like fusion protein comprises at least three biologically active portions of an LER-like protein.
  • the term "operatively-linked" is intended to indicate that the LER-like polypeptide(s) and/or the non-LER-like polypeptide are fused in-frame with one another.
  • the non-LJR-like polypeptide can be fused to the N-terminus or C-terminus of the LER-like polypeptide.
  • the fusion protein is a GST-LJR-like fusion protein in which the LIR-like sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences.
  • Such fusion proteins can facilitate the purification of recombinant LIR-like polypeptides.
  • the fusion protein is an LER-like protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of LER-like can be increased through use of a heterologous signal sequence.
  • the fusion protein is an I -R-like-immunoglobulin fusion protein in which the LER-like sequences are fused to sequences derived from a member of the immunoglobuiin protein family.
  • the ILIR-like-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an LIR-like ligand and an LER- like protein on the surface of a cell, to thereby suppress LIR-like-mediated signal transduction in vivo.
  • the LIR-like-immunoglobulin fusion proteins can be used to affect the bioavailability of an LIR-like cognate ligand.
  • Inhibition of the LIR-like ligand/LIR- like interaction can be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival.
  • the I-ER-like-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-LIR-like antibodies in a subject, to purify LIR-like ligands, and in screening assays to identify molecules that inhibit the interaction of LIR- like with an LIR-like ligand.
  • 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, e.g., Ausubel, et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An LER-like-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the LIR-like protein.
  • the isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequence set forth as any one of SEQ TD NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58, 66, or 68-74 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ED NO: 1-2, 4-5, 7, 16- 18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 or the corresponding full length or mature protein.
  • Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in the SEQ ED NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58, 66, or 68-74 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.
  • the invention also provides biologically active or immunologically active variants of any of. -he amino air _.e r-:er_ces set forth " es SEQflD NO: ,376. S-I3.T9 -2, ⁇ 2-1-25. 33- ⁇ 4. 5 7 . 4o. 42-45. 50. 52-5S. c6. or-6S-74 c r "
  • Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58, 66, or 68-74.
  • Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.
  • Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. A er. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
  • the present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.
  • the protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.
  • the mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.
  • the sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.
  • proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which it is expressed.
  • Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
  • the present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic ⁇ ⁇ c l fragments e: the . present m ' ven'-icn.
  • degenerate variant is intended nucleotide r.ecie-y ⁇ cie ' " ragment of the pr ⁇ -sent invent: n 'e.g.. . ⁇ n ⁇ ;et. due to tne cieg ⁇ r.eraev of the geneei: cede, encode . identical polypeptide sequence.
  • Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
  • the amino acid sequence can be synthesized using commercially available peptide synthesizers.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies • against the native polypeptide. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.
  • a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.
  • One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.
  • the invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.
  • the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the-encoded p.oiv "eptid ⁇ . T*e p ⁇ 'ypep ⁇ e can be recovered from the c.b ure. eo_v7e e .. Tfe_ia the -cel'.ere medium. - ⁇ ' from a Iv ⁇ t ⁇ .
  • re p are from the. best Cip ' s.ar.ci further -.rifled. - Preferred embodiments mciude those m • * hrch -the protei . produce:: -by such process is a full length or mature form of the protein.
  • the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.
  • One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion- exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer- Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular. Biology.
  • Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.
  • the purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.
  • the molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell animal death or prolonged survival of the animal/cells.
  • the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.
  • toxins e.g., ricin or cholera
  • the toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 3, 6, 8-13, 19, 22, 24-28, 33-34, 37, 40, 42-45, 50, 52-58, 66, or 68-74.
  • the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized somatic c germ cells containing-a nueieotide sequence encoding the
  • modifications, in the peptide or DNA sequence can be made by those skilled in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.
  • Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine- containing variant for biological activity. This type of analysis determines the importance of the substituted amino acid(s) in biological activity. Regions of the protein that are important for protein function cay be determined by the eMATRIX program.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBatTM kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), inco ⁇ orated herein by reference.
  • an insect cell capable of expressing a polynucleotide of the present invention is "transformed.” . ' ⁇ ' - . _
  • the prcce of- ⁇ he i-nventit-r. may be 'prepare,; by cuituring :r..r.sfcrme ⁇ ' h_est _?? ' !:$ -under suiture.coneuions y.i, eble :.. e.yyess _hee : ⁇ combinant-rcrotein.
  • the resiliting expressed protein may then be purified from such culture ⁇ i.e., from culture medium or - cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA SepharoseTM
  • hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
  • immunoaffinity chromatography immunoaffinity chromatography
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • epitope FLAG®
  • Kodak New Haven, Conn.
  • HPLC steps employing hydrophobic RP-H ⁇ LC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • the polypeptides of the invention include analogs (variants).
  • the polypeptides of the invention include LER-like analogs.
  • LIR-like polypeptide of the invention This embraces fragments of LIR-like polypeptide of the invention, as well LIR-like polypeptides which comprise one or more amino acids deleted, inserted, or substituted. Also, analogs of the LIR-like polypeptide of the , -mv ⁇ ttor. erpbrace fusions of the LIR-like polypeptides .or modifications of the LIR-like ,_cc'ypeps. ⁇ ce . erein - e L!R---. e-po " >pent:ee or ar. ⁇ .og- : s -fused to ⁇ - _" ⁇ ⁇ r m-e e:y cr - moieties _e" .
  • moieties which may be fused to the LIR-like polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to neurons, e.g., antibodies to central nervous system, or antibodies to receptor and ligands expressed on neuronal cells.
  • moieties which may be fused to LER-like polypeptide include therapeutic agents which are used for treatment, for example anti -depressant drugs or other medications for neurological disorders.
  • LER-like polypeptides may be fused to neuron growth modulators, and other chemokines for targeted delivery.
  • Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WT), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et al., Nucleic Acids Res. vol. 25, pp.
  • the invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.
  • Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no.
  • polypeptides of the invention in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.
  • Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.
  • the polypeptides of the present invention can be inhibite ' d by using targeted deletion methods, or the insertion of a r.gg'dtiv e regulatoryei ⁇ me ⁇ t such as ⁇ s-'lencer. v..nich-is'tissue specific.
  • DNA sequences allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.
  • Cells can be - modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous ' romoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.
  • cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under" the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.
  • gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
  • regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
  • sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.
  • These sec enoes include po ⁇ y signals. mRNA stability-elemerts. spVce site-, leader plague -secuer ces for-en ! "_.r.c , ⁇ g-or mocityng.trarsport cr sec-reticn p?epert:es of the p-irem. r ether sequences -'.rich- tlteror improve tr.e " fun tie- cr t ⁇ b_et_. of pmtein :r RNA ' molecules.
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell- type specificity than the naturally occurring elements.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
  • transgenic animals Animals m which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as "knockout" animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.
  • Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be ca ⁇ ied out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.
  • the homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
  • the polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express functional LER-like polypeptide or that express a variant of LIR-like polypeptide. Such animals are useful as models for studying the in vivo activities of LIR-like polypeptide as well as for studying modulators of the LIR-like polypeptide.
  • one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi,
  • Transgenic animals can be prepared es desc ⁇ bed m U.S. Patent No. 5,557,032, inco ⁇ orated herein by reference.
  • Transgenic animals are useful to determine 0 the roles polypeptides of the invention play in biological processes, and preferably in disease states.
  • Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.
  • Transgenic animals, preferably non-human mammals are produced using methods as described in U.S. Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference. 5 Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.
  • Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.
  • the 0 homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.
  • polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or 0 vectors suitable for introduction of DNA).
  • the mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.
  • compositions of the invention include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or r ⁇ i e.p : ' .es of the i vention (-incl- ⁇ d * r-g full length n--ctsin.-matur ⁇ .protein and " ⁇ - tie nc.->: ⁇ c ⁇ .- 1, x:x s thereof e or compounds and ether ⁇ ebstances that mod-.late-rhe -Ovemll act: -i-y ef tee targer' ene products, e-tner _: tne level of " ⁇ rget gcre, p.o.e.
  • S c modulators include polypeptides. ⁇ n ⁇ ' gs. (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.
  • the polypeptides of the present invention may likewise be involved in cellular activation or in one*of the other physiological pathways described herein.
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for ⁇ x ple. that described - Gvuris et al . Cell 7 - 91-S03 ( ' 1993". " . to identifv t?o " _ - ⁇ clerf. e * encoding the other-protein v. ' :h .wh.ic . bm.emg cccurs-cr tc .de'n.:-. " '. :rh' c. CrS of the binding interact: cr
  • polypeptides provided " ay t.ne present invention can similarly be used m assays to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonist, of the binding interaction.
  • polypeptides of the invention are also useful for making antibody substances that are specifically immunoreactive with LER-like proteins.
  • Antibodies and portions thereof e.g., Fab fragments
  • Fab fragments which bind to the polypeptides of the invention can be used to identify the presence of such polypeptides in a sample. Such determinations are carried out using any suitable immunoassay format, and any polypeptide of the invention that is specifically bound by the antibody can be employed as a positive control.
  • Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon sourceTuse as a n ⁇ trogen ⁇ so ⁇ .rCB ⁇ rttd ' 'es ⁇ a_ ⁇ rsource " of-carbohydratel In.sech cases the peivp ⁇ p-tide-crpiplyn cleoti e of the-invsnticn car/be added te :he» eed sf-a p-.rt-Ci.l-_r crganism-er c..n be administere -as a separate solid or'ii-ycd preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is culture
  • polypeptides of the invention can be used as molecular weight markers, and as a food supplement.
  • a polypeptide consisting of SEQ ED NO: 3, for example, has a molecular mass of approximately 34 kDa in its unprocessed and unglycosylated state.
  • Protein food supplements are well known and the formulation of suitable food supplements including polypeptides of the invention is within the level of skill in the food preparation art.
  • a polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.
  • Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin- ⁇ , Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • a polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.
  • Administration of the polypeptide of the invention to stem cells in vivo or ex vivo may maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering, damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.
  • the ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases
  • tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others
  • organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.
  • exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LDF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MU -alpha), G- CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).
  • SCF stem cell factor
  • LDF leukemia inhibitory factor
  • Flt-3L Flt-3 ligand
  • any of the interleukins recombinant soluble IL-6 receptor fused to IL-6
  • macrophage inflammatory protein 1-alpha MU -alpha
  • stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.
  • Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Patent No. 5,690,926).
  • Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.
  • polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.
  • the polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.
  • the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.
  • ⁇ __or. ⁇ i ohes the use ' T " e eei - ⁇ yce- specr.c promotei u-iu g ⁇ _.e_ect..bie m- ker
  • stem cells ca 'be .nduced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L.
  • directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.
  • stem cells In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.
  • Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.
  • the ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi- solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).
  • a polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders. Even marginal biological activity in support of colony forming cells or of factor- dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent in supporting the growth and prclrfepation of megakaryocyte_ra d ""..Suuer.fl.
  • ace cr or cc-r-piimer .-y to platelet transfusions: an ⁇ or m supporting the grov tn an proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
  • stem cell disorders such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria
  • compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; EQrayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R.
  • a polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.
  • a polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.
  • Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention.
  • tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.
  • Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing __ damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament A bcne ⁇ ir-etner tissues, ⁇ nu repa. ⁇ g defects to tendon or ligament tissue De rr ⁇ ⁇ > - ⁇ Alc- ⁇ , ' :.
  • compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an approp ⁇ ate matrix and/or sequestering agent as a carrier as is well known in the art.
  • compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.
  • compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • compositions of the present invention may also be involved in the generation oi -regeneration -of other tissues, such as organs (including, fcr-e ample, pancreas. -liver, intestme. >. ra.. c ' e'> ' _.meo-r. skeletal or earaac r.--. ascular imciu ⁇ mg vascv.-tr er. o'-rer.u i tissue, or tor p-orr_ot.r.g ne gro .. ct cei.s ccmpr.sm such tissues.
  • Part of the uesired effects ma> be oy inhibition or modulation of "Verotic scarring may allow normal tissue to regenerate.
  • a polypeptide of the present invention may also exhibit angiogenic activity.
  • a composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues and conditions resulting from systemic cytokine damage.
  • a composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or f inhibiting the growth of tissues described above.
  • compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those describee in: Intemational Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Pate Publication No. WO91/07491 (skin, endothelium).
  • Assays for wound healing activity include, without limitation, those described i Winter, Epidermal Wound Healing, pp. 71-112 (Maibach, H. I. andRovee, D. T., eds.) Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
  • a polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assa are described herein.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such activities.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCTD)), e.g., in regulating (up or down) growth and proliferation of T and or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCTD severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HTV) as well as bacterial or fungal infections, or may result from autoimmune disorders More specifically, infectious diseases causes -by viral, " e ⁇ cter. ⁇ l.
  • proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocy-osis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.
  • allergic reactions and conditions e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocy-osis, allergic rhinitis, hypersensitivity pneumonitis, urticaria,
  • a protein (or antagonists thereof) of the present invention may also be treatable using a protein (or antagonists thereof) of the present invention.
  • the therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol.73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ.
  • the functions of activated T cells may be inhibited by suppressing T cell -responses or by inducing specific tolerance in T cells, or both. "" Inmunosuppres ⁇ 1 . en or " T. ce"'i'.-esp «n--es is ger.er '.;. c ⁇ -£tive. ron-antigen-specr ⁇ c.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • B lymphocyte antigen functions such as, for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • the administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the irnmunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental Immunology. Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect cf therapeutic-eompositions of the invention on the devel pment ⁇ ⁇ tm . disease , - - "
  • Blocking err gen function also be theiapeut-.ca.lv ⁇ e; A for t: eating autoimmune eiseases. Man) autoimmune CiiOrclers ar ⁇ the result oi inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRIJlpr lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis. ⁇
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells ' express all or a portion of the protein on their " Surface, and rein tf ⁇ duce -the -transfected ceils m:e be patienrrThe-i ⁇ fected cells v cu ' .d now be capable of-celr- ermg a - co ⁇ timulutcry signal to. and thereo ' y activate. T cells in vivo.
  • a polypeptide of the present -invention a) provide the r.ecessar -stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
  • tumor cells which lack MHC class I or MHC class H molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic- domain truncated portion) of an MHC class I alpha chain protein and ⁇ 2 microglobulin protein or an MHC class II alpha chain protein and an MHC class U beta chain protein to ' thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class 11 associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in 1-rnmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Iirimunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; ⁇ naba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 5 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry I4:591-S97, 1993: Gorczyca et ai.. ⁇ ntemationaFJ ⁇ Hial of Oncology 1:639-648. 1992.
  • a polypeptide of the present invention may also exhibit activin- or inhibin-related activities.
  • a polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.
  • Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a polypeptide of the -present invention alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • polypeptide of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.
  • a polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.
  • polypeptide of the invention may, among other means, be measured by the following methods.
  • Assays for activin/inhibin activity include, without limitation, those described in:
  • a polypeptide of the.pres ⁇ nt invention raay-be.irt oived in chemotactic or " c ' r.eme iretic acti-v.tv tor -mammalian ceils, including, - ' cr ev-c p ' e mor.ocy.es. fibroblasts. neutrophil ⁇ . T-ce!ls. mast ceils, eosinop ' ⁇ iis. epithelial and cr ehcotce ⁇ ..: • ceils.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such attributes.
  • Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic compositions e.g. proteins, antibodies, binding partners, or modulators of the invention
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.
  • compositions may be useful in treatment of .v rious coagulation disorders (including hereditary disorders, such as nemophiiias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al, J. Clin. Pharmacol. 26:131-140, 1986; Burdicket al., Thrombosis Res. 45:413-419, 1987; Humphrey et al, Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988. '
  • Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.
  • Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.
  • Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic etastases, blood cell malignancies :ncl ⁇ dmg-mult p!e and c-hronic ieukeiruas. and ⁇ ymp ⁇ em ⁇ s head _r.d "ee.e aree s nc.-.r ne me-.:", cancer, " arm >.
  • Polypeptides, polynucleotides, or modulators of polypeptides of the invention may be administered to treat cancer.
  • Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermo herapy, and laser therapy, and may provide a beneficial effect,' e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.
  • composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.
  • An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti -cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine.
  • Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), dacarbazine, Dactinomycin, Daunorubicin HCl, -Doxeru iein HG1. Es-r ⁇ mu ⁇ fine.-phosph-ue sodium. Eto. ⁇ oside (V 1:6-213).
  • Floxuridins are 5- Fl ⁇ orouraeii • 5-Fu>. Fluta ide. Hvdrc.'.urea-'hydro ycarbamide " . Ifesfamfee. In * e r fei'cn Alpha-2a. Interferon Alph ⁇ -2b. L ⁇ uproiid ⁇ acetate iLHRH-releas g factor cm-fiog.. Lomustine, Mechlorethamine HCl (nitrogen mustard), Meiphalan, Merc ⁇ ptopurine.
  • MTX Methotrexate
  • Mitomycin Mitoxantrone HCl
  • Octreotide Plicamycin
  • Procarbazine HCl Streptozocin
  • Tamoxifen citrate Thioguanine, Thiotepa
  • Vinblastine sulfate Vincristine sulfate
  • Amsacrine Azacitidine
  • Hexamethylmelamine Interleukin-2
  • Mitoguazone Pentostatin
  • Semustine Teniposide
  • Vindesine sulfate Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2
  • therapeutic compositions of the invention may be used for prophylactic treatment of cancer.
  • hereditary conditions and/or environmental situations e.g. exposure to carcinogens
  • In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.
  • These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl. Can.
  • Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs. 5.7.12 RECEPTOR/LIGAND ACTIVITY
  • a polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.
  • a polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their jig ⁇ nds. receptor kinases and.tner .:g ⁇ r-t ⁇ .
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a polypeptide of the invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1- 7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med.
  • polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).
  • Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.
  • polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods.
  • radioisotopes include, but are not limited to, tritium and carbon-14 .
  • colorimetric.molec. ⁇ i ⁇ s include, but are not limited to, fluorescent molec-ules such as fluoresc ⁇ nr.ne, or rhodamine cr other ccicr ⁇ r ":-:ric moleeuies. examples of include, but are . et limited, to ncin. . .
  • This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.
  • the polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.
  • Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial Ubraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.
  • Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.
  • the sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.
  • Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 252:63-68 (1998).
  • the binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.
  • the toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity 5 of the binding molecule for a polypeptide of the invention.
  • the binding molecules may be ' complexed with imaging agents for targeting and imaging purposes.
  • the invention also provides methods to detect specific binding of a polypeptide 0 e.g. a ligand or a receptor.
  • a polypeptide 0 e.g. a ligand or a receptor.
  • the art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells, or dibybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind
  • Liga n ds for receptor polypeptides of the invention can also be identified by adding ' exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical 0 except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) are then compared.
  • an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).
  • BIAcore 5 assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
  • downstream intracellular signaling molecules in the signaling cascade 0 of the polypeptide of the invention can be determined.
  • a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified is produced in a host cell.
  • the cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.
  • Known downstream proteins 5 involved in intracellular signaling can then be assayed for expected modifications i.e. phosphorylation.
  • Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.
  • compositions of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or . promote an infiammatorv response.
  • Ccrrcpositions with such activities can " be used to treat A infl ⁇ m ⁇ torv- een-i:. ⁇ ons including' e-hrc-nic'-sr acute.
  • compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.
  • conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegen
  • Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.
  • leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
  • Nervous system disorders involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorder- which e ⁇ uit -n either a disconnection of a ⁇ ons.
  • ⁇ a diminution or " degeneration -'- eu rys er s ⁇ ye'ir.a ⁇ on Ne; .ous i-vstem lesions wh.eh my be treate in a pat:e"t society 1 r edmg numan and r.cn- iman mammalian patients ⁇ according to the i ention include but are not limited to the following lesions of either the central spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
  • ischemic lesions in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
  • degenerative lesions in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
  • neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and
  • demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple ⁇ 5 ⁇ - ⁇ sclerosis, huma -immunodeficiency virus-associated velopathy. transverse my ⁇ loo-ithy er r.iv etiologies, .e m.u-.foc:.. " ere Ce n t al ⁇ o ⁇ r.e • mvehr.o.vsis.
  • Therapeutics which are useful according to the " i . en h on or treatment of ⁇ nervous system disorder may be selected by testing for biological activity in promoting 10 the survival or differentiation of neurons.
  • therapeutics which elicit any of the following effects may be useful according to the invention:
  • Such effects may be measured by any method known in the art.
  • increased survival of neurons may be measured by the method set 20 forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci.
  • neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron 25 dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may 30 affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease). . . _ - " . .
  • a polypeptide or the invention ⁇ v a ' c exhibit _ ,e or more of tne following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing
  • polymorphisms make possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.
  • Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment . appropriately. '-F ⁇ r-ex-amp-le. the existence o " f a pol-ymorphism associ ted with a
  • predisjDOsiti A ⁇ to inflammation er.at.toi-mnlune disease makes po.ssib ' . ⁇ rr.e-Jagnosis of this condition in humans by identifying the presence of the polvmorphism.
  • Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.
  • the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).
  • allele-specific oligonucleotide hybridization in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch
  • a single nucleotide extension assay in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides.
  • traditional restriction fragment length polymorphism analysis using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism
  • the array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.
  • any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.
  • a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.
  • the immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system.
  • the experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at, 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129.
  • Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tJ-ibe-reulos-is'm.o.- ⁇ -nplete Fr ⁇ unds adjuvant (CFA ⁇
  • the route ofinjection can vary, but r ⁇ es F ⁇ W be mcec r ed at " e base of the ta.- ⁇ . -.ryar. ad,'... art m ⁇ t r ⁇
  • the polype tide is et-rninistered phosphate oufferec solution ⁇ ?BS ⁇ at a dose of about 1-5 mg.
  • Tee control consists of aammiste ⁇ ng PBS only.
  • the procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.
  • an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.
  • compositions including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides
  • therapeutic applications include, but are not limited to, those exemplified herein.
  • One embodiment of the invention is the administration of an effective amount of the LIR-like polypeptides or other composition of the invention to individuals affected fr a disease or disorder that can be modulated by regulating the soluble immunoglobuiin receptor of the invention. While the mode of administration is not particularly important parenteral administration is preferred. An exemplary mode of administration is to delive an intravenous bolus.
  • the dosage of LIR-like polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.
  • the amount of polypeptide administered per dose will be in the range of about O.Ol ⁇ g/kg to 100 mg kg of body weight, with the referred dose being about 0.4. ⁇ g kg.tQ V0 mg kg of. patient body weight.
  • parenteral administration LIR- like -pciyne-D ide ⁇ of .he..nvsn:.em will-be formulated Jr. an injectable form-combined. with a pharmaceutically aceeptable . parentera! -vehicle..
  • Such vehicles are well known in the art and examples include water, saline. Ringer " s solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
  • the vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the ait.
  • a protein or other composition of the present invention may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or exci ⁇ ient(s) at doses to treat or ameliorate a variety of disorders.
  • a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF. IL-1, ⁇ L-2, B -3, IL-4, ⁇ L-5, IL-6, D -?, IL-8, IL-9, IL-10, IL-11, ⁇ L-12, IL-13, IL-14, IL- 15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • cytokines cytokines, lymphokines, or other hematopoietic factors
  • proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet- derived growth factor (PDGF), fransforming growth factors (TGF- ⁇ and TGF- ⁇ ), insulinlike growth factor (IGF), as well as cytokines described herein.
  • EGF epidermal growth factor
  • PDGF platelet- derived growth factor
  • TGF- ⁇ and TGF- ⁇ fransforming growth factors
  • IGF insulinlike growth factor
  • the .pharmaceutical composition may further contain other agents which either enhance -the activity of the protein o other active ingredient or complement its acti-v ; ty or ' . -ese in treatment. ' Such .-.dditionaj factors- and'or agents may be include .1 in the .- . '
  • protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti- thrombotic factor, or anti-inflammatory agent (such as IL-lRa, IL-1 Hyl, IL-1 Hy2, anti- TNF, corticosteroids, immunosuppressive agents).
  • IL-lRa IL-1 Hyl
  • IL-1 Hy2 anti- TNF
  • corticosteroids immunosuppressive agents
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).
  • Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack . Publishing Co., Easton, PA, latest edition.
  • a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g. , treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective dose When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether adrninistered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein or other active ingredient of the present vention is administered to a mammal having ⁇ condition to be treated.
  • Protein or other acti'.e mgredient of the-pr ⁇ sent in -ent.en may pe admire -.tered in ucccreai.ee wrm ;he method cf the invention eimer alone or m -combination vw.n ether .therapies such as treatments employing cytokines, lymph ⁇ kihes or other hematopoietic factors.
  • protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intiameduUary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • the compounds may be administered topically, for example, as eye drops.
  • a targeted drug delivery system for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
  • the polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.
  • the determination of a suitable foute ' of ⁇ ' administratio -an ' an effective dosage lOr-a-particalar in-iiea:ion is within .he-level cf skill in the art.
  • Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
  • compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
  • protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain, a solid carrier such as a gelatin or an adjuvant.
  • a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.
  • protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • the preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone. agar. or alginic acid or a salt thereof such as sodium alginate.
  • Dr gee ceres are provided-with suitable coatings. For tnis purpose.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols..
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the. active compounds in water-soluble form: Additionally,- suspensions of the. active ' compound., may be. repared as appropriate, oi-yirje ⁇ .ion -suspensions. ' . -. ' .
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame ' oil. or svnthetic fatty acid esters, such as ethyl oleate or- triglycerides, or " liposomes.
  • -Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation. of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a co- solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the co-solvent system may be the VPD co- solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co- solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be va ⁇ ed:O.rerb;ocompatible polymers -may replace polyethylene glycol. e.g.,p ⁇ Iyvinyl pvrrohdc . ne: ._rd other ' sugars cr p-elysaecha ⁇ ees substi.tute-for dextrose. Alternatively, ctr.er deliver) sv stem?
  • hydrop ' re'rrc pharm ⁇ ce ⁇ tical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein or other active ingredient stabilization may be employed.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.
  • Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, tii-dkylamine, dialkylamine, mono-ilkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.
  • the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.
  • the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
  • B lymphocytes will respond to antigen through their surface immunoglobuiin receptor.
  • T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
  • TCR T cell receptor
  • MHC and structurally related proteins including those encoded by class I and class 13 MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen ⁇ " components could also be supplied as purified ' MHC -peptide c ⁇ mplexes alone ⁇ with co- s. ⁇ m ⁇ l- ⁇ te c ymc!ec: ⁇ les' ⁇ .hat oar. direct ' .) s'gr ⁇ i T ceils Alternative ' .) a . oc;es e'e to bind surface Immunoglobuiin ahe other moiecu.es o ⁇ " B cells as well as ar.t.ecaies able to bind the TCR and other molecules on T ecus can be combined with the pharmaceutical composition of the invention.
  • the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
  • the amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about 0.1 ⁇ g to about 10 mg, more preferably about 0.1 ⁇ to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.
  • the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of
  • the e ⁇ miposition may desir ⁇ b " ) " be encapsulated or injected in a viscces form for ⁇ e v ery to y.p ⁇ -:_e- of b ⁇ ">e. .cartilage or tissue damage.
  • Topical adrninistra::cn may be sui'abie for ound healing and tissue repair.
  • Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as 0 described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage 5 and optimally capable of being resorbed into the body.
  • matrices may be formed of - materials presently in use for other implanted medical applications.
  • Potential 0 matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well- defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are 5 nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle 0 shape, and biodegradability.
  • a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as ⁇ l ylc ⁇ llulcses, (including including methylc ⁇ iiulose. e ⁇ h)icellul?se.-h)dr ⁇ y.eth)ice:I-.; ->se. hvdrox-vprepylcelh ⁇ lo ⁇ e h droy pro.p l- rr.ethvlcell-.lese. P.i car-bo-.vm ⁇ thyce ⁇ lulese.-.he-most- preferre being caticr.lc saits-of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5- 20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins or other active ingredient of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and TGF- ⁇ ), and insulin-like growth factor (IGF).
  • EGF epidermal growth factor
  • PDGF
  • the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredient of the present invention.
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • IGF I insulin like growth factor I
  • the addition of other known growth factors, such as IGF I may also effect the dosage.
  • Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
  • Poiynucleotitle ⁇ of the present invention can also be used for gene therapy.
  • Jes can re ' ⁇ t.oc.uced e-mer m v.vo or ex vi :n:o c ⁇ hs or expression m a mammaVn sue ec: Pcvnecleotides of the in .entic'n may also be administered by ether known methods for introduction of nucleic acid into a cell or organism (.including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half -maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic -index and it can be expressed as the ratio between LD;n ⁇ "d ED 0. -
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10- 90% of the time, preferably between 30-90% and most preferably between 50-90%.
  • an exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 ⁇ g/kg to 100 mg kg of body weight daily, with the preferred dose being about 0.1 ⁇ g/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.
  • the amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • the pack may, for example, comprise metal or plastic foil, suc.n as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • ANTIBODIES Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention.
  • Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments thereof, and humanized forms or fully human forms, such as those produced in transgenic animals.
  • the invention further provides a hybridoma that produces an antibody according to the invention.
  • Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which maybe mediated by the protein.
  • Any animal which is known to produce antibodies can be immunized with a peptide or polypeptide of the invention.
  • Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide.
  • One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection.
  • the protein that is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity.
  • Methods of increasing the antigenicity of a protein include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or ?-galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ?-galactosidase
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Agl4 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybiidomas with an ELISA assay, western blot analysis, or ra ⁇ oimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)).
  • Hybiidomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention. ⁇ For polvclonal antibodies, antibody-containing antiserum is isolated from the
  • Antibodies can oe delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as Fl ' i ' C or rhodamine, etc.), paramagnetic atoms, etc.
  • the labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed.
  • the antibodies may also be used directly in therapies or other diagnostics.
  • the present invention further provides the above-described antibodies immobilized on a solid support.
  • solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D.M. et al., "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D.
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.
  • 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 nvbrdoma technique., see Kozbfir. et a!.. -19S3 Immunol Today ⁇ . ⁇ 2 ' ⁇ ⁇ nd.the ' EBV . o ⁇ .eckr.iq ⁇ e to procuc ⁇ 'nliman mcnec'or. ⁇ i antibodies >'see Cole, et al..
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybiidomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by teansforming 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 immunoglobuiin loci into transgenic animals, e.g., mice in which the endogenous immunoglobuiin 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 immunoglobuiin 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 erflbocc enfof such a nonhuman animal is a mouse, and is termed the - :Xenom ⁇ use S ⁇ I ...s d.scioseU V PCT publications O 96- 3-3735 and WO « - ⁇ 096 " .
  • This a ⁇ mai'nroduees B cells ich secrete fully-human immanoglobu. s.
  • the antibodies can be obtained directly from tiie 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 hybiidomas producing monoclonal antibodies.
  • 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 immunoglobuiin heavy chain is disclosed in U.S. Patent No. 5,939,598.
  • 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.
  • 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 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.
  • 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.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobuiin 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 immunoglobuiin 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 of the 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 can be fused to immunoglobuiin constant domain sequences.
  • the f-. ⁇ '.on prefer ⁇ b'y is with a imm ⁇ noglubu n be ⁇ vy-ch ⁇ c cemp-'iing r: ,e ⁇ s: $s. of the r-.nge.
  • DNAs encoding the immunoglobulm heavy-chain fusions and, if desired, the immunoglobulm light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • DNAs encoding the immunoglobulm heavy-chain fusions and, if desired, the immunoglobulm light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • 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 ammo acid side chains from the interfa ⁇ e of the 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 difhiol 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 of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. . " "
  • 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 domains of one fragment are forced to pair with the complementary VT, and VH domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V domains of one fragment are forced to pair with the complementary VT, and VH domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • 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.
  • an anti-antigenic arm of an immunoglobuiin 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. D28. or -By), or Fc -receptors for IgG rFc ⁇ R). such as-ftr R ⁇ CGO6 F0.
  • 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).
  • TF tissue factor
  • 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 H3V 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 cross-linking 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 hav ⁇ -enhanced complement-lysis and ADCC capabilities. See Stevenson e: ⁇ i.. " Anti-C ⁇ nc ⁇ r Drug -Design. 3.. 219-13- ) ⁇ -l9S9>. - "
  • 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, .
  • m ⁇ deccin A chain alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPIJ, 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 radioconjugated antibodies. Examples include Bi, I, In, 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-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidy
  • aricin 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 can be conjugated to a "receptor" (such " s * i.-ept-? fdin) for utilization in-tumorrpretargeting -.v erei-n-th ⁇ ar tiredy.
  • a nucleotide sequence of the present invention can be recorded on computer readable media.
  • computer readable media refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.
  • sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file* stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • a skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide s ⁇ qL-e-nee ' -iRfor -jati . ⁇ n.Q.f.the:pre's ⁇ nt invention.. . - ..' . - - . ' • - ⁇ ' •
  • ORFs open reading frames
  • Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.
  • the minimum hardware means of the computer- based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.
  • data storage means refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
  • search means refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural " motif with the -sequence information stored within the data storage means. Searcn mean' are-used to identify fragments or-regie-s of a-krowry ec_-icr.ee wh.ch match a particular target sequence or target motif.
  • a v-trie-v cf'kr.own algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).
  • a target sequence can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.
  • the most preferred sequence length of a target sequence is from about 10 to 100 amino acids, or from about 30 to 300 nucleotide residues.
  • searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing may be of shorter length.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzyme active sites and signal sequences.
  • Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
  • fragments of the present invention can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.
  • Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are desig ed to be -complementary to a,fe'g-i'on of the gene involved in
  • Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.
  • the present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.
  • methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.
  • Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.
  • methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.
  • such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for bindmg of the nucleic acid probes or antibodies to components within the test sample.
  • Incubation conditions depend on tne format employed m the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.
  • any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
  • test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
  • kits which contain the necessary reagents to carry out the assays of the present invention.
  • the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.
  • a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allows one to efficiently transfer reagents from one compartment to another compartment such, that the samples and reagents are not 5 cross-contaminated, -and-the agents or solutions of each container can be added in a " quantit ⁇ -iv ⁇ fashion if om e-iie co pa ⁇ me ⁇ t ' to andther.
  • Such containers will inci-.de a ' •container w h.ich will accept the test:sam ⁇ e.
  • a container which contains the antibodies ' used in the assay containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the
  • detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the 20 polypeptide of the invention is involved in the immune response, for imaging sites of ii-flammation or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778.
  • Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.
  • the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set 30 forth in the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63- 65, or 67, or bind to a specific domain of the polypeptide encoded by the nucleic acid.
  • said method comprises the steps of:
  • .•therefpr ⁇ . such.meth ⁇ ds f ⁇ Y-identirlying-co ⁇ npeund ⁇ hat i ⁇ .d to ' .polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
  • such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.
  • Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex- is detected, a compound that binds a polypeptide of the invention is identified.
  • Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).
  • compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).
  • Compounds, such as compounds identified via the methods of the invention can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.
  • the agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.
  • the agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. -For random screening, agents such -as peptides. carbohydrates, pharmaceutical
  • agents and the'like are selected at random and are assayed for ' their abi'ity tc- ind :o" ⁇ e protein encoded by the ORF of the present invention.
  • Aitemati-veiy. agents may be rationally selected or designed.
  • an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the particular protein.
  • one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp.289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.
  • one class of agents of the present invention as broadly described, can be .used to control gene expression through binding to one of the ORFs or EMFs of the present invention.
  • DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.
  • Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.
  • Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix ehgonec-,eotide and other DNA binding agents
  • Agents which bind to a protein encceed by one of the ORFs of the present invention can be used as a diagnostic agent.
  • Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.
  • Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.
  • the hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ JD NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67.
  • a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.
  • PCR as described in US Patents Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.
  • probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.
  • the probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.
  • nucleic acid sequences include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.
  • vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.
  • the nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.
  • the nucleotide sequence provided herein may be mapped to-a chromosome or specific regions'of a chromosome using well known " ' genetic anwi'or ehrom9SomaLfnapping:teehnisr.es. These techniques include _m S J hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988)
  • chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.
  • Oligonucleotides i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.
  • One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.
  • L-c-mobilization can be achieved using passive adsorption (fnouye & Hondo, 1990 J. Clin.
  • CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.
  • CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5'-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen etal, (1991) Anal Biochem 198(1) 138-42.
  • CovaLink NH strips for covalent binding of DNA molecules at the 5'-end has been described (Rasmussen et al., 1991).
  • a phosphoramidate bond is employed (Chu et al., 1983 Nucleic Acids 11(18) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred.
  • the phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.
  • the oligonucleotide terminus must have a 5 -end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes. More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold 0.1 M 1- methylimidazole, pH 7.0 (l-Me--m ), is then added to a final concentration of 10 mM 1- Melm 7 .
  • a ss DNA solution is then dispensed into CovaLink NH strips (75 ul well) standing on ice.
  • This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3 -reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.
  • the oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.
  • Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.
  • An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.
  • addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) S.cience 251(4995) 767-73, incorporated herein by reference.
  • Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal Biochem 169(1) 104-8; all references being specifically incorporated herein.
  • oligonucleotide to link an oligonucleotide to a nylon support, as described by Van Ness et al. (1991), requires activation of the nylon surface via alkylation and selective activation of the 5'-amine of oligonucleotides with cyanuric chloride.
  • One particular way to prepare support bound oligonucleotides is to utilize the light- generated synthesis described by Pease et al., (1994) Proc. Natl. Acad. Sci USA 91(11) 5022-6. These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).
  • oligonucleotide probes in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5'-protected N-acyl-deo ⁇ ynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.
  • a matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.
  • the nucleic acids may be obtained from any appropriate source, such as cDNAs. • 5 genomie-D ' NA-. chromosomal DNA. -micro-dissected chromosome " bands.-Cosmid or YAC inserts, and RNA, including mRNA ithout any amplification steps.
  • cDNAs • 5 genomie-D ' NA-. chromosomal DNA. -micro-dissected chromosome " bands.-Cosmid or YAC inserts, and RNA, including mRNA ithout any amplification steps.
  • Sambroo ' k at al. (19S9) .describes three protocols for the isoiafi ' on-of-high molecular weight ' DNA from mammalian cells (p. 9.14-9.23).
  • DNA fragments may be prepared as clones in M13, plasmid or lambda vectors 10 and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.
  • nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 15 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.
  • Low pressure shearing is also appropriate, as described by Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6.
  • DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.
  • a lever device allows controlled application of low to intermediate pressures to the cell. The results of 20 these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.
  • the restriction endonuclease Cv z ' J ⁇ normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.
  • Atypical reaction conditions, which alter the specificity of this enzyme (Cv ' JP*) yield a quasi-random distribution of DNA 30 fragments form the small molecule pUC19 (2688 base pairs).
  • Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.
  • Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm 2 , depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.
  • Each of the subarrays may represent replica spotting of the same samples.
  • a selected gene segment may be amplified from 64 patients.
  • the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8 x 12 cm membrane.
  • Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm 2 and there may be a 1 mm space between subarrays. • Another approach is to use membranes or plates (available from NUNC, Naperville, Illinois) which may be partitioned by physical spacers e.g.
  • the present invention is ⁇ -ll.:s. ate in the following examples.
  • Upon-c nsider ⁇ ticn of the present disclosure one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of 10 the following examples.
  • the present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present 15 preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.
  • a plurality of novel nucleic acids were obtained from cDNA libraries prepared from human leukocyte mRNA (GIBCO Laboratories) (SEQ ID NO: 1, SEQ ID NO: 16); 5 from infant brain mRNA (Columbia University) (SEQ ID NO: 35); from human mammary gland RNA (Invitrogen) (SEQ ID NO: 47); and from bone marrow mRNA (Clontech) (SEQ ID NO: 63) using standard PCR, sequencing by hybridization sequence signature analysis, and Sanger sequencing techniques.
  • the inserts of the library were amplified with PCR using primers specific for vector sequences flanking the inserts.
  • the insert ' .- ⁇ was identified :as anovel sequence net-previously obtained fr ⁇ rn " t-hi5 library and n.cy ' previously-reporte -in-public databases.
  • the sequences were designated as SEQ ID NO: 1, l'6, 35, 47, ' and 63. " ⁇ '" '
  • SEQ ID NO: 2 17, 48, and 64 were assembled using SEQ ID NO: 1, 16, 47, or 63 as a seed, respectively. Then a
  • the nearest neighbor result for the assembled contigs were obtained by a FASTA version 3 search against Genpept release 114, using FASTXY algorithm.
  • FASTXY is an improved version of FASTA alignment which allows in-codon frame shifts.
  • the nearest 5 neighbor result showed the closest homologue for each assemblage from Genpept (and contains the translated amino acid sequences for which the assemblage encodes).
  • the nearest neighbor results is set forth below:
  • the nucleotide -seGuence-within the ' assembled ee ti-.s-tr.at codes for- ⁇ i_mal peotide sequences and their cleavage sites can be determined from using Neural Network SignalP VI.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark).
  • the process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication " Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites" Protein Engineering, vol. 10, no. 1, pp.
  • the predicted amino acid sequences for SEQ ID NO: 17, 48, and 64 were obtained by using a software program called FASTY (available from http://fasta:bioch. virginia.edu) which selects a polypeptide based on a comparison of translated novel polynucleotide to known polynucleotides (W.R. Pearson. Methods in " Enzy ology. l$3"63-9S-fl99GV.ncorpcr _.ed her ⁇ m b) referenced
  • a polypeptide (SEQ ID NO: 3) was predicted to be encoded by SEQ ID NO: 2 as set forth below.
  • the polypeptide was predicted using a software program called BLASTX which selects a polypeptide based on a comparison of translated novel polynucleotide to known polynucleotides.
  • the initial methionine starts at position 114 of SEQ TD NO: 2 and the putative stop codon, TAG, begins at position 1029 of the nucleotide sequence.
  • a polypeptide (SEQ ID NO: 6) was predicted to be encoded by SEQ ID NO: 5 as set forth below.
  • the polypeptide was predicted using a software program called BLASTX which selects a polypeptide based on a comparison of translated novel polynucleotide to known polynucleotides.
  • the initial methionine starts at position 280 of SEQ ID NO: 5 and the putative stop codon, TAG, begins at position 766 of the nucleotide sequence.
  • the leukocyte immunoglobuiin receptor-like polypeptide of SEQ 3D NO: 3 is an approximately 305-amino acid protein with a predicted molecular mass of approximately 3-!- kDa unglycosylated.
  • n Altschul -S F. et al . J. Mol. Biol 21:403- 10 (1990 '. rerem incorpcratee-by reference indicate that SEQ TD NO" 3 is homologous to leukocyte immunoglobuiin receptors like IRCla and NK cell inhibitory receptors like PIGR-1.
  • Protein database search with eMATRIX software (Stanford University, Stanford CA) further show that a portion of SEQ ID NO: 3 (i.e. SEQ 3D NO: 8) is homologous to poly Ig receptors.
  • Figure 1 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ 3D NO: 2 (i.e. SEQ 3D NO: 3) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human IRCla protein SEQ 3D NO: 14, indicating that the two sequences share 55% similarity over 297' amino acid residues and 37% identity over the same 297 amino acid residues. .
  • Figure 2 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ 3D: 2 (i.e. SEQ 3D NO: 3) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LJR-like") and human PIGR-1 (Patent Application No. EP897981) SEQ 3D NO: 15, indicating that the two sequences share 63% similarity over 176 amino acid residues and 53% identity over the same 176 amino acid residues.
  • SEQ 3D NO: 3 i.e. SEQ 3D NO: 3
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LJR-like
  • human PIGR-1 Patent Application No. EP897981
  • a predicted soluble, secreted splice variant of SEQ 3D NO: 3 is SEQ 3D NO: 6. It is an approximately 162 amino acid protein with a predicted molecular mass of approximately 18 kDa unglycosylated. Protein database searches with the B3 ASTX algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated by reference) indicate that SEQ 3D NO: 6 is homologous to leukocyte immunoglobuiin receptors like IRCla and NK cell inhibitory receptor like PIGR-1.
  • SEQ 3D NO: 7 i.e. SEQ 3D NO: 9
  • Figure 3 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ 3D: 5 (i.e. SEQ 3D NO: 6) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human IRCla protein SEQ 3D NO: 14, indicating that the two sequences share 57% similarity over 135 amino acid residues and 38 ⁇ identity over the same 135 amino acid residues.
  • Figure - 1 shows the " BLASTX mmo ae.d sequence alignment between the protein " encoded by SEQ ED NO: 5 i e.
  • SEQ ID NO 6' leukocyte immunoglobuiin receptor-like polypeptide also identified as “LIR-like ' ') and human PIGR-1 (Patent Application No. EP897981) SEQ 3D NO: 15, indicating that the two sequences share 67% similarity over 145 amino acid residues and 59% identity over the same 145 amino acid residues.
  • a predicted approximately thirty residue signal peptide is encoded from approximately residue 1 to residue 30 of both SEQ ID NO: 3 and SEQ 3D NO: 6 (SEQ 3D NO: 10). The extracellular portion is useful on its own.
  • SEQ 3D NO: 12 is the peptide resulting when the signal peptide is removed from SEQ 3D NO: 3.
  • SEQ 3D NO: 13 is the peptide resulting when the signal peptide is removed from SEQ 3D NO: 6.
  • a predicted approximately fourteen residue transmembrane peptide is encoded from approximately residue 170 to residue 193 of SEQ 3D NO: 3 (SEQ 3D NO: 11). This can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). One of skill in the art will recognize that the actual transmembrane region may be different than that predicted by the computer program.
  • both the membrane-bound and soluble IJR-like polypeptide is expected to have a poly Ig receptor domain at residues 82 - 129 of SEQ 3D NO: 3 and residues 82 - 129 of SEQ 3D NO: 6 (SEQ 3D NO: 8 and SEQ 3D NO: 9, respectively).
  • SEQ 3D NO 8 has serine in the position 129 while SEQ ID NO: 9 contains proline in the same position.
  • Poly immunoglobuiin receptor domain (3i ⁇ )NQ3SNRT3r VTMEDLMKTDADTYWCGrEKTGNDLGVTVQVTIDPAS designated as SEQ 3D NO: 8) p-value of 3.628e-9, DM01688B 2 (identification number correlating to signature); located at residues 82-129 of SEQ 3D NO: 3 and
  • Poly immunoglobuiin receptor domain (3- DNQ3SNRT3r VTMEDLMKTDADTYWCG3EKTGNDLGVTVQVTIDPAP designated as SEQ 3D NO: 9) p-value of 2.5e-10, DM01688B 2 (identification number correlating to signature); located at residues 82-129 of SEQ 3D NO: 6.
  • a polypeptide (SEQ 3D NO: 19) was predicted to be encoded by SEQ 3D NO: 18 as set forth below.
  • the polypeptide was predicted using software programs called BLASTX/FASTY which selects a polypeptide based on a comparison of translated novel polynucleotide to known polypeptides.
  • the initial methionine ATG starts at position 169 of the nucleotide sequence and the putative stop codon, TAG, ends the coding region at position 879.
  • Leukocyte immunoglobuiin receptor-like polypeptide SEQ 3D NO: 19 is an approximately 236-amino acid protein with a predicted molecular mass of approximately 263-Da unglycosylated.
  • Protein database searches with the BLASTP algorithm Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al, J. Mol. Biol. 21 :403- 10 (1990), herein incorporated by reference
  • SEQ 3D NO: 19 is homologous to leukocyte immunoglobuiin receptors (LTRs) and immunoglobulin-like protein IGSF-1.
  • LTRs leukocyte immunoglobuiin receptors
  • IGSF-1 immunoglobulin-like protein
  • SEQ 3D NO: 19 is homologous to killer cell inhibitory receptors.
  • Figure 5 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ 3D NO: 18 (i.e. SEQ ID NO: 19) leukocyte immunoglobuiin receptorlike polypeptide (also identified as "LIR-like") and putative inhibitory receptor (Rojo et al, (1997-) J. Immunol., 158, 9-12) SEQ 3D NO: 29, indicating that the two sequences share 5 l c .similarity over 145.amino aeid residues .and 33% identity over the same 145 ⁇ mino acid residues.
  • Figure 6 shows the BLASTP ammo cid sequence alignment between the protein encoded by SEQ 3D NO: 18 (i.e. SEQ ID NO: 19) leukocyte immunoglobuiin receptor- like polypeptide (also identified as "LIR-like") and human GP49 HM18 polypeptide (Patent Application No. WO9809638) SEQ 3D NO: 30, indicating that the two sequences share 50% similarity over 123 amino acid residues and 34% identity over the same 123 amino acid residues.
  • SEQ 3D NO: 18 i.e. SEQ ID NO: 19
  • leukocyte immunoglobuiin receptor- like polypeptide also identified as "LIR-like
  • human GP49 HM18 polypeptide Patent Application No. WO9809638
  • a polypeptide (SEQ 3D NO: 22) was predicted to be encoded by SEQ 3D NO: 21 as set forth below.
  • the polypeptide was predicted using a software program called BLASTX which selects a polypeptide based on a comparison of translated novel polynucleotide to known polynucleotides.
  • the initial methionine starts at position 176 of SEQ ID NO: 21 and the putative stop codon, TAA, begins at position 773 of the nucleotide sequence.
  • SEQ 3D NO 22 is a soluble, secreted splice variant of SEQ 3D NO: 19. It is an approximately 199 amino acid protem with a predicted molecular mass of approximately 22 kDa unglycosylated. Protein database searches with the B3 ASTP algorithm (Altschul S.F. et al., J. Mol. Evol.36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol.21:403- 10 (1990), herein incorporated by reference) indicate that SEQ 3D NO: 22 is homologous to leukocyte immunoglobuiin receptors (J IRs) and immunoglobulin-like protein IGSF-1. Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, CA) further shows that SEQ 3D NO: 22 is homologous to killer cell inhibitory receptors.
  • J IRs leukocyte immunoglobuiin receptors
  • IGSF-1 immunoglob
  • Figure 7 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ 3D NO: 21 (i.e SEQ 3D NO: 22) leukocyte immunoglobuiin receptor- like polypeptide (also identified as "L3R-like”) and immunoglobulin-like protein IGSFl (Mazzarella et al, (1998) Genomics 48, 157-162) SEQ 3D NO: 31, indicating that the two sequences share 53% similarity over 209 amino acid residues and 38% identity over the same 209 amino acid residues.
  • SEQ 3D NO: 21 i.e SEQ 3D NO: 22
  • leukocyte immunoglobuiin receptor- like polypeptide also identified as "L3R-like
  • IGSFl immunoglobulin-like protein IGSFl
  • Figure 8 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ 3D NO: 21 (i.e SEQ 3D NO: 22) leukocyte immunoglobuiin receptor- like polypeptide Calso identified as "LIR ike * ') and human L-R-pbm36-2 prot ⁇ in .
  • SEQ 3D NO: 21 i.e SEQ 3D NO: 22
  • leukocyte immunoglobuiin receptor- like polypeptide C also identified as "LIR ike * '
  • human L-R-pbm36-2 prot ⁇ in .
  • a predicted approximately sixteen residue signal peptide is encoded from approximately residue 1 to residue 16 of both SEQ 3D NO: 19 and SEQ 3D NO: 22 (SEQ 3D NO: 25).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • SEQ ID NO: 27 is the peptide resulting when the predicted signal peptide is removed from SEQ 3D NO: 19.
  • SEQ 3D NO: 28 is the peptide resulting when the predicted signal peptide is removed from SEQ 3D NO: 22.
  • a predicted approximately twenty-three residue transmembrane peptide is encoded from approximately residue 135 to residue 157 of SEQ 3D NO: 19 (SEQ 3D NO: 26). This can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • One of skill in the art will recognize that the actual transmembrane region may be different than that predicted by the computer program.
  • LIR-like polypeptides of SEQ 3D NO: 19 and 22 were determined to have a region at residues 26 - 97 with characteristic motifs to the killer cell inhibitory receptor domain (SEQ 3D NO: 24).
  • 3D NO: 37 and 40 is an approximately 230-amino acid protein with a predicted molecular mass of approximately 26 kDa unglycosylated. Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, CA) further shows that both SEQ 3D NO: 37 and 40 and is homoiogous to P58 killer cell inhibitory receptor.
  • Figure 9 shows the GeneAtlas amino acid sequence alignment between the protein encoded by SEQ 3D NO: 36 and 39 (i.e SEQ 3D NO: 37 and 40, respectively) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human P58 killer cell inhibitory receptor protein, pdb Identification No. Ib6u, SEQ 3D NO: 46, indicating that the two sequences share 33% similarity over 103 amino acid residues and 19.4% identity over the same 103 amino acid residues.
  • SEQ 3D NO: 36 and 39 i.e SEQ 3D NO: 37 and 40, respectively
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like
  • pdb Identification No. Ib6u SEQ 3D NO: 46
  • a predicted approximately fifteen residue signal peptide is encoded from approximately residue 1 to residue 15 of both SEQ 3D NO: 37 and SEQ 3D NO: 40 (SEQ 3D NO: 43).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • a predicted approximately twenty-seven residue transmembrane peptide is encoded from approximately residue 116 to residue 143 of SEQ 3D NO: 37 and SEQ 3D NO: 40 (SEQ 3D NO: 44).
  • the transmembrane portion may be useful on its own. This can be confirmed by expression in m-irnmalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • One of skill in the art will recognize that the actual transmembrane region may be different than that predicted -by the computer program.
  • the leukocyte immunoglobuii rec ⁇ tOr-i ⁇ e,polypep. ⁇ de cf SEQ D NO: -50 is-an approximately 201 -amino acid protein with a predicted molecular mass of approximately 22 kDa unglycosylated.
  • Protein database searches with the BLASTX algorithm Altschul S.F. et al., J. Mol. Evol.36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol.21:403- 10 (1990), herein incorporated by reference
  • SEQ 3D NO: 50 is homologous to leukocyte immunoglobuiin receptors like CMRF35, NK cell inhibitory receptor, and human PIGR-2 receptor.
  • Figure 10 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ 3D NO: 49 (i.e. SEQ 3D NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human CMRF 35 protein (Jackson et al, (1992) Eur. J. Immunol., 22, 1157-1163) SEQ 3D NO: 60 , , indicating that the two sequences share 62% similarity over 217 amino acid residues and 48% identity over the same 217 amino acid residues.
  • Figure 11 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e.
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "I-TR-like”
  • human Natural Killer inhibitory receptor protein SEQ 3D NO: 61
  • Figure 12 shows the BLASTX amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e. SEQ ID NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and human PIGR-2 protein (Patent Application No. EP905237) SEQ 3D NO: 62, indicating that the two sequences share 58% siinilarity over 205 amino acid residues and 47% identity over the same 205 amino acid residues.
  • SEQ ID NO: 49 i.e. SEQ ID NO: 50
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like
  • human PIGR-2 protein Patent Application No. EP905237
  • Figure 13 shows the GeneAtlas amino acid sequence alignment between the protein encoded by SEQ ID NO: 49 (i.e SEQ 3D NO: 50) leukocyte immunoglobuiin receptor-like polypeptide (also identified as "LIR-like") and T cell receptor, pdb Identification No. Ib88, SEQ 3D NO: 59, indicating that the two sequences share 29.5% similarity over 112 amino acid residues and H.6%. identity over the same 112 amin' ⁇ acid residues.
  • SEQ ID NO: 49 i.e SEQ 3D NO: 50
  • leukocyte immunoglobuiin receptor-like polypeptide also identified as "LIR-like
  • a predicted approximately twenty-four residue transmembrane peptide is encoded from approximately residue 167 to residue 191 of SEQ 3D NO: 50 (SEQ 3D NO: 56).
  • the transmembrane portion may be useful on its own. This can be confirmed by expression in mammalian cells.
  • the transmembrane peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • One of skill in the art will recognize that the actual transmembrane region may be different than that predicted by the computer program.
  • SEQ 3D NO: 50 LIR-like polypeptide is expected to have two poly-Ig receptor domains.
  • Poly Immunoglobuiin receptor domain 3TD33PGDLTFTVTl-ENLTADDAGKYRCG3AT-LQEDG- ⁇ G-rI PDPFFQ designated as (SEQ 3D NO: 53) p-value of 4.504e-9, DM01688B 2 (identification number correlating to signature); located at residues 85-132 of SEQ 3D NO: 50.
  • a polypeptide (.SEQ ID NO: 66) was predicted to be encoded by SEQ 3D NO: 65 as set forth below.
  • the polypeptide was predicted using software programs called BLASTX, which selects a polypeptide based on a comparison of translated novel polynucleotide to known polypeptides.
  • the initial methionine ATG starts at position 35 of the nucleotide sequence and the putative stop codon, TGA, ends the coding region at position 850.
  • the leukocyte immunoglobuiin receptor-like polypeptide of SEQ 3D NO: 66 is an approximately 271 -amino acid protein with a predicted molecular mass of approximately 30.4 kDa unglycosylated.
  • Protein database searches with the BLASTP algorithm (Altschul S.F. et al., J. Mol. Evol. 36:290-300 (1993) and Altschul S.F. et al., J. Mol. Biol. 21:403-10 (1990), herein incorporated by reference) indicate that SEQ 3D NO: 66 is homologous to human platelet glycoprotein VI-2 and FcR-3I protein.
  • Figure 14 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ 3D NO: 65 (i.e.
  • SEQ ID NO: 66 leukocyte immunoglobuiin receptor-like polypeptide (also identified as "J R-like") and human platelet glycoprotein VI-2 protein (Ezumi et al, (2000) Biochem. Biophys. Res. Commun. 277, 27-36) SEQ 3D NO: 75, indicating that the two sequences share 50% similarity over residues 1-219 of SEQ 3D NO: 66, and 37% identity over the same residues 1-219 of SEQ 3D NO: 66.
  • Figure 15 shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ 3D NO: 65 (i.e. SEQ 3D NO: 66) leukocyte immunoglobuiin receptor-tike polypeptide (also identified as "LIR-like") and human FcR-3I protein (Patent Application No. WO9831806), SEQ 3D NO: 76, indicating that the two sequences share 51 % similarity over residues 27-262 of SEQ 3D NO: 66 and 37% identity over the same amino acid residues 27-262 of SEQ 3D NO: 66.
  • a predicted approximately sixteen-residue signal peptide is encoded from approximately residue 1 through residue 16 of SEQ 3D NO: 66 (SEQ 3D NO: 71).
  • the extracellular portion is useful on its own. This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.
  • the signal peptide region was ' . predicted using the Kyte-Doolittle hydrophobocity prediction algorithm -(J. Mol Biol. " ' -15 . " po 105-31 i 198 1. incorporated herein by reference; _ n .d also -.sirg'.h Neural Net ork S.gn'ai? VI. I -program ⁇ ' rcm Center- or Biological Sequence Analysis. The Technical University of Denmark).
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • a predicted approximately twenty four-residue transmembrane region is encoded from approximately residue 231 through residue 254 of SEQ 3D NO: 66 (SEQ 3D NO: 73).
  • the transmembrane portion may be useful on its own.
  • the transmembrane region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference).
  • Kyte-Doolittle hydrophobocity prediction algorithm J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference.
  • One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
  • SEQ 3D NO: 66 LIR-like polypeptide is expected to have three poly-Ig receptor domains.
  • GSLPKPSLSAWPSSWPANSNVTLRCWTPARGVSFN designated as (SEQ 3D NO: 68) p-value of 6.625e-10, PD01652A (identification number correlating to signature); located at residues 24 - 59 of SEQ 3D NO: 66.
  • GGI designated as (SEQ 3D NO: 69) p-value of 1.836e-9, PD01652B (identification number correlating to signature); located at residues 14 - 65 of SEQ 3D NO: 66.
  • PD01652B identification number sorrelatin-t to sianature-., 'oca.ed at residues Ml - 162-of SEQ-ID NO- 66.
  • EXAMPLE 6 A. Expression of SEO TD NO: 3. 6, 19, 22, 37, 40. 50. and 66 in cells
  • CHO cells or other suitable cell types are grown in DMEM (ATCC) and 10% fetal bovine serum (FBS) (Gibco) to 70% confluence. Prior to transfection the media is changed to DMEM and 0.5% FCS. Cells are transfected with cDNAs for SEQ 3D NO: 3, 6, 19, 22, 37, 4050, 66, or with pBGal vector by the FuGENE-6 transfection reagent (Boehringer). In summary, 4 ⁇ l of FuGENE-6 is diluted in 100 ⁇ l of DMEM and incubated for 5 minutes. Then, this is added to 1 ⁇ g of DNA and incubated for 15 minutes before adding it to a 35 mm dish of CHO cells.
  • FBS fetal bovine serum
  • the CHO cells are incubated at 37°C with 5% C0 2 . After 24 hours, media and cell lysates are collected, centrifuged and dialyzed against assay buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mM CaCl 2 and MgCl 2 .
  • Human cDNA libraries are used as sources of expressed genes from tissues of interest (adult bladder, adult brain, adult heart, adult kidney, adult lymph node, adult liver, adult lung, adult ovary, adult placenta, adult rectum, adult spleen, adult testis, bone marrow, thymus, thyroid gland, fetal kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).
  • Gene- specific primers are used to amplify portions of the SEQ ID NO: 1-2, 4-5, 7, 16-18, 20- 21, 23, 35-36, 38-39, 41, 47-49, 51, 63-65, or 67 sequences from the samples.
  • Amplified products are separated on an agarose gel, transferred and chemically linked to a nylon filter.
  • the filter is then hybridized with aradioactively labeled ( 33 P-dCTP) double- stranded probe generated from SEQ 3D NO: 1-2, 4-5, 7, 16-18, 20-21, 23, 35-36, 38-39, 41, 47-49. 51, 63-65. or 67 using a Klenow polymerase, random-prime method.
  • the filters are washed (high stringency) and used to expose a phosphorimaging scr ⁇ n for several hours. Bands indicate the presenc ⁇ of cDNA inc-iudmg SEQ ID NO: 1-2. -5.7. 16-1S. 20-21. 23. 35-36. 3S-39. 41, J-7-49. 51. 6X65. or 67 sequences in ⁇ spee-ific - library , and thus mRNA expression in the corresponding celi type or tissue.

Abstract

L'invention concerne de nouveaux polynucléotides et des polypeptides codés par lesdits polynucléotides ainsi que leurs mutants ou variants qui correspondent à un nouveau polypeptide humain sécrété semblable à un récepteur de leucocyte de type immunoglobuline. Ces polynucléotides renferment des séquences d'acide nucléique isolées de banques d'ADNc préparées à partir d'une banque d'ADNc préparée à partir d'un ARNm de leucocyte humain (Laboratoires GIBCO) (SEQ ID NO:1, SEQ ID NO:16); d'un ARNm du cerveau d'un nourrisson (Columbia University) (SEQ ID NO: 35); d'un ARNm de glande mammaire humaine (Invitrogène) (SEQ ID NO: 47); et d'un ARNm de moelle osseuse (Clontech) (SEQ ID NO: 63). D'autres aspects de l'invention concernent des vecteurs contenant des procédés de production de polypeptides humains sécrétés semblables à LIR, ainsi que des anticorps propres à ces polypeptides.
PCT/US2001/049435 2000-12-29 2001-12-28 Procedes et materiaux concernant des polypeptides et polynucleotides semblables a des recepteurs de leucocytes de type immunoglobuline (lir) WO2002066600A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050704A1 (fr) * 2002-11-29 2004-06-17 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Agents diagnostiques et therapeutiques
EP1628991A2 (fr) * 2003-04-14 2006-03-01 Nuvelo, Inc. Procedes de therapie et de diagnostic reposant sur le ciblage de cellules qui expriment une proteine du type recepteur de type immoglobuline de cellule tueuse.
EP1712563A1 (fr) * 2004-01-27 2006-10-18 Medical and Biological Laboratories Co., Ltd. Methode pour isoler des monocytes
WO2017009712A1 (fr) * 2015-07-13 2017-01-19 Compugen Ltd. Compositions de hide1 et méthodes associées

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050704A1 (fr) * 2002-11-29 2004-06-17 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Agents diagnostiques et therapeutiques
EP1628991A2 (fr) * 2003-04-14 2006-03-01 Nuvelo, Inc. Procedes de therapie et de diagnostic reposant sur le ciblage de cellules qui expriment une proteine du type recepteur de type immoglobuline de cellule tueuse.
EP1628991A4 (fr) * 2003-04-14 2008-07-23 Nuvelo Inc Procedes de therapie et de diagnostic reposant sur le ciblage de cellules qui expriment une proteine du type recepteur de type immoglobuline de cellule tueuse.
EP1712563A1 (fr) * 2004-01-27 2006-10-18 Medical and Biological Laboratories Co., Ltd. Methode pour isoler des monocytes
JPWO2005070964A1 (ja) * 2004-01-27 2008-01-17 株式会社医学生物学研究所 単球の単離方法
EP1712563A4 (fr) * 2004-01-27 2008-05-28 Medical & Biol Lab Co Ltd Methode pour isoler des monocytes
WO2017009712A1 (fr) * 2015-07-13 2017-01-19 Compugen Ltd. Compositions de hide1 et méthodes associées
EP3971211A1 (fr) * 2015-07-13 2022-03-23 Compugen Ltd. Compositions hide1 et procédés

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