WO2002074906A2 - Proteines de mammiferes lp et reactifs associes - Google Patents

Proteines de mammiferes lp et reactifs associes Download PDF

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Publication number
WO2002074906A2
WO2002074906A2 PCT/US2002/005093 US0205093W WO02074906A2 WO 2002074906 A2 WO2002074906 A2 WO 2002074906A2 US 0205093 W US0205093 W US 0205093W WO 02074906 A2 WO02074906 A2 WO 02074906A2
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Prior art keywords
seq
coding portion
primate
polypeptide
mature coding
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PCT/US2002/005093
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English (en)
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WO2002074906A3 (fr
Inventor
Bernard Yaovi Amegadzie
Margret Barbara Basinski
Dayue Chen
Chongxi Huang
Gerald Patrick Keleher
Douglas Raymond Perkins
Paul Robert Junior Rosteck
Scott William Rowlinson
Patanjali Raghavacharya Sankhavaram
Eugene Thomas Seno
Eric Wen Su
Yu Zhi
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Eli Lilly And Company
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Priority to EP02719036A priority Critical patent/EP1434783A4/fr
Priority to US10/480,172 priority patent/US20040152885A1/en
Priority to AU2002250143A priority patent/AU2002250143A1/en
Publication of WO2002074906A2 publication Critical patent/WO2002074906A2/fr
Publication of WO2002074906A3 publication Critical patent/WO2002074906A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) exhibiting identity over a length of at least about 12 contiguous amino acids to a corresponding sequence of SEQ ID NO: Y; a natural sequence LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) of SEQ ID NO: Y or Table 1, 2, 3, 4, 5, 6, 7 or.8; a fusion protein comprising LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) sequence.
  • the wash conditions are at 55° C and/or 300 mM salt; 60° C and/or 150 mM salt; the identity is over a stretch is at least 55 or 75 nucleotides.
  • the invention provides a method of modulating physiology or development of a cell or tissue culture cells comprising introducing into such cell an agonist or antagonist of an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346).
  • an LP LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346
  • LP318a(cl6hDGL) exhibits amino acid sequence similarity to a rodent (e.g., mouse) protein designated mDGLl.
  • the gene (mdgll) encoding the mDGLl protein is located on a small segment of mouse chromosome 16, which is highly homologous to a segment of human chromosome 22.
  • the mouse and human chromosomal regions on, respectively, chromosomes 16 and 22 may represent ortho- or paralog segments (particularly, the C22ql 1 region of human chromosome 22; see, e. g., Botta, et al, 1997 Mammalian Genome.
  • the CTAFS, VCFS, DiGeorge, Cat Eye, and CATCH 22 syndromes are often associated with anomalous developmental characteristics of the cardiovascular and/or nervous systems, and/or anomalous development of the face and head (see, e.g. Momma, et al, 1999 Ped Cardio 20: 97-102; Hong, R, 1998, Seminars in Hematology, 35: 282-290). It has been suggested that the DiGeorge syndrome is associated with a basic embryological defect (e.g, inadequate development of the facial neural crest tissues) (see, e.g. Hong, 1998).
  • CATCH 22 syndromes For example, it has recently been demonstrated that nodal signaling is required for the proper development of laterally asymmetric structures in the brain (e.g, in the dorsal diencephalon, specifically, habenular nucleii and pineal structures) (see, e.g. Concha, et al, 2000 Neuron 28: 399-409; and Liang, et al, 2000 Development 127:5101- 112).
  • genes involved in the nodal pathway such as, e.g, squint, cyclops, lefty, antivin, and pitx2 are also responsible for localizing components of the dorsal diencephalon to the left side of the brain in vertebrates.
  • LP318a(cl6hDGL)'s homology to proteins involved in blood coagulation e.g, plasma kallikrein, coagulation factor XI, and plasminogen
  • apple domains which have been shown to be involved in binding other members of the coagulation cascade (such as, e.g, kininogen, and factor Xlla) suggest that LP318a(cl6hDGL) may also be participate in the blood coagulation system.
  • LP318a(cl6hDGL) may also participate in inflammatory processes.
  • One is based on the observation that after injury there is typically a simultaneous activation of the innate immune response and the coagulation system.
  • the second piece of evidence supporting this view is the expression data for LP318a(cl6hDGL), which is primarily in IL-5 activated eosinophils, and eosinophils exhibiting hyper-eosinophilia, and in asthma patients Thus, supporting the linkage between the immune system and the coagulation system.
  • LP318b(cl6hDGL) Additional interesting sections of LP318b(cl6hDGL) are the discovered portions of LP318b(cl6hDGL) from Asn-8 to Ser-17; Ser-33 to Pro-40; His-67 to Glu-80; Phe-85 to Glu-97; Leu-102 to Ala-109; Asp-110 to Thr-119; Lys-123 to Ser-130; and Gly-137 to His- 145. These fragments were discovered based on analysis of antigenicity plots.
  • coil structures are Ile-14 to Pro-16; Asp-21 to Asp-27; Glu-37 to Arg-38; Ala-46 to Thr-50; Arg-63 to Asn-68; Leu-85 to Lys-88; Lys-100 to Thr- 104; Ser-123 to Asp-127; Asn-132 to Ser-135; Asp-140 to Glu-142; Lys-167 to Gln-168; Lys- 212 to Thr-222; Thr-246 to Leu-255; Tyr-264 to Ala-268; Pro-275 to Gly-278; Arg-308 to Asp-317; Thr-343 to Asp-347; Val-361 to Thr-366; Gln-381 to Pro-390; Met-398 to Ser-400; and Ile-409 to Lys-413.
  • An individual YWTD island is predicted to fold into a higher order structure designated a six-bladed beta-propeller, which is composed of six similar subunits (see, e.g., Murzin, et al. 1996 J. Mol. Biol. 247:536-540).
  • Each beta sheet of the beta propeller has an almost identical tertiary structure (but see below suggesting that the blade positions may be more conserved between than within propellers) and the beta sheets are radially arranged about a pseudosymmetrical axis ultimately yielding a compact higher order structure that is cylindrical or toroidal-like in shape and that brings neighboring modules (e.g , EGF-like domains) into close proximity.
  • an LP288 variant designed to block its internabzation e.g, but mutating or abobshing an Lxxy or tSxV motif in its cytoplasmic portion
  • an LP288 variant designed to block its internabzation would be restricted in its signabng options and vice versa.
  • LP288 may form intraceUular complexes with proteins known to be involved in binding intraceUular motifs (e.g, such as PZD domains) of members of this signabng pathway (such as, e.g, vertebrate homologs of the binding partners of Frizzled proteins (e.g, disheveled (dsh), prickled, inturned, fuzzy, and multiple-wing-hair proteins) since it has also been shown that the terminal S/TxV motif of members of the frizzled gene family interact directly with PDZ domains found vertebrate intracellular molecules.
  • intraceUular motifs e.g, such as PZD domains
  • Frizzled proteins e.g, disheveled (dsh), prickled, inturned, fuzzy, and multiple-wing-hair proteins
  • LP288 YWTD repeats Nos. 1-6
  • LP288 & LIPIDS Evidence suggests that LRPs have the abibty to mediate ceUular uptake of bpophibc molecules, such as, for example, apoE-containing and remnant-bke bpoproteins.
  • bpophibc molecules such as, for example, apoE-containing and remnant-bke bpoproteins.
  • a non-bmiting example of such a technique to employ are the methods described in Sugiyama, et al. 2000 Biochemistry 39:15817-15825, which is incorporated herein by reference for the assay techniques described therein.
  • LP289 sequence (SEQ ID NO: 7) is expressed in the foUowing number of LIFESEQ GOLDTM database tissue and cDNA bbraries: Digestive System 1/151; Genitaba, Male 2/118; Germ CeUs 1/5; Hemic and Immune System 3/166; Liver 1 /34; Respiratory System 1/95; Sense Organs 1/10; and Nervous System 17/221.
  • a predicted mature LP289 sequence is as follows:
  • a further alternate predicted mature LP289 sequence is as follows: QSLEFNSPADNYTVCEGDNATLSCFIDEHVTRVAWLNRSNILYAGNDR TSDPRVRLLINTPEEFSILITEV GLGDEGLYTCSFQTRHQPYTTQVYLIVHVPARIV ISSPVTV EGGNVNLLCLAVGRPEPTVTWRQLRDGFT SEGEILEISDIQRGQAGEYECVTHNGV SAPDSRRVLVTVNYPPTITDVTSARTALGRAALLRCEAMAVPPA DFQ YKDDRLLSSGTAEGL1WQTERTRS LLFANVSARHYGNYTCRAANRLGASSASMRLLRPGSLEN* An Alternate P289 Mature Sequence (292aa) :
  • a Variant LP289 ( 291aa ) : The structural features of an LP289 variant represent a soluble counterpart to a non-soluble LP289 version encompassed herein, wherein a difference of a soluble LP289 is at the LP289 C-terminus in which a GPI- anchored binding site is absent.
  • Bio agents are those that can act on a ceU, organ or organism, including, but not bmited to, pharmaceutical agents and gene debvery agents.
  • Numerous targetable debvery vehicles are known, including bposomes, ghost ceUs and polypeptide matrices (see, for example, Huang et al, Proc. Natl Acad. Sci. USA, 84, 7851-7855, 1987; Kreuter, Infection 19 Supp. 4, 224-228, 1991, or Michel et al. Research in Virology, 144, 263-267, 1993).
  • Such targeting agents are bound covalently or noncovalently to a biological agent or a vehicle for debvering biological agents that can act on a cell, organ, or organism, including, but not bmited to, pharmaceutical agents and gene debvery agents.
  • biological agents that can be usefully targeted to, e.g, the bmbic system include, neuro transmitter biosynthetic enzymes (such as tyrosine hydroxylase), neurotransmitter transporters (such as the GABA transporter), neurotransmitter receptors (such as type la, lb, II or III dopamine receptors, a and (3 adrenergic receptors and 5-HT receptors), neurotrophic and growth factors (such as NGF, BDNF, NT-3, NT-4, NT-5, TGF13, basic FGF and GDNF), neurotrophic factor receptors, protein kinases (such as MAP kinases and protein kinase C) and protein phosphatases.
  • neuro transmitter biosynthetic enzymes
  • ceUs are examined within 24 hours to determine the presence or absence of neurite growth, a weU-differentiated neural morphology, cyto-architecture, and arborization. CeUs can also be examined for these criteria when they are pre-treated with LP289 antibody, or soluble LP289 to determine if the length and/or number of neurites are significantly modified.
  • LP319b (SEQ ID NO: 14)
  • the underlined portion is a predicted signal sequence (Met-1 to Ser-30)
  • a predicted SP cleavage site is between
  • the added sequence is indicated by underlining.
  • IgLONs IgLONs
  • those known in the art e.g., assay methods to determine binding activities of suspected IgLONs such as neurite outgrowth, homo- or heterophibc binding, axonal pathfinding, opiod-bke binding, e.g, the assays described in, e.g, Flachisuka, et al. 1996 Neurochem. Int. 28:373-379 such as which is incorporated by reference herein for such assay teachings
  • one skiUed in the art would be able to test LP319a or LP319b for IgLON-bke activities without undue experimentation (e.g, using common assay techniques and commerciaUy available reagents).
  • LP319a Additional interesting sections of LP319a are the discovered portions of LP319a from about Arg-8 to about Val-23, from about Asn-41 to about Trp-50, from about Leu-51 to about Asp-62 (LNRSNILYAGND); from about Val-70 to about Val-84; from aboutVal-84 to about Phe-99; from about Pro-117 to about Glu-131; from about Gly- 132 to about Arg-144; from about Trp-151 to about lle-164; from about Glu-166 to about Glu-177; from about Thr-198 to about Ala-211, from about Arg-212 to about Pro-225; from about Trp-226 to about Ile-236.
  • ion flow such as, e.g, calcium, sodium, potassium, or chloride ions
  • modulation of an ionic flow through a membrane and/or a pore, wherein said membrane is on a epithebal ceU, modulation of an ionic flow on a membrane and/or a pore of a microbe; mediation of innate immunity; modulation of chloride ion flow; creation of an ionic pore in a membrane; create an ionic pore in a membrane, wherein said membrane is of an epithebum, wherein said epithebum is a broncho epithebum; wherein said epithebum is in a lung; creating
  • the invention further provides a method for detecting an inflammatory pathology in a subject by determining the amount of LP321 in a biological sample from the subject and comparing that amount to an amount present in a normal subject.
  • a method can be used to determine the presence of an inflammatory pathology such as an inflammatory bowel disease, pancreatitis, a maUgnant condition, an infection, or an ileititic condition.
  • the invention also provides a method for treating an inflammatory sydrome, condition, state or disease in a subject by administering an LP321, an LP321 variant, an LP321 pharmaceutical composition, an LP321 binding agent, or an LP321 hetero- or an LP321 homomultimer to a subject having such a condition, state or disease.
  • a cryptdin such as, for example, cyrptdin 1, cyrptdin 2, cyrptdin 3, cyrptdin 4, cyrptdin 5, cyrptdin 6, or any combination thereof; or with a defensin, such as, for example, HD-1, HD- 2, HD-3, HD-4, HD-5, HD-6, HNP-1 , HNP-2, HNP-3 (or any combination thereof), or a thionin; or any combination thereof.
  • a cryptdin such as, for example, cyrptdin 1, cyrptdin 2, cyrptdin 3, cyrptdin 4, cyrptdin 5, cyrptdin 6, or any combination thereof
  • a defensin such as, for example, HD-1, HD- 2, HD-3, HD-4, HD-5, HD-6, HNP-1 , HNP-2, HNP-3 (or any combination thereof), or a thionin; or
  • an antimicrobial activity of a cryptdin, or a cryptdin-bke peptide can be determined against various pathogens.
  • various microorganisms can be grown to an appropriate concentration, mixed with an appropriate medium (such as, for example, an agarose-trypticase soy mediums), and contacted with an LP321 or a cryptdin, or a defensin to assess an antimicrobial activity.
  • An antimicrobial activity is apparent, for example, from clear zones that typicaUy surround a cryptdin or cryptdin-Uke composition (e.g, such as an LP321) that is placed in an agar for a diffusion assay.
  • an LP321 or LP321 variant may be more active than rabbit NP-1 at a concentration above 100 ug/ml or more active than NP-1 when compared at 100 ug/ml and 300 ug/ml. Higher concentrations may be more effective than the same concentration of NP-1 at inhibiting the growth of S. aureus and of wild type and mutant strains of S. typhimurium. An inhibition of S. aureus is interpreted as indicating that an LP321 or LP321 variant peptide inhibits bacterial growth.
  • LP317 is a novel primate (e.g, human) polypeptide (SEQ ID NO: 18) that exhibits similarity to a defensin family of proteins. SpecificaUy, LP317 is a novel member of the gamma-thionin family of proteins. These defensins exhibit remarkable structural sir la ⁇ ty to sco ⁇ ion neurotoxins and insect defensins, which are generaUy recognized a having antibiotic, antifungal, antitumor, antineoplastic and antiviral activities.
  • Some peptides known to belong to the defensin family include, e.g.: Rabbit defensins and corticostatins: CS-I (NP-3A), CS-II (NP-3B), CS-III, (MCP-1), CS-IV (MCP- 2), NP-4, and NP-5; Guinea-pig neutrophil defensin (GPNP); Human neutrophil defensins 1 to 4 and intestinal defensins 5 and 6; Mouse smaU bowel cryptdins 1 to 5 and; Rat NP-1 to
  • a predicted mature LP317 sequence is as follows: GTTTT VAEARVCMGKSQHHSFPCISDRLCSNECVKEDGG TAGYCHLRYCRCQKAC
  • Certain cationic peptide variants are acetylated at the N-terminal amino acid, and/or amidated (or esterified) at the C-terminal amino acid.
  • a cationic peptide variant encompassed by the invention can be modified by incorporation of homoserine/homoserine lactone at the C-terminal amino acid.
  • an LP cationic peptide variant encompassed herein exhibits at least 50%, and preferably, greater than 60, 70, 80, 85, 87, or 90% of an activity of a corresponding naturaUy occurring LP peptide of the invention as determined by any art known assay or an assay described or referenced herein.
  • LP283 and its spbce variants are novel primate (e.g, human) polypeptide (SEQ ID NO: 20, 21, 22, & 23) members of the epidermal growth factor (EGF) superfamily.
  • EGF epidermal growth factor
  • the EGF superfamily comprises a diverse group of proteins that function as secreted signabng molecules, growth factors, and components of the extraceUular matrix involved in, for example, ceU-ceU, and/or ceU-matrix adhesion.
  • fibropeUins are secreted glycoproteins that form physical associations to provide a protein substratum of the apical lamina, a component of the hyabne layer that surrounds sea urchin embryos (DelgadiUo-Reynoso et al. 1989 J. Mol. Evol. 29:314-327; Burke, et al. 1998 CeU Adhes Commun 5:97-108).
  • LP283 nucleic acid sequence (SEQ ID NO: 19) is expressed in the foUowing LIFESEQ GOLDTM database tissue and cDNA bbraries: Embryonic Structures 1/23; Endocrine System 3/63; Genitaba, Female 2/113; Hemic and Immune System 3/166; Musculoskeletal System 1/50; Nervous System 4/221; Sense Organs 1 /10; and Urinary Tract 2/66. Sequence encoding LP283 and its spbce variants (LP344, LP345, & LP346) has been locabzed to human chromosome region 6p21.1-21.33.
  • LP283 splice variants are LP283 splice variants.
  • LP283 splice variant also known as LP344 (SEQ ID NO 21)
  • Exon 7 normally encodes the LP283 portion from about Gly-238 to about Asn-253 (GERRLEQHIPTQA SN).
  • LP283 splice variants are LP283 splice variants. Another such LP variant is listed below Applicants discovered that this LP283 variant (also known as LP345, (SEQ ID NO: 22)) is the result of loss of processing of exon 7 and exon 16 from genomic LP283 sequence (see below). Exon 7 normally encodes the LP283 portion from about Gly-238 to about Asn-253 (GERRLEQHIPTQAVSN). Exon 16 normally encodes the LP283 portion from about Ser-654 to about Ala-706
  • LP283 sequence (SCPQGTYYHGQTEQCVPCPAGTFQEREGQLSCDLCPGSDAHGPLGATNVTTCA)
  • the alternate predicted mature LP283 sequence (LP345,) is as follows. GSGRVPGLCLLVLLVHARAAQYSKAAQDVDECVEGTDNCHIDAICQNTPRSYKCICKSGYTGDGKHCKDVD ECEREDNAGCVHDCVNIPGNYRCTCYDGFHLAHDGHNCLDVDECAEGNGGCQQSCV M GSYECHCREGFFL SDNQHTCIQRPEEGMNCMNKNHGCAHICRETPKGGIACECRPGFELTKNQRDCKLTCNYGNGGCQHTCDDTE QGPRCGCHIKFVLHTDGKTCIETCAV NGGCDSKCHDAATGVHCTCPVGFMLQPDRKTCKDIDECRLN GGC DHICRNTVGSFECSCKKGYKLLINERNCQDIDECSFDRTCDHICVNTPGSFQCLCHRG
  • Exon 7 (see above) and exon 16 are missing in LP346.
  • Exon 16 in LP344 is missing in LP345, .
  • particularly interesting LP 283 segments are LP secondary structures (e.g., such as a heUx, a strand, or a coil).
  • Particularly interesting LP345, coil structures are the foUowing: from about Met-1 to about Pro-7; from about Glu-35 to about Cys-40; from about Asn-48 to about Ser-52; from about Ser-59 to about Asp-70; from about Arg-76 to about Cys-82; from about Asn-88 to about Asn-92; from about Tyr-98 to about Phe-101; from about H ⁇ s-105 to about Cys-110; from about Glu-118 to about Cys-123; from about Met-131 to about Ser-133; from about Cys-138 to about Gly-141; from about Ser-145 to about His-149; from about Gln-153 to about Asn-160; from about Asn-163 to about Gly-167; from about Glu-174 to about Gly- 179
  • Particularly interesting heUx structures are from about His-17 to about Ala-27; from about Leu-554 to about Glu-555; from about Arg-573 to about Leu-580; from about Leu-574 to about Lys-589; from about Tyr-866 to about Arg-875; from about Gln-886 to about Phe-899; and from about Ser-932 to about Leu-937.
  • Some non- limiting examples of functions an LP283, LP283 variant, or an LP283 binding agent is Ukely to participate in are, for example, those such as: a ceU adhesion; ceU-matrix adhesion; neural development, such as, e.g., brain development, sense organ development, such as, for example, the eye; Umb development; protein-protein interactions; protein-extraceUular matrix interactions; chemotaxis; metaUoproteinase activity, added hair growth/hair replacement, cause breast cancer and embryogenesis.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • Total NT Seq refers to the total number of nucleotides in a polynucleotide sequence identified by an "LP No.”
  • the nucleotide position of SEQ ID NO: X of the putative start codon (methionine) is identified as "5' NT of Start Codon.”
  • Sirmlarly, the nucleotide position of SEQ ID NO: X of a predicted signal sequence of an LP protein or polypeptide is identified as "5' NT of First AA of Signal Pep.”
  • Such a polypeptide includes, e.g., naturaUy occurring polypeptides that are isolated, recombinantiy produced polypeptides, syntheticaUy produced polypeptides, or polypeptides produced by any combination of these methods Means for preparing such polypeptides are weU understood in the art.
  • An LP polypeptide (or fragment thereof) may be in the form of, a mature polypeptide, a secreted protein (including the mature form), or it may be a fragment thereof, or it may be a part of a larger polypeptide or protein, such as, e g., a fusion protein.
  • an LP polypeptide e.g , additional amino acid sequence that contains, e.g., secretory or leader sequences, pro- sequences, sequences that aid in purification, such as, e.g., multiple histidine residues, or an additional sequence for stabihty during recombinant production.
  • additional amino acid sequence that contains, e.g., secretory or leader sequences, pro- sequences, sequences that aid in purification, such as, e.g., multiple histidine residues, or an additional sequence for stabihty during recombinant production.
  • An LP polypeptide (or fragment thereof) is preferably provided in an isolated or recombinant form, or it may be preferably substantially purified.
  • a recombinandy produced version of an LP polypeptide of the invention, including a secreted polypeptide, can be substantiaUy purified using techniques described herein or otherwise known in the art, such as, e.g., the single-step purification method (Smith and Johnson (1988) Gene 67(1):31- 40).
  • An LP polypeptide (or fragment thereof) can also be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, e.g., using an antibody of the invention raised against a secreted protein.
  • the polynucleotide can contain the nucleotide sequence of the fuU length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as weU as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • An "LP polynucleotide” also encompasses, e.g., those polynucleotides that stably hybridize, under stringent hybridization conditions to an LP sequence of a table herein, or to a sequence contained in SEQ ID NO:X.
  • altered nucleic acid sequences encoding LP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as LP or a polypeptide with at least one functional characteristic of LP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oUgonucleotide probe of the polynucleotide encoding LP, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding LP.
  • substantial similarity exists when the segments wiU hybridize under selective hybridization conditions, to a strand, or its complement, typicaUy using a sequence derived from SEQ ID X.
  • selective hybridization will occur when there is at least about 55% similarity over a stretch of at least about 30 nucleotides, preferably at least about 65% over a stretch of at least about 25 nucleotides, more preferably at least about 75%, and most preferably at least about 90% over about 20 nucleotides. See Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • nucleic acid sequences of polypeptides are substantiaUy identical is that the polypeptide encoded by the first nucleic acid is immunologicaUy cross reactive with the polypeptide encoded by the second nucleic acid.
  • Another indication that two nucleic acid sequences are substantiaUy identical is that the two molecules hybridize to each other under stringent conditions. "Homologous" polynucleotide sequences, when compared, exhibit significant similarity (e.g., sequence identity at the nucleotide level).
  • standards for determining homology between nucleic acid molecules use art known techniques which examine, e.g., the extent of structural similarity or sequence identity between polynucleotide sequences; and/or that determine a phylogenetic relationship (e.g., whether compared sequences are orthologs or paralogs); and/or that are based on the abiUty of sequences to form a hybridization complex.
  • Hybridization conditions are described in detail herein.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions.
  • Specific hybridization is an indication that two nucleic acid sequences share a high degree of similarity and/or identity. Specific hybridization complexes form under permissive anneaUng conditions and remain hybridized after "washing.” Washing is particularly important in determining the stringency of the hybridization process, typicaUy, with more stringent conditions aUowing less non-specific binding (e.g., binding between polynucleotide sequences that demonstrate less sequence identity or similarity). Permissive conditions for anneaUng of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve a desired stringency, and therefore, a particular hybridization specificity.
  • Stringent conditions when referring to homology or substantial similarity and/or identity in the hybridization context, wiU be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typicaUy those controUed in hybridization reactions.
  • Stringent temperature conditions wiU usuaUy include temperatures in excess of about 30°C, more usuaUy in excess of about 37°C, typicaUy in excess of about 40°C, characteristicaUy in excess of about 42°C, routinely in excess of about 45°C, usuaUy in excess of about 47°C, preferably in excess of about 50°C, more typicaUy in excess of about 55°C, characteristicaUy in excess of about 60°C, preferably in excess of about 65°C, and more preferably in excess of about 70°C.
  • Stringent salt conditions wiU ordinarily be less than about 500 mM, usuaUy less than about 450 mM, even more usuaUy less than about 400 mM, more usuaUy less than about 350 mM, even more usuaUy less than about 300 mM, typicaUy less than about 250 mM, even more typicaUy less than about 200 mM, preferably less than about 100 mM, and more preferably less than about 80 mM, even down to less than about 20 mM.
  • the term "about” includes, e.g., a particularly recited molarity (e.g., 400 mM), and/or a molarity that is greater or lesser than that of the stated molarity by, e.g., three, five, seven, nine, eleven or fifteen miUimolar (e.g., 389 mM or 415 mM). It is to be remembered that the combination of parameters is more important than the measure of any single parameter (see, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370).
  • a nucleic acid probe that binds to a target nucleic acid under stringent conditions to form a stable hybridization complex is said to be specific for said target nucleic acid.
  • hybridization under stringent conditions should give a signal of at least 2-fold over background, more preferably a signal of at least 3 to 5-fold over background or more.
  • a hybridization probe is more than 11 nucleotides in length and is sufficientiy identical (or complementary) to the sequence of the target nucleic acid (over the region determined by the sequence of the probe) to bind the target under stringent hybridization conditions to form a detectable stable hybridization complex.
  • hybridization complex refers to a complex formed between two nucleic acid molecules by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or gt analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobuized on a sohd support (such as, e.g., without kmitation, paper, plastic, a membrane, a filter, a chip, a pin, glass, or any other appropriate substrate to which ceUs or their nucleic acids can be complexed with either covalendy or non-covalentiy).
  • a sohd support such as, e.g., without kmitation, paper, plastic, a membrane, a filter, a chip, a pin, glass, or any other appropriate substrate to which ceUs or their nucleic acids can be complexed with either covalendy or non-covalentiy.
  • a non- Umiting example of a high stringency condition of the invention comprises including a wash condition of 68°C in the presence of about 0.2X SSC and about 0.1% SDS, for 1 hour.
  • temperatures of about 67°C, 63°C, 61°C, 59°C, 57°C, 53°C, 51°C, 49°C, 47°C, 43°C, or 41 °C may be used.
  • SSC concentration may be varied from about 0 1 to 2.0X SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block nonspecific hybridization
  • Such blocking reagents include, for instance, sheared, and denatured salmon sperm DNA at about 100-200 ug/ml.
  • Organic solvent such as, e.g , formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for a RNA:DNA hybridization.
  • Another non-bmiting example of a stringent hybridization condition comprises, e.g., an overmght incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodmm citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, foUowed by washing the filters in O.lx SSC at about 65°C
  • nucleic acid molecules that hybridize to an LP polynucleotide sequence at lower stringency hybridization conditions.
  • washes are performed foUowing stringent hybridization at higher salt concentrations (e.g.
  • blocking reagents include, e.g., Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commerciaUy avaUable proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of a hybridization conditions described herein.
  • an "isolated" nucleic acid is a nucleic acid molecule or a polynucleotide sequence (e.g., an RNA, DNA, cDNA, genomic DNA, or a mixed polymer) which is substantiaUy separated from other biologic components that naturally accompany a native sequence (e.g., proteins and flanking genomic sequences from the originating species).
  • the isolated LP sequence is free of association with components that can interfere with diagnostic or therapeutic uses for the sequence including, e.g., enzymes, hormones, and other proteinaceous or non-proteinaceous agents.
  • the term embraces a polynucleotide sequence removed from its naturaUy occurring environment.
  • an isolated polynucleotide sequence could comprise part of a vector or a composition of matter, or could be contained within a ceU, and stiU be "isolated" because the vector, composition of matter, or ceU is not the original environment of the polynucleotide sequence.
  • the term encompasses recombinant or cloned DNA isolates, chemicaUy synthesized analogs, or analogs biologicaUy synthesized using heterologous systems.
  • the term includes both double- stranded and single-stranded embodiments. If single-stranded, the polynucleotide sequence may be either the "sense" or the "antisense" strand.
  • a substantiaUy pure molecule includes isolated forms of the molecule.
  • An isolated nucleic acid molecule wiU usuaUy contain homogeneous nucleic acid molecules, but, in some embodiments, it wiU contain nucleic acid molecules having minor sequence heterogeneity. TypicaUy, this heterogeneity is found at the polymer ends or portions of the LP sequence that are not critical to a desired biological function or activity.
  • isolated does not refer to genomic or cDNA Ubraries, whole ceU total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole ceU genomic DNA preparations, or other compositions where the art demonstrates no distinguishing features of a LP polynucleotide sequence of the present invention.
  • the present invention encompasses a mature LP protein, including a polypeptide or protein that is capable of being directed to the endoplasmic reticulum (ER), a secretory vesicle, a ceUular compartment, or an extraceUular space typicaUy, e.g., as a result of a signal sequence, however, a protein released into an extraceUular space without necessarily having a signal sequence is also encompassed.
  • ER endoplasmic reticulum
  • a secretory vesicle e.g., as a result of a signal sequence
  • a protein released into an extraceUular space without necessarily having a signal sequence is also encompassed.
  • GeneraUy the polypeptide undergoes processing, e.g., cleavage of a signal sequence, modification, folding, etc., resulting in a mature form (see, e.g., Alberts, et al. (1994) Molecular Biology of The CeU, Garland Pub
  • the invention also embraces polypeptides that exhibit similar structure to an LP polypeptide (e.g., one that interacts with an LP protein specific binding composition).
  • binding compositions e.g., antibodies, typicaUy bind an LP protein with high affinity, e.g., at least about 100 nM; usuaUy, better than about 30 nM; preferably, better than about 10 nM; and more preferably, at better than about 3 nM.
  • An LP polypeptide can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques that are weU known in the art. Such modifications are weU described in basic texts and in more detailed monographs, as weU as in a voluminous research Uterature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • substantially pure refers to LP nucleic acid or LP protein or polypeptide that are removed from their natural environment and are isolated and/or separated from other contaminating proteins, nucleic acids, and other biologicals. Purity may be assayed by standard methods, and wiU ordinarily be at least about 50% pure, more ordinarily at least about 60% pure, generaUy at least about 70% pure, more generaUy at least about 80% pure, often at least about 85% pure, more often at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in most preferred embodiments, at least 99% pure. Similar concepts apply, e.g., to LP antibodies or nucleic acids of the invention.
  • the temperature at use is greater than about 18° C and more usually greater than about 22° C.
  • the temperature wiU usuaUy be about room temperature or warmer, but less than the denaturation temperature of components in the assay.
  • the temperature wiU usuaUy be body temperature, typicaUy about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the size and structure of the polypeptide should generaUy be in a substantiaUy stable state, and usuaUy not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubiUty, or associated with Upids or detergents in a manner which approximates natural Upid bilayer interactions.
  • the solvent wiU have a neutral pH, typicaUy between about 5 and 10, and preferably about 7.5.
  • a detergent wiU be added, typicaUy a mild non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3-[3-cholamidopropyl)- dimethylammonio]-l -propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • the signal sequence identified by the above analysis may not necessarily predict a naturaUy occurring signal sequence.
  • a naturally occurring signal sequence may be further upstream from a predicted signal sequence.
  • a predicted signal sequence will be capable of directing the secreted protein to the ER.
  • the present invention encompasses a mature LP polypeptide or protein produced by expression of a polynucleotide sequence Usted in a Table herein or an LP polynucleotide sequence of SEQ ID NO: X.
  • These LP polypeptides (and fragments thereof), and the polynucleotides encoding them, are also encompassed by the present invention.
  • the present invention encompasses nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide sequence that is at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, e.g., a polynucleotide coding sequence of SEQ ID NO: X (or a strand complementary thereto); a nucleotide sequence encoding a polypeptide of SEQ ID NO: Y; and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., a fragment as defined herein).
  • Polynucleotides that stably hybridize to a polynucleotide fragment (as defined herein) under stringent hybridization conditions or lower stringency conditions, are also encompassed by the invention, as are polypeptides (or fragments thereof) encoded by these polynucleotides.
  • a polynucleotide sequence having at least some "percentage identity,” (e.g., 95%) to another polynucleotide sequence means that the sequence being compared (e.g., the test sequence) may vary from another sequence (e.g. the referent sequence) by a certain number of nucleotide differences (e.g., a test sequence with 95% sequence identity to a reference sequence can have up to five point mutations per each 100 contiguous nucleotides of the referent sequence).
  • determining if a particular nucleic acid molecule or polynucleotide sequence exhibits at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an LP polynucleotide sequence can be accompUshed using any art known method.
  • Variants encompassed by the present invention may contain alterations in the coding regions, non-coding regions, or both. Moreover, variants in which 1-2, 1-5, or 5-10 amino acids are substituted, deleted, or added in any combination are also preferred.
  • at least about includes, e.g., a specificaUy recited value (e.g., 20nt), and a value that is larger or smaUer by one or more nucleotides (e.g., 5, 4, 3, 2, or 1), at either terminus or at both termini.
  • a polynucleotide fragment has use that includes without Umit; e.g., diagnostic probes and primers as discussed herein.
  • polypeptide fragment or segment encompasses an amino acid sequence that is a portion of SEQ ID NO: Y.
  • Protein and/or polypeptide fragments or segments may be "free-standing,” or they may comprise part of a larger polypeptide or protein, of which the fragment or segment forms a portion or region, e.g., a single continuous region of SEQ ID NO: Y connected in a fusion protein.
  • the pluraUty wiU be at least two, more usuaUy at least three, and preferably four, five, six, seven, eight, mne, ten, or even more. While length minima are stipulated, longer lengths (of various sizes) may be appropriate (e.g., one of length seven, and two of lengths of twelve).
  • Preferred polypeptide fragments include, e.g., the secreted protein as weU as the mature form.
  • polypeptide fragments include, e g , the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, can be deleted from the carboxy terminus of the secreted protein or mature form.
  • any combination of the above amino and carboxy terminus deletions are preferred.
  • polynucleotides encoding these polypeptide fragments are also preferred.
  • polypeptide fragments or segments that characterize structural or functional domains, such as, fragments, or combinations thereof, that comprise e.g., alpha-hehx, and alpha-heUx forming regions, beta-sheet, and beta-sheet-forming regions, turn, and turn-forming regions, coil, and coil-forming regions, hydrophiUc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, loop regions, hairpin domains, beta-alpa-beta motifs, heUx bundles, alpha/beta barrels, up and down beta barrels, jeUy roU or Swiss roU motifs, transmembrane domains, surface- forming regions, substrate binding regions, transmembrane regions, Unkers, immunogenic regions, epitopic regions, and high antigenic index regions.
  • the present invention further encompasses a polynucleotide sequence encoding an epitope of a polypeptide sequence of the invention (such as, e.g , a sequence disclosed in SEQ ID NO: X or a Table herein), a polynucleotide sequence of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and a polynucleotide sequence that stably hybridizes to a complementary strand under stringent hybridization conditions or lower stringency hybridization conditions as defined herein.
  • a polynucleotide sequence encoding an epitope of a polypeptide sequence of the invention such as, e.g , a sequence disclosed in SEQ ID NO: X or a Table herein
  • a polynucleotide sequence of the complementary strand of a polynucleotide sequence encoding an epitope of the invention and a polynucleotide sequence that stably hybridizes
  • the yeast Pichia pastons is used to express a polypeptide of the present ⁇ nvent ⁇ on(or fragment thereof) in an eukaryotic system (see, e.g., EUis, et al., Mol. CeU. Biol. 5:1111-21 (1985); Koutz, et al, Yeast 5: 167-77 (1989); Tschopp, et al., Nucl. Acids Res. 15:3859-76 (1987)).
  • a polypeptide of the invention, (or fragment thereof) may be modified at random or at predetermined positions within the molecule and may include, e.g., one, two, three, or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • a preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" means that in polyethylene glycol preparations, some molecules wiU weigh more and some wiU weigh less, than the stated molecular weight).
  • polyethylene glycol may be covalendy bound through amino acid residues via a reactive group, such as, e.g., a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • Amino acid residues having a free amino group may include, e.g., lysine residues, and N-terminal amino acid residue.
  • An LP polynucleotide sequence (or fragment thereof) can be used in numerous ways, e.g., such as a reagent.
  • the foUowing descriptions are non- limiting examples of ways to use an LP polynucleotide sequence (or fragment thereof).
  • an LP polynucleotide sequence (or fragment thereof) is useful for chromosome identification.
  • chromosome markers There exists an ongoing need to identify new chromosome markers, since few chromosome-marking reagents, based on actual sequence data (repeat polymo ⁇ hisms), are presendy available.
  • Each polynucleotide of the present invention can therefore, be used as a chromosome marker.
  • a kit in another specific embodiment, includes, e.g., two polynucleotide probes defining an internal region of an LP polynucleotide sequence, where each probe has one strand containing a 31 mer-end internal to a region the polynucleotide.
  • the level in the first sample is measured or estimated from an individual having, or suspected of having, a disease, syndrome, disorder or condition and comparing that level to a second level, wherein the second level is obtained from an individual not having and/or not being suspected of having a disease, syndrome, disorder or condition.
  • the second level is determined by averaging levels from a population of individuals not having or suspected of having a disease, syndrome, disorder, or condition.
  • a “biological sample” is intended to mean herein any sample comprising biological material obtained from, using, or employing, e.g., an organism, body fluid, exudate, lavage product, waste product, ceU (or part thereof), ceU Une, organ, biopsy, tissue culture, or other source originating from, or associated with, a Uving ceU, tissue, organ, or organism, which contains, e.g., a polypeptide (or fragment thereof), a protein (or fragment thereof), a mRNA (or fragment thereof), or polynucleotide sequence (or fragment thereof) of the present invention, including, e.g., without Umitation, a sample such as from, e.g., hair, skin, blood, saUva, semen, vomit, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum, urine, fecal matter, a lavage product, etc.
  • a sample such as from, e.g., hair, skin, blood,
  • a polypeptide (or fragment thereof) can be used to assay a protein level, e.g., of a secreted protein, in a sample, e.g., such as a bodily fluid by using antibody-based techmques.
  • a protein level e.g., of a secreted protein
  • a sample e.g., such as a bodily fluid
  • antibody-based techmques protein expression in a tissue can be studied by an lmmunohistological method (see, e.g., Jalkanen, et al. (1985) J. CeU Biol. 101:976-985; Jalkanen, et al. (1987) J. CeU Biol. 105:3087-303096).
  • Recombinant LP or LP antibodies can be purified and admimstered to a subject for treatment. These reagents can be combined for use with additional active or inert ingredients, e.g., in conventional pharmaceuticaUy acceptable carriers or diluents, e.g., immunogemc adjuvants, along with physiologicaUy innocuous stabiUzers and excipients. These combinations can be sterile filtered and placed into dosage forms as by lyophiUzation in dosage vials or storage in stabiUzed aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • the quantities of reagents necessary for effective treatment wiU depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicaments admimstered. Thus, treatment dosages should be titrated to optimize safety and efficacy.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents.
  • Animal testing of effective doses for treatment of particular disorders wiU provide further predictive indication of human dosage.
  • Various considerations are described, e.g., in Gilman, et al.
  • PharmaceuticaUy acceptable carriers wiU include water, saUne, buffers, and other compounds described, e.g., in the Merck Index. Merck & Co., Rahway, NJ.
  • Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typicaUy less than about 10 ⁇ M concentrations, usuaUy less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier.
  • Slow release formulations, or a slow release apparatus wiU often be utiUzed for continuous admimstration.
  • Each carrier should be both pharmaceuticaUy and physiologicaUy acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) admimstration.
  • the formulations may convementiy be presented in unit dosage form and may be prepared by any methods weU known in the art of pharmacy. See, e.g., Gilman, et al.
  • deUvery systems are known and can be used to admimster, e.g., a composition, formulation, antibody polypeptide (or fragment thereof), or polynucleotide (or fragment thereof) of the invention.
  • deUvery can use Uposomes, microparticles, microcapsules, recombinant ceUs, receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J Biol. Chem. 262:4429-4432), inclusion of a nucleic acid molecule as part of a retroviral or other vector, etc.
  • Methods of admimstration include, e g., without Umit, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • An LP can be useful in ameUorating, treating, preventing, modulating, and/or diagnosing a disease, disorder, syndrome, or condition of the immune system, by, e.g., activating or inhibiting the proUferation, differentiation, or mobiUzation (chemotaxis or directed movement) of an immune ceU.
  • TypicaUy immune ceUs develop through a process caUed hematopoiesis, producing myeloid (platelets, red blood ceUs, neutrophds, and macrophages) and lymphoid (B and T lymphocytes) ceUs from pluripotent stem ceUs.
  • An LP can be useful in ameUorating, treating, preventing, modulating, and/or diagnosing a disease, disorder, syndrome, and/or a condition of a hematopoietic ceU.
  • An LP could be used to increase or inhibit the differentiation or proUferation of a hematopoietic ceU, including a pluripotent stem ceU such an effect can be implemented to treat, prevent, modulate, or ameUorate a disease, disorder, syndrome, and/or a condition associated with a decrease in a specific type of hematopoietic ceU.
  • An example of such an immunologic deficiency, disease, disorder, syndrome, and/or condition includes, e.g., without Umitation, a blood condition (e.g.
  • increasing hemostatic or thrombolytic activity can treat or prevent a blood coagulation condition such as e.g., afibnnogenemia, a factor deficiency, a blood platelet disease (e.g. thrombocytopema), or a wound resulting from e.g., trauma, surgery, etc.
  • a composition of the invention can be used to decrease hemostatic or thrombolytic activity or to inhibit or dissolve a clotting condition.
  • Such compositions can be important in a treatment or prevention of a heart condition, e.g., an attack infarction, stroke, or mycardial scarring.
  • An LP can be used to modulate, ameUorate, treat, prevent, and/or diagnose a hype ⁇ roUferative disease, condition, disorder, or syndrome (such as, e.g., a neoplasm) via direct or indirect interactions.
  • a hype ⁇ roUferative disease, condition, disorder, or syndrome such as, e.g., a neoplasm
  • a hype ⁇ roUferative disease, condition, disorder, or syndrome such as, e.g., a neoplasm
  • a hype ⁇ roUferative disease, condition, disorder, or syndrome such as, e.g., a neoplasm
  • a hype ⁇ roUferative disease, condition, disorder, or syndrome such as, e.g., a neoplasm
  • a desired effect using a composition of the invention may also be accompUshed either by, e.g., enhancing an existing immune response, or by initiating a new immune response.
  • hype ⁇ roUferative conditions include, e.g., without Umit hypergammaglobuUnemia, lymphoproUferative conditions, paraproteinemias, purpura, sarcoidosis, Hamartoma, Sezary Syndrome, Waldenstron's MacroglobuUnemia, Gaucher's Disease syndrome, histiocytosis, and other hype ⁇ roUferative states.
  • One preferred embodiment utilizes an LP to inhibit aberrant ceUular division, through a polynucleotide deUvery technique.
  • the present invention provides a method for treating, preventing, modulating, ameUorating, preventing, inhibiting, and/or diagnosing ceU proUferative diseases, disorders, syndromes, and/or conditions described herein by inserting into an abnormaUy proUferating ceU a composition of the present invention, wherein said composition beneficiaUy modulates an excessive condition of ceU proUferation, e.g., by inhibiting transcription and/or translation.
  • Another embodiment comprises administering one or more active copies of an LP polynucleotide sequence to an abnormaUy proUferating ceU.
  • an LP polynucleotide sequence is operably Unked in a construct comprising a recombinant expression vector that is effective in expressing a polypeptide (or fragment thereof) corresponding to the polynucleotide of interest.
  • the construct encoding a polypeptide or fragment thereof is inserted into a targeted ceU utilizing a retrovirus or an adenoviral vector (see, e.g., Nabel, et al. (1999) Proc. Nad. Acad. Sci. USA 96: 324-326).
  • An LP sequence may be useful in repressing the expression of a gene or an antigenic composition, e.g., an oncogenic retrovirus.
  • repressing the expression of a gene is meant, e.g., the suppression of the transcription of a 'gene', the degradation of a 'gene' transcript (pre-message RNA), the inhibition of spUcing of a 'gene', the destruction of mRNA, the prevention of a post-translational modification of a polypeptide, the destruction of a polypeptide, or the inhibition of a normal function of a protein.
  • Local administration to an abnormaUy proUferating ceU may be achieved by any art known method or technique discussed herein including, e.g., without Umit to transfection, electroporation, microinjection of ceUs, or in vehicles (such as a Uposome, Upofectin, or a naked polynucleotide).
  • Encompassed deUvery systems include, without Umit, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Nad. Acad. Sci. U.S.A. 85:3014); vaccinia virus systems (Chakrabarty, et al., Mol.
  • cell proliferative condition any human or animal disease, syndrome, disorder, condition, or state, affecting any ceU, tissue, any site or any combination of organs, tissues, or body parts, which is characterized by a single or multiple local abnormal proUferation of ceUs, groups of ceUs, or tissues, whether benign or maUgnant.
  • Any amount of LP may be administered as long as it has a desired effect on the treated ceU, e.g., a biologicaUy inhibiting effect on an abnormaUy proUferating ceU.
  • a desired anti-angiogenic effect may be achieved indirecdy, e.g., through the inhibition of hematopoietic, tumor-specific ceUs, such as, e.g., tumor-associated macrophages (see e.g., Joseph, et al. (1998) J Natl. Cancer Inst. 90(21): 1648-53).
  • hematopoietic, tumor-specific ceUs such as, e.g., tumor-associated macrophages
  • a desired anti- angiogenic effect may be achieved directly, (e.g., see Witte, et al., (1998) Cancer Metastasis Rev. 17(2): 155-61).
  • an LP may induce apoptosis via other mechanisms, such as, e.g., through the activation of a pathway that subsequently activates apoptosis, or through stimulating the expression of a protein(s) that activates an apoptotic pathway, either alone or in combination with smaU molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (see e.g., Mutat Res 400 (l-2):447-55 (1998), Med Hypotheses. 50(5): 423-33 (1998), Chem Biol Interact. Apr 24; 111-112:23-34 (1998), J Mol Med. 76(6): 402-12(1998), Int J Tissue React; 20 (1):3-15 (1998), which are all hereby incorporated by reference for these teachings).
  • smaU molecule drugs or adjuvants such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins
  • An LP is useful in inhibiting ceU metastasis either directiy as a result of administering a polynucleotide or polypeptide (or fragment thereof), or an agonist or antagonist thereto, (as described elsewhere herein), or indirectly, such as, e.g., by activating or increasing the expression of a protein known to inhibit metastasis, such as, e.g., an alpha integrin, (see, e.g., Cur. Top Microbial Immunol 1998; 23 1: 125-4 1, which is hereby inco ⁇ orated by reference for these teachings).
  • a desired effect can be achieved either alone using an LP or in combination with e.g., a smaU molecule drug or an adjuvant.
  • An LP or a protein fusion thereto, is useful in enhancing the immunogenicity and/or antigenicity of a proUferating ceU or tissue, either directly, (such as would occur if e.g., an LP polypeptide (or fragment thereof) 'vaccinated' the immune system to respond to a proUferative antigen or immunogen), or indirectly, (such as in activating, e.g., the expression a of protein known to enhance an immune response (e.g. a chemokine), to an antigen on an abnormaUy proUferating ceU).
  • An LP may be used to, modulate, ameUorate, effect, treat, prevent, and/or diagnose a cardiovascular disease, disorder, syndrome, and/or condition.
  • cardiovascular abnormaUties such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome peripheral artery disease, syndrome, such as Umb ischemia.
  • Additional cardiovascular disorders encompass, e.g., congenital heart defects which include, e.g., aortic coarctation, car triatriatum, coronary vessel anomaUes, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of faUot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as e.g., aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of FaUot, and ventricular heart septal defects.
  • congenital heart defects which include, e.g., aortic coarctation, car triatriatum, coronary vessel anomaUes, crisscross heart, dextrocardi
  • cardiovascular disorders include, e.g., arrhythmias including, e.g., sinus arrhythmia, atrial fibriUation, atrial flutter, bradycardia, extra systole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre- excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, and ventricular fibriUation tachycardias.
  • arrhythmias including, e.g., sinus arrhythmia, atrial fibriUation, atrial flutter, bradycardia, extra systole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre- excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, and ventricular
  • Tachycardias encompassed with the cardiovascular condition described herein include, e.g., paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal re-entry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal re-entry tachycardia, sinus tachycardia, Torsades de Pointes Syndrome, and ventricular tachycardia.
  • Myocardial conditions associated with cardiovascular disease include, e.g., myocardial diseases such as, e.g., alcohoUc cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
  • myocardial diseases such as, e.g., alcohoUc cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and my
  • Cardiovascular diseases also encompassed herein include, e.g., vascular diseases such as e.g., aneurysms, angiodysplasia, angiomatosis, baciUary angiomatosis, Hippel-Lindau Disease syndrome, Klippel-Trenaunay- Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic disease, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive disease, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disease, diabetic angiopathies, diabetic retinopathy, emboUsm, thrombosis, erythromeialgia, hemorrhoids, hepatic veno-occlusive disease syndrome, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary
  • Cardiovascular conditions further include, e.g., aneurysms such as, e.g., dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iUac aneurysms.
  • aneurysms such as, e.g., dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iUac aneurysms.
  • Arterial occlusive cardiovascular conditions include, e.g., arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease syndrome, renal artery obstruction, retinal artery occlusion, and thromboangiitis obUterans.
  • Cerebrovascular cardiovascular conditions include, e.g., carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral emboUsm and thrombosis, carotid artery thrombosis, sinus thrombosis, WaUenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient cerebral ischemia), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
  • cerebral amyloid angiopathy cerebral aneurysm
  • cerebral anoxia cerebral arteriosclerosis
  • cerebral arteriovenous malformation cerebral artery disease
  • cerebral emboUsm and thrombosis carotid artery thrombo
  • EmboUc cardiovascular conditions include, e.g., air emboUsms, amniotic fluid emboUsms, cholesterol emboUsms, blue toe syndrome, fat emboUsms, pulmonary emboUsms, and thromboemboUsms.
  • Thrombotic cardiovascular conditions include, e.g., coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, WaUenberg's syndrome, and thrombophlebitis.
  • the naturaUy occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences typicaUy predominate (see, e.g., Rastine j ad, et al., CeU 56345-355 (1989)).
  • angiogenesis is st ngendy regulated, and deUmited spatiaUy and temporaUy.
  • pathological angiogenesis such as, e.g., during soUd tumor formation, these regulatory controls fail and unregulated angiogenesis can become pathologic by sustaining progression of many neoplastic and non-neoplastic diseases.
  • admimstration of an LP provides for the treatment, ameUoration, modulation, diagnosis, and/or inhibition of a disease, disorder, syndrome, and/or condition associated with neovascularization.
  • This method is of particular value to a prophylactic treatment of a condition known to result in the development of a hypertrophic scar or a keloid (e.g, burns), and is preferably initiated after the proUferative phase of scar formation has had time to progress (approximately, e.g, 14 days after the initial injury), but before hypertrophic scar or keloid development.
  • the present invention also provides methods for ameUorating, treating, preventing, and/or diagnosing neovascular diseases of the eye, including e.g, corneal graft neovascularization, neovascular glaucoma, proUferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
  • ocular diseases, disorders, syndromes, and/or conditions associated with neovascularization that can be modulated ameUorated, treated, prevented, and/or diagnosed with an LP include, e.g, without Umit; neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of premature macular degeneration, corneal graft neovascularization, as weU as other inflammatory eye diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization (see, e.g, reviews by Waltman, et al, (1978) Am. J. Ophthal.
  • neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising administering to a patient a therapeutically effective amount of an LP composition to the cornea, such that the formation of blood vessels is inhibited or delayed.
  • the cornea is a tissue that normaUy lacks blood vessels. In certain pathological conditions however, capiUaries may extend into the cornea from the pericorneal vascular plexus of the Umbus.
  • Topical therapy may also be useful prophylacticaUy in corneal lesions that are known to have a high probabiUty of inducing an angiogenic response (such as, e.g, a chemical burn).
  • the treatment (Ukely in combination with steroids) may be instituted immediately to help prevent subsequent compUcations.
  • an LP composition may be injected directiy into the corneal stroma using microscopic guidance by an ophthalmologist.
  • the preferred site of injection may vary with the mo ⁇ hology of the individual lesion, but the goal of the administration is to place a composition of the invention at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea).
  • methods for treating or preventing neovascular glaucoma, comprising administering to a patient a therapeuticaUy effective amount of an LP to the eye, such that the formation of blood vessels is inhibited.
  • the composition may be administered topicaUy to the eye to treat or prevent early forms of neovascular glaucoma.
  • the composition may be implanted by injection into the region of the anterior chamber angle.
  • the composition may also be placed in any location such that the composition is continuously released into the aqueous humor.
  • the composition may be admimstered topicaUy, via intravitreous injection and/or via intraocular implants.
  • Additional, diseases, disorders, syndromes, and/or conditions that can be modulated, ameUorated, treated, prevented, and/or diagnosed with an LP include, e.g, without Umitation, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound heaUng, granulations, hemophiUc joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
  • an amount of an LP sufficient to block embryo implantation is admimstered before or after intercourse and fertiUzation have occurred, thus providing an effective method of birth control, possibly a "morning after" method.
  • An LP may also be used in controlUng menstruation or administered either as a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
  • An LP may be utiUzed in a wide-variety of surgical procedures.
  • a compositions in the form of, e.g, a spray or film
  • a compositions may be utiUzed to coat or spray an area before removal of a tumor, to isolate normal surrounding tissues from maUgnant tissue, and/or to prevent the spread of disease to surrounding tissues
  • an LP composition e g, in the form of a spray
  • an LP composition may be deUvered via endoscopic procedures to coat tumors, or inhibit angiogenesis in a desired locale.
  • an anti-angiogenic composition of the invention is admimstered directly to a tumor excision site (e.g, appUed by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic composition).
  • an anti-angiogenic composition may be incorporated into a known surgical paste before admimstration
  • an anti-angiogenic composition of the invention is appUed after hepatic resections for maUgnancy, and after neurosurgical operations
  • admimstration can be to a resection margin of a wide variety of tumors, including e.g, breast, colon, brain, and hepatic tumors.
  • anti-angiogenic compositions may be admimstered to the site of a neurological mmor after excision, such that the formation of new blood vessels at the site is inhibited Diseases at the Cellular Level Diseases associated with increased ceU survival or the inhibition of apoptosis that could be modulated, ameUorated, treated, prevented, and/or diagnosed by an LP include, e.g, cancers (such as, e.g, foUicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, e.g, but without Umit, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, gUoblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endotheUoma, osteoblastoma, osteoclastoma, osteosarcoma, chondro
  • an LP is used to inhibit growth, progression, and/or metastases of cancers such as, in particular, those Usted herein.
  • Additional diseases, states, syndromes, or conditions associated with increased ceU survival that could be modulated, ameUorated, treated, prevented, or diagnosed by an LP include, e.g, without Umitation, progression, and/or metastases of maUgnancies and related disorders such as leukemia including acute leukemias (such as, e.g, acute lymphocytic leukemia, acute myelocytic leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) and chrome leukemias (e.g, chrome myelocytic, chronic granulocytic, leukemia, and chrome lymphocytic leukemia)), polycythemia Vera, lymph
  • Diseases associated with increased apoptosis that could be modulated, ameUorated, treated, prevented, and/or diagnosed by an LP include, e.g, AIDS, conditions (such as, e.g, Alzheimer's disease syndrome, Parkinson's disease syndrome, Amyotrophic lateral sclerosis, Retimtis pigmentosa, CerebeUar degeneration and brain tumor, or pnon associated disease); autoimmune conditions (such as, e.g, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biUary cirrhosis, Behcet's disease syndrome, Crohn's disease syndrome, polymyositis, systemic lupus erythematosus, immune-related glomeruloneph ⁇ tis, and rheumatoid arthritis); myelodysplastic syndromes (such as aplastic anemia), graft v.
  • conditions such as, e.g, Alzheimer's disease syndrome, Parkinson's disease syndrome, Amy
  • an LP composition may be cUnicaUy useful in stimulating wound heaUng including e.g , surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from exposure heat or chemicals, abnormal wound heaUng conditions associated with e.g, uremia, malnutrition, vitamin deficiency and wound heaUng compUcations associated with systemic treatment with steroids, radiation therapy, anti-neoplastic drugs, and anti-metaboUtes.
  • wound heaUng including e.g , surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, ve
  • An LP can be used to promote skin strength and to improve the appearance of aged skin. It is beUeved that an LP wiU also produce changes in hepatocyte proUferation, and epitheUal ceU proUferation in, for example, the lung, breast, pancreas, stomach, smaU intestine, and large intestine.
  • EpitheUal ceU proUferation can be effected in epitheUal ceUs such as, e.g, sebocytes, hair foUicles, hepatocytes, type II pneumocytes, mucin-producing goblet ceUs, and other epitheUal ceUs or their progenitors which are contained within the skin, lung, Uver, and gastrointestinal tract.
  • Inflammatory bowel diseases such as Crohn's disease and ulcerative coUtis, are diseases that result in destruction of the mucosal surface of the smaU or large intestine, respectively.
  • an LP could be used to promote resurfacing of a mucosal surface to aid more rapid heaUng and to prevent progression of inflammatory bowel disease resulting in a desired effect, e.g, such as on the production of mucus throughout the gastrointestinal tract and the protection of intestinal mucosa from injurious substances that are ingested or foUowing surgery.
  • An LP could be used to treat a condition associated with the under expression of an LP polynucleotide sequence or an LP polypeptide of the present invention (or fragment thereof), or an agonist or antagonist thereto.
  • an LP could be used to prevent and heal damage to the lungs due to various pathological states, such as, e.g, stimulating proUferation and differentiation to promote repair of alveoU and bronchiolar epitheUum.
  • pathological states such as, e.g, stimulating proUferation and differentiation to promote repair of alveoU and bronchiolar epitheUum.
  • emphysema inhalation injuries, that (e.g, from smoke inhalation) and burns, which cause necrosis of the bronchiolar epitheUum and alveoU could be effectively ameUorated, treated, prevented, and/or diagnosed using a polynucleotide or polypeptide of the invention (or fragment thereof), or an agomst or antagomst thereto.
  • an LP could be used to stimulate the proUferation of and differentiation of type II pneumocytes, to help treat or prevent hyaUne membrane diseases, such as e.g, infant respiratory distress syndrome and bronchopulmonary displasia, (in premature infants).
  • An LP could stimulate the proUferation and/or differentiation of a hepatocyte and, thus, could be used to aUeviate or treat a Uver condition such as e.g, fulminant Uver failure (caused, e.g, by cirrhosis), Uver damage caused by viral hepatitis and toxic substances (e.g., acetaminophen, carbon tetrachlo ⁇ de, and other known hepatotoxins).
  • a Uver condition such as e.g, fulminant Uver failure (caused, e.g, by cirrhosis), Uver damage caused by viral hepatitis and toxic substances (e.g., acetaminophen, carbon
  • an LP could be used treat or prevent the onset of diabetes meUitus.
  • an LP could be used to maintain the islet function so as to aUeviate, modulate, ameUorate, delay, or prevent permanent manifestation of the disease.
  • an LP could be used as an auxiUary in islet ceU transplantation to improve or promote islet ceU function.
  • Nervous system diseases, disorders, syndromes, states, and/or conditions that can be modulated, ameUorated, treated, prevented, and/or diagnosed with an LP composition include, e.g, without Umitation, nervous system injuries diseases, disorders, states, syndromes, and/or conditions that result in either a disconnection or misconnection of an axon or dendnte, a diminution or degeneration of a cell (or part of a ceU) of the nervous system (such as, e g , without Umitation, neurons, astrocytes, microgUa, macrogUa, oUgodendrogUa, Schwann ceUs, and ependymal ceUs), demyeUnation or improper mylenation, neural ceU dysfunction (such as, e.g, failure of neurotransmitter release or uptake), or interference with mylenization.
  • any art known method can be used to: measure increased neuronal survival (such as, e.g, described in Arakawa, et al. (1990) J. Neurosci. 10:3507-3515); detect increased or decreased sprouting (such as, e.g, described in Pestronk, et al. (1980) Exp. Neurol. 70:65-82; Brown, et al. (1981) Ann. Rev. Neurosci.
  • motor neuron diseases, disorders, syndromes, and/or conditions that may be modulated, ameUorated, treated, prevented, and/or diagnosed using an LP composition include, e.g, without Umitation, infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or maUgnancy (that may affect motor neurons as weU as other components of the nervous system), as weU as conditions that selectively affect neurons such as, e.g, without Umitation, Amyotrophic lateral sclerosis progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poUomyeUtis post poUo syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Mane-Tooth Disease). Infectious Disease
  • An LP composition can be used to modulate, ameUorate, treat, prevent, and/or diagnose an effect of an infectious agent in a subject or associated with a condition. For example, by increasing an immune response e g, particularly increasing the proUferation and differentiation a of B and/or a T ceU, infectious diseases may be modulated, ameUorated, treated, prevented, and/or diagnosed.
  • the immune response may be increased either by enhancing an existing immune response, or by initiating a new immune response.
  • an LP may also directly inhibit an infectious agent, without necessarily eUciting an immune response.
  • Viruses are a type of an infectious agent that can cause diseases, disorders, syndromes, and/or conditions that may be modulated, ameUorated, treated, prevented, and/or diagnosed using an LP composition of the invention.
  • viruses include, e.g, without Umitation, the foUowing DNA and RNA viruses and viral famiUes: ⁇ rbovirus, Adenovindae, Arenavindae, Artenvirus, Birnaviridae, Bunyavindae, Caliciviridae, Circovindae, Coronavindae, Dengue, EBV, HIV, Flavivtridae, Hepadnavindae (Hepatitis), Herpesvindae (such as, e.g, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g,
  • Paramyxov ndae Morbilhvirus, abdovmdae
  • Orthomyxovindae e.g., Influenza A, Influenza B, and para ⁇ nfluen a
  • Pap lomavirus Papovavmdae
  • Parvovindae Picomav ridae
  • Poxvindae such as, e.g.
  • viruses of these famiUes can cause a variety of undesired conditions, including, but not Umited to: e.g, arthritis, bronchioUitis, respiratory syncytial virus, encephaUtis, eye infections (e.g , conjunctivitis, keratitis), chrome fatigue syndrome, hepatitis (e.g, of type A, B, C, E, Chrome Active, or Delta), Japanese BencephaUtis, Jumn, Chikungunya, Rift VaUey fever, yeUow fever, memngitis, oppormmstic infections (e.g, AIDS), pneumoma, Burkit
  • An LP can be used to modulate, ameUorate, treat, prevent, and/or diagnose any of these symptoms or diseases.
  • an LP composition is used to modulate, ameUorate, treat, prevent, and/or diagnose e.g, memngitis, Dengue, EBV, and/or hepatitis (e.g, hepatitis B).
  • an LP is admimstered to a subject that is non-responsive to one or more currently estabUshed commerciaUy available, hepatitis vaccines.
  • an LP can be used to modulate, ameUorate, treat, prevent, and/or diagnose AIDS or an AIDS-related syndrome or condition.
  • Actinomycetales e.g, Corynebactenum, Mycobacterium, Norcardia
  • Cryptococcus neoformans e.g., Aspergillosis
  • Baallaceae e.g., Anthrax
  • Salmonella typht, and Salmonella paratyphi Salmonella typht, and Salmonella paratyphi
  • Serratta Yerstnta
  • Erystpelothrix Heltcobacter, Legtonellosis
  • Eeptospirosis asterta
  • Mycoplasmatales Mycobacterium leprae
  • Vibrio cholerae Vibrio cholerae
  • Netssenaceae e.g, Ac ⁇ netobacter,Gonorrhea, Memgococcal
  • Meisserta meningttidis Pasteurellacea Infections (e g, Actmobacillus, Heamophtlus (e.g, Heamophtlus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydtaceae, Syphilis, Shigella spp.,Staphylococcal, Memngiococcal, Pneumococcal and Streptococcal (e.g. Streptococcus pneumontae and Group B Streptococcus).
  • These bacterial or fungal famiUes can cause the foUowing diseases, disorders, conditions, syndromes, or symptoms including, e.g, without Umitation, bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g, AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease syndrome, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease syndrome, Cat-Scratch Disease syndrome, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g, meningitis types A and B), Chlamydia, SyphiUs, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, BotuUsm, gangrene,
  • an LP composition can be used to modulate, ameUorate, treat, prevent, and/or diagnose: tetanus, Diptheria, botuUsm, and/or meningitis type B.
  • An LP composition of the invention can be used e.g, to differentiate a ceU, tissue; or biological structure, de-differentiate a ceU, tissue; or biological structure; cause proUferation in ceU or a zone (similar to a ZPA in a Umb bud), have an effect on chemotaxis, remodel a tissue (e.g, basement membrane, extra ceU matrix, connective tissue, muscle, epitheUa), or lmtiate the regeneration of a tissue, organ, or biological structure (see, e.g. Science (1997) 276:59-87).
  • a tissue e.g, basement membrane, extra ceU matrix, connective tissue, muscle, epitheUa
  • lmtiate the regeneration of a tissue, organ, or biological structure see, e.g. Science (1997) 276:59-87).
  • Regeneration using an LP composition of the invention could be used to repair, replace, remodel, or protect tissue damaged by, e.g, congenital defects, trauma (such as, e.g, wounds, burns, incisions, or ulcers); age; disease (such as, e.g, osteoporosis, osteoarthritis, periodontal disease syndrome, or Uver failure), surgery, (including, e.g, cosmetic plastic surgery); fibrosis; re-perfusion in j ury; or cytokine damage.
  • trauma such as, e.g, wounds, burns, incisions, or ulcers
  • age disease
  • disease such as, e.g, osteoporosis, osteoarthritis, periodontal disease syndrome, or Uver failure
  • surgery including, e.g, cosmetic plastic surgery
  • fibrosis re-perfusion in j ury
  • cytokine damage e.g, cytokine damage.
  • Tissues that can be regenerated include, e.g, without Umitation, organs (e.g , pancreas, Uver, intestine, kidney, epitheUa, endotheUum), muscle (smooth, skeletal, or cardiac), vasculature (including vascular and lymphatics), nervous system tissue, ceUs, or structures; hematopoietic tissue; and skeletal (bone, cartilage, tendon, and Ugament) tissue.
  • organs e.g , pancreas, Uver, intestine, kidney, epitheUa, endotheUum
  • muscle smooth, skeletal, or cardiac
  • vasculature including vascular and lymphatics
  • nervous system tissue ceUs, or structures
  • hematopoietic tissue hematopoietic tissue
  • skeletal (bone, cartilage, tendon, and Ugament) tissue Preferably, regeneration occurs with Uttle or no scarring.
  • Regeneration also may include, e.g,
  • an LP composition may increase the regeneration of an aggregation of special ceU types, a tissue, or a matrix that typicaUy is difficult to heal. For example, by increasing the rate at which a tendon/Ugament heals after damage. Also encompassed is using an LP prophylacticaUy to avoid damage (e.g, in an interstitial space of a joint or on the cartalagenous capsule of a bone).
  • Specific diseases that could be modulated, ameUorated, treated, prevented, and/or diagnosed using an LP composition include, e.g, without Umitation, tendinitis, carpal tunnel syndrome, and other tendon or Ugament defects
  • Examples of non-heaUng wounds include, wounds that would benefit form regeneration treatment, e.g, without Umit pressure ulcers, ulcers associated with vascular insufficiency, surgical wounds, and traumatic wounds.
  • nerve and brain tissue also could be regenerated using an LP.
  • Such nervous system conditions that could be modulated, ameUorated, treated, prevented, and/or diagnosed using an LP composition include, e.g , without Umitation, central and peripheral nervous system diseases, neuropathies, or mechamcal and traumatic conditions (e.g., spinal cord disorders or syndromes, head trauma, cerebrovascular disease syndrome, and stoke).
  • diseases associated with peripheral nerve injuries include, e.g, without Umitation, peripheral neuropathy (e.g, resulting from chemotherapy or other medical therapies), locaUzed neuropathies, and central nervous system diseases (e.g., Alzheimer's disease syndrome, Parkinson's disease syndrome, Huntington's disease syndrome, Amyotrophic lateral sclerosis, and Shy-Drager syndrome).
  • AU could be ameUorated, treated, prevented, and/or diagnosed using an LP.
  • An LP may have an effect on a chemotaxis activity.
  • chemotactic molecules can attract or mobiUze (but may also repeal) ceUs (e.g, monocytes, fibroblasts, neutrophils, T-ceUs, mast ceUs, eosinophils, epitheUal and/or endotheUal ceUs) or ceU processes (e.g, filopodia, psuedopodia, lameUapodia, dendntes, axons, etc.) to a particular site (e.g, such as inflammation, infection, site of hyperproUferation, the floor plate of the developing spinal cord, etc.).
  • ceUs e.g, monocytes, fibroblasts, neutrophils, T-ceUs, mast ceUs, eosinophils, epitheUal and/or endotheUal ceUs
  • ceU processes e.g, filopodia, psuedopodia, lameUapodia, dendntes, axons
  • such mobiUzed ceUs can then fight off and/or modulate a particular trauma, abnormaUty, condition, syndrome, or disease.
  • An LP may have an effect on a chemotactic activity of a ceU (such as, e.g, an attractive or repulsive effect).
  • a chemotactic molecule can be used to modulate, ameUorate, treat, prevent, and/or diagnose inflammation, infection, hyperproUferative diseases, disorders, syndromes, and/or conditions, or an immune system disorder by increasing the number of ceUs targeted to a particular location in the body.
  • a chemotactic molecule can be used to attract an immune ceU to an injured location in a subject.
  • An LP that had an effect on a chemotactant could also attract a fibroblast, which can be used to modulate, ameUorate, and/or treat a wound. It is also contemplated that an LP may inhibit a chemotactic activity to modulate, ameUorate, treat, prevent, and/or diagnose a disease, disorder, syndrome, and/or a condition.
  • a preferred kit for determining the concentration of, e.g, a LP protein in a sample would typicaUy comprise a labeled compound, e.g, binding partner or antibody, having known binding affinity for the LP protein, a source of LP protein (naturaUy occurring or recombinant), and a means for separating the bound from free labeled compound, for example, a soUd phase for immobiUzing the LP protein. Compartments containing reagents, and instructions, wiU normaUy be provided.
  • Another diagnostic aspect of this invention involves use of oUgonucleotide or polynucleotide sequences taken from the sequence of a LP protein.
  • sequences are used as probes for detecting levels of the LP protein message in samples from natural sources, or patients suspected of having an abnormal condition, e.g, cancer or developmental problem.
  • the preparation of both RNA and DNA nucleotide sequences, the labeUng of the sequences, and the preferred size of the sequences has received ample description and discussion in the Uterature.
  • a kit may include, e.g, a recombinantly produced or chemicaUy synthesized polypeptide antigen.
  • the polypeptide antigen of the kit may also be attached to a soUd support.
  • the detecting means of the above-described kit includes, e.g, a soUd support to which said polypeptide antigen is attached.
  • Such a kit may also include, e.g, a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen is detected by binding of the reporter-labeled antibody.
  • the claimed invention include an isolated or recombinant nucleic acid molecule comprising a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence of at least about: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, or 150 contiguous nucleotides of a sequence of SEQ ID NO:X wherein X is any integer as defined in a Table herein.
  • inventions include an isolated or recombinant nucleic acid molecule comprising a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence of at least about: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, or 150 contiguous nucleotides of a mature coding portion of SEQ ID NO:X wherein X is any integer as defined in a Table herein.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is include, e.g. in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the Clone
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included, e.g, in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3' nucleotide of the Clone Sequence as defined for SEQ ID NO:X in a Table herein.
  • polynucleotide sequence that is at least 95% identical to one, exhibits 95% sequence identity to at least: 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polynucleotide fragments 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, or 150 contiguous nucleotides in length of the mature coding portion of SEQ ID NO:X, wherein any one such fragment is at least 21 contiguous nucleotides in length.
  • nucleic acid molecule comprising a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence of at least about: 200, 250, 300, 350, 400, 450, or 500 contiguous nucleotides of the mature coding portion of SEQ ID NO:X.
  • an isolated or recombinant nucleic acid molecule comprising a polynucleotide sequence that is at least 95% identical to a sequence of at least about: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, or 150 contiguous nucleotides in at least one nucleotide sequence fragment of SEQ ID NO:X, wherein the length of at least one such fragment is about 200, 250, 300, 350, 400, 450, or 500 contiguous nucleotides of SEQ ID NO:X.
  • Another preferred embodiment is an isolated or recombinant nucleic acid molecule comprising a polynucleotide sequence that is at least 95% identical to a sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the 5' Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position o the 3' Nucleotide of a Clone Sequence as defined for SEQ ID NO:X in a Table herein.
  • a further preferred embodiment is an isolated or recombinant nucleic acid molecule comprising a polynucleotide sequence, which is at least 95% identical to the complete mature coding portion of SEQ ID NO:X or a species variant thereof.
  • an isolated or recombinant nucleic acid molecule comprising polynucleotide sequence that hybridizes under stringent hybridization conditions to a mature coding portion of a polynucleotide o the invention (or fragment thereof), wherein the nucleic acid molecule that hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
  • the kit generaUy includes, e.g, a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystaUization, and others. See, e.g, Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification,” Methods in Enzymology vol. 182, and other volumes in this series; CoUgan, et al. (1995 and supplements) Current Protocols in Protein Science John Wiley and Sons, New York, NY; P. Matsudaira (ed.) (1993) A Practical Guide to Protein and Peptide Purification for Microsequencing.
  • OIAexpress The High Level Expression and Protein Purification System QUIAGEN, Inc., Chatsworth, CA. Standard immunological techniques are described, e.g, in Hertzenberg, et al. (eds. 1996) Weir's Hanbook of Experimental Immunology vols. 1-4, BlackweU Science; CoUgan (1991) Current Protocols in Immunology Wiley/ Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.
  • Standard methods are used to isolate fuU length genes from a cDNA Ubrary made from an appropriate source, e.g, human ceUs.
  • the appropriate sequence is selected, and hybridization at high stringency conditions is performed to find a fuU length corresponding gene using standard techniques.
  • the fuU length, or appropriate fragments, of human genes are used to isolate a corresponding monkey or other primate gene.
  • a fuU length coding sequence is used for hybridization.
  • Similar source materials as indicated above are used to isolate natural genes, including genetic, polymorphic, aUeUc, or strain variants. Other species variants are also isolated using similar methods. With a positive clone, the coding sequence is inserted into an appropriate expression vector.
  • This may be in a vector specificaUy selected for a prokaryote, yeast, insect, or higher vertebrate, e.g, mammaUan expression system.
  • Standard methods are appUed to produce the gene product, preferably as a soluble secreted molecule, but wiU, in certain instances, also be made as an intraceUular protein.
  • IntraceUular proteins typicaUy require ceU lysis to recover the protein, and insoluble inclusion bodies are a common starting material for further purification. With a clone encoding a vertebrate LP protein, recombinant production means are used, although natural forms may be purified from appropriate sources.
  • the protein is secreted into the medium, and the soluble product is purified from the medium in a soluble form.
  • inclusion bodies from prokaryotic expression systems are a useful source of material.
  • TypicaUy the insoluble protein is solubiUzed from the inclusion bodies and refolded using standard methods. Purification methods are developed as described herein. The product of the purification method described above is characterized to determine many structural features. Standard physical methods are appUed, e.g, amino acid analysis and protein sequencing. The resulting protein is subjected to CD spectroscopy and other spectroscopic methods, e.g,
  • Tissue distribution of mRNA expression of a polynucleotide of the present invention is determined using protocols for Northern blot analysis, described (among others) by, e.g, Sambrook, et al.
  • a cDNA probe produced using common techniques is labeled with P 32 using the Rediprime DNA labeUng system (Amersham Life Science), according to manufacturer's instructions. After labeUng, the probe is purified using CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified, labeled probe is then used to examine various human tissues for mRNA expression.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • PTU90-1 Express Hyb 1711 hybridization solution
  • blots are mounted and exposed to film (overnight at -70 °C), and the films are subsequendy developed according to standard procedures.
  • Example 3 Chromosomal Mapping of an LP Polynucleotide An oUgonucleotide primer set is designed according to the sequence at the 5' end of a
  • SEQ ID NO:X identified sequence.
  • This primer preferably spans about 100 nucleotides.
  • This primer set is then used in a polymerase chain reaction under the foUowing set of conditions: 30 seconds, 95 °C; 1 minute, 56 °C; 1 minute, 70 °C. This cycle is repeated 32 times foUowed by one 5-minute cycle at 70 °C.
  • Human, mouse, and hamster DNA is used as template in addition to a somatic ceU hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reaction is analyzed on either 8% polyacrylamide gels or 3.5 % agarose gels. Chromosome mapping is determined by the presence of an approximately lOObp PCR fragment in a particular somatic ceU hybrid.
  • the foUowing protocol produces a supernatant containing an LP polypeptide (or fragment thereof) to be tested.
  • This supernatant can then be used in a variety of screening assays (such as, e.g, those taught herein).
  • Distribute the solution over each weU a 12-channel pipetter may be used with tips on every other channel).
  • the transfection reaction is terminated, preferably by spUtting tasks (as above) at the end of the incubation period.
  • Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each weU.
  • Incubate at 37 °C for 45 or 72 hours depending on the media used (1 %BSA for 45 hours or CHO-5 for 72 hours).
  • On day four using a 300 ul multichannel pipetter, aUquot 600 ⁇ l in one 1 ml deep weU plate and the remaining supernatant into a 2 ml deep weU. The supernatants from each weU can then be used in an assay taught herein.
  • the activity when activity is obtained in an assay described herein using a supernatant, the activity originates either from the polypeptide (or fragment thereof) directiy (such as, e.g, from a secreted protein or fragment thereof) or by the polypeptide (or fragment thereof) inducing expression of another ⁇ rote ⁇ n(s), which is/are then released into the supernatant.
  • the invention provides a method of identifying a polypeptide (or fragment thereof) in a supernatant characterized by an activity in a particular assay taught herein.
  • Example 5 Construction of a GAS Reporter Construct
  • One signal transduction pathway involved in ceUular differentiation and proUferation is a Jaks-STATS pathway.
  • Activated proteins in a Jaks-STATS pathway have been shown to bind to gamma activation site "GAS” elements or interferon-sensitive responsive element ("ISRE"), which are located, e.g, in the promoter region of many genes.
  • GAS gamma activation site
  • ISRE interferon-sensitive responsive element
  • GAS and ISRE elements are recognized by a class of transcription factors caUed Signal Transducers and Activators of Transcription, or "STATS.”
  • STATS Transcription factors
  • the Statl and Stat3 members of the STATS family are present in many ceU types, (as is Stat2) probably, because the response to IFN- alpha is widespread.
  • Stat4 is more restricted to particular ceU types though, it has been found in T helper class I ceUs after their treatment with IL-12.
  • Stat 5 onginaUy designated mammary growth factor
  • Stat 5 is activated in tissue culture ceUs by many cytokines.
  • Jaks represent a distinct family of soluble tyrosine kinases and include, e.g, Tyk2, Jakl, Jak2, and Jak3 These Jak kinases display sigmficant sequence similarity to each other and, generaUy, are catalyticaUy inactive in resting ceUs. However, Jaks are catalyticaUy activated by a wide range of receptors (summarized in the Table below, adapted from Schidler and DarneU (1995) Ann.
  • Class 1 includes, e.g, receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; while Class 2 includes, e.g, IFN-a, IFN-g, and IL-10.
  • the Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding T ⁇ -Ser-Xxx-T ⁇ -Ser).
  • Jaks are typicaUy activated and, in turn, subsequently activate STATS, which translocate and bind to GAS transcriptional elements (located in the nucleus of the ceU). This entire process of sequential activation is encompassed in a typical Jaks-STATS signal transduction pathway.
  • activation of a Jaks-STATS pathway (reflected by binding of a GAS or ISRE element) is used to indicate that an LP polypeptide (or fragment thereof) is involved in the proUferation and/or differentiation of a ceU.
  • growth factors and cytokines are examples of proteins that are known to activate a Jaks-STATS pathway. Consequently, by using a GAS element Unked to a reporter molecule, an activator of a Jaks-STATS pathway is identified.
  • the 5' primer contains four tandem copies of the GAS binding site found in the IRF1 promoter, which has previously been shown to bind STATS after induction by a range of cytokines (see, e g, Rothman, et al. (1994) Immunity 1:457-468). Although, however, it is possible to use other GAS or ISRE elements.
  • the 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an Xhol site. The sequence of the 5' primer is:
  • the downstream primer which is complementary to the SV40 promoter and is flanked with a Hind III site, is- 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:25).
  • PCR ampUfication is performed using the SV40 promoter template present in a B- gakpromoter plasmid (Clontech).
  • the resulting PCR fragment is digested with Xhol/Hind III and subcloned into BLSK2- (Stratagene).
  • the reporter molecule is a secreted alkaUne phosphatase (SEAP).
  • SEAP secreted alkaUne phosphatase
  • any appUcable reporter molecule is used instead of SEAP without undue experimentation.
  • art known methods such as, e.g, without Umitation, chloramphenicol acetyltransferase (CAT), luciferase, alkaUne phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein (detectable by an antibody or detectable binding partner) could be substituted for SEAP.
  • the synthetic GAS-SV40 promoter element is subcloned into a pSEAP-Promoter vector (Clontech) using Hindlll and Xhol. This, effectively, replaces the SV40 promoter with the ampUfied GAS:SV40 promoter element to create a GAS-SEAP vector.
  • the resulting GAS-SEAP vector does not contain a neomycin resistance gene it is not a preferred embodiment for use in mammaUan expression systems.
  • the GAS-SEAP cassette is removed (using Sail and Notl) from the GAS-SEAP vector and inserted into a backbone vector contaimng a neomycin resistance gene, such as, e.g, pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create a GAS-SEAP/Neo vector.
  • a neomycin resistance gene such as, e.g, pGFP-1 (Clontech)
  • the GAS-SEAP/Neo vector can also be used as a reporter molecule for GAS binding as taught in an assay as described herein Similar constructs is made using the above description and replacing GAS with a different promoter sequence.
  • reporter-molecules containing NFK-B and EGR promoter sequences are appUcable. AdditionaUy, however, many other promoters is substituted using a protocols described herein, e.g, SRE, IL-2, NFAT, or Osteocalcin promoters is substituted, alone or in combination with another (e.g., GAS/NF- KB/EGR, GAS/NF-KB, I1-2/NFAT, or NF-KB/GAS).
  • a protocols described herein e.g, SRE, IL-2, NFAT, or Osteocalcin promoters is substituted, alone or in combination with another (e.g., GAS/NF- KB/EGR, GAS/NF-KB, I1-2/NFAT, or NF-KB/GAS).
  • ceU Unes is used to test reporter construct activity, such as, e.g, without Umitation, HELA (epitheUal), HUVEC (endotheUal), Reh (B-ceU), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte ceU Unes.
  • HELA epidermal
  • HUVEC endotheUal
  • Reh B-ceU
  • Saos-2 osteoblast
  • HUVAC aortic
  • Cardiomyocyte ceU Unes can be performed (without undue experimentation) by adopting a method as described, e.g, in Ho, et al. (1995) Mol. CeU. Biol. 15:5043-5-53.
  • the foUowing protocol is used to assess T-ceU activity by identifying factors and/or determining whether a supernate (described herein) contaimng an LP polypeptide (or fragment thereof) modulates the proUferation and/or differentiation of a T-ceU.
  • T-ceU activity is assessed using a GAS/SEAP/Neo construct.
  • a factor that increases SEAP activity indicates an ability to activate a Jaks-STATS signal transduction pathway.
  • One type of T-ceU used in this assay is, e.g, a Jurkat T-ceU (ATCC Accession No.
  • ceUs can also be used such as, e.g, without Umitation, Molt-3 ceUs (ATCC Accession No. CRL-1552) or Molt-4 ceUs (ATCC Accession No. CRL-1582).
  • Jurkat T-ceUs are lymphoblastic CD4+ Thl helper ceUs.
  • approximately 2 milUon Jurkat ceUs are transfected with a GAS-SEAP/Neo vector using DMRIE-C (Life Technologies) in a transfection procedure as described below.
  • Transfected ceUs are seeded to a density of approximately 20,000 ceUs per weU and any resulting transfectant (resistant to 1 mg/ml genticin) is subsequendy selected. Resistant colonies are then expanded and tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is then estabUshed.
  • the foUowing method yields a number of cells sufficient for 75 weUs (each containing approximately 200 ul of ceUs).
  • the method can be modified easily (e.g, it can either be scaled up or performed in multiples to generate sufficient numbers of ceUs for multiple 96 weU plates).
  • Jurkat ceUs are maintained in RPMI + 10% serum with 1 % Pen-Strep.

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Abstract

L'invention concerne des molécules d'acides nucléiques isolées codant pour des polypeptides provenant d'un humain, ainsi que des réactifs associés (notamment des anticorps spécifiques aux polypeptides purifiés). L'invention concerne également des procédés d'utilisation desdits réactifs, ainsi que des kits diagnostiques.
PCT/US2002/005093 2001-03-16 2002-03-01 Proteines de mammiferes lp et reactifs associes WO2002074906A2 (fr)

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GB2381790A (en) * 2001-09-26 2003-05-14 Glaxo Group Ltd LDL-receptor polypeptides
WO2006009241A1 (fr) * 2004-07-22 2006-01-26 Eisai Co., Ltd. MARQUEUR DE CELLULE PRÉCURSEUR DE NEURONE, PRODUISANT DE LA DOPAMINE Lrp4/CORINE
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JP2007306919A (ja) * 2004-07-22 2007-11-29 Eisai R & D Management Co Ltd Ｌｒｐ４／Ｃｏｒｉｎドーパミン産生ニューロン前駆細胞マーカー
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US9994816B2 (en) 2004-07-22 2018-06-12 Eisai R&D Management Co., Ltd. Lrp4/corin dopamine-producing neuron precursor cell marker
US8067161B2 (en) 2005-08-18 2011-11-29 Eisai R&D Management Co., Ltd. Dopaminergic neuron proliferative progenitor cell marker Nato3
US9453840B2 (en) 2011-07-27 2016-09-27 Kyoto University Markers for dopaminergic neuron progenitor cells
EP2905622A1 (fr) * 2014-02-07 2015-08-12 Institut D'Investigaciones Biomédiques August Pi i Sunyer Diagnostic d'une maladie neurologique
CN105934673A (zh) * 2014-02-07 2016-09-07 奥古斯特·皮·松耶尔生物医学研究所 神经学疾病的诊断
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WO2015118115A1 (fr) * 2014-02-07 2015-08-13 Institut D'investigaciones Biomèdiques August Pi I Sunyer Diagnostic d'une maladie neurologique
US20180335439A1 (en) * 2014-02-07 2018-11-22 Institut D'investigaciones Biomèdiques August Pi I Sunyer Diagnosis of a neurological disease
CN105934673B (zh) * 2014-02-07 2020-11-27 奥古斯特·皮·松耶尔生物医学研究所 神经学疾病的诊断
US10962554B2 (en) 2014-02-07 2021-03-30 Institut D'investigaciones Biomèdiques August Pi I Sunyer Diagnosis of a neurological disease

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