EP1434783A2 - Proteines de mammiferes lp et reactifs associes - Google Patents

Proteines de mammiferes lp et reactifs associes

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Publication number
EP1434783A2
EP1434783A2 EP02719036A EP02719036A EP1434783A2 EP 1434783 A2 EP1434783 A2 EP 1434783A2 EP 02719036 A EP02719036 A EP 02719036A EP 02719036 A EP02719036 A EP 02719036A EP 1434783 A2 EP1434783 A2 EP 1434783A2
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EP
European Patent Office
Prior art keywords
seq
coding portion
primate
polypeptide
mature coding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02719036A
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German (de)
English (en)
Other versions
EP1434783A4 (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 Co
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Eli Lilly and Co
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Publication of EP1434783A2 publication Critical patent/EP1434783A2/fr
Publication of EP1434783A4 publication Critical patent/EP1434783A4/fr
Withdrawn legal-status Critical Current

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Classifications

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

Definitions

  • the present invention generally relates to compositions related to proteins
  • it provides purified genes, polynucleotide sequences, proteins, polypeptides, antibodies, binding compositions, and related reagents useful, e.g , in the diagnosis, treatment, and prevention of cell proliferaUve, autoimmune /inflammatory, cardiovascular, neurological, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of such proteins.
  • Protein transport and secretion are essen ⁇ al for cellular function. Protein transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted. Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane.
  • Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues
  • Secreted proteins are generally synthesized as inactive precursors that are acdvated by post-transladonal processing events during transit through the secretory pathway Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. Examples of secreted proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling.
  • Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiauon factors, enzymes, neuropepudes, and vasomediators (reviewed in Alberts, et al. (1994) Molecular Biology of The Cell, Garland Publishing, New York, NY, pp. 557-560, 582-592.).
  • the present invention is based in part upon the discovery of LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) proteins and/or polypeptides.
  • the invention provides substantially pure, isolated, and/or recombinant LP protein or peptide (LP318a, LP318b, LP288, LP289, LP343, LP319a,
  • 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 portion is at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous amino acid residues in length.
  • the LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346): LP318a comprises a mature sequence of Table 1; LP318b comprises a mature sequence of Table 2; LP288 comprises a mature sequence of Table 3; LP289 or LP343 comprises a mature sequence of Table 4; LP319a or LP319b comprises a mature sequence of Table 5; LP321 comprises a mature sequence of Table 6; LP317 comprises a mature sequence of Table 7; and LP283 LP344, LP345, or LP346 comprises a mature sequence of Table 8
  • compositions comprising: a sterile LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) protein or peptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • a sterile LP LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346
  • the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • the invention further provides a fusion protein, comprising: mature protein comprising sequence of Table 1, 2, 3, 4, 5, 6, 7 or 8 a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another LP LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) protein or peptide.
  • a detection or purification tag including a FLAG, His6, or Ig sequence
  • reagents also make available a kit comprising such an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) protein or polypeptide, and: a compartment comprising the protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • LP LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346
  • the invention further provides a binding compound comprising an antigen binding portion from an antibody, which specificaUy binds to a natural LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) protein or polypeptide, wherein: the protein or polypeptide is a primate protein; the binding compound is an Fv,
  • the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide comprising sequence of Table 1, 2, 3, 4, 5, 6, 7, or 8 is raised against a mature LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) is immunoselected; is a polyclonal antibody; binds to a denatured LP, LP1, LP2, LP3, LP4,
  • kits include those containing the binding compound, and: a compartment comprising the binding compound; and/or instructions for use or disposal of reagents in the kit. Many of the kits will be used for making a qualitative or quantitative analysis.
  • compositions comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
  • the present invention further provides an isolated or recombinant LP nucleic acid encoding a protein or peptide or fusion protein described above, wherein: the LP protein and/or polypeptide is from a mammal, including a primate; or the LP nucleic acid: encodes an antigenic peptide sequence from an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) of Table 1, 2, 3, 4, 5, 6, 7, or 8 encodes a plurality of antigenic peptide sequences from an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) of Table 1, 2, 3, 4, 5, 6, 7, or 8 encodes
  • the invention provides a cell or tissue comprising such a recombinant LP nucleic acid.
  • Preferred cells include: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
  • kit embodiments include a kit comprising the described LP nucleic acid, and: a compartment comprising the LP nucleic acid; a compartment further comprising an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346) protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • the kit is capable of making a qualitative or quantitative analysis.
  • LP nucleic acid embodiments include those which: hybridize under wash conditions of at least 42°C, 45°C, 47°C, 50°C, 55°C, 60°C, 65°C, or 70°C and less than about 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 100 mM, to an LP of SEQ ID NO: X that exhibit identity over a stretch of at least about 30, 32, 34, 36, 38, 39, 40, 42, 44, 46, 48, 49, 50, 52, 54, 56, 58, 59, 75, or at least about 150 contiguous nucleotides to an LP (LP318a, LP318b, LP288, LP289, LP343, LP319a, LP319b, LP321, LP317, LP283, LP344, LP345, or LP346).
  • 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
  • Polynucleotide sequences encoding an LP of the present invention are analyzed with respect to the tissue sources from which they were derived.
  • Various cDNA library/tissue information described herein is found in the cDNA library/tissues of the LIFESEQ GOLDTM database (Incyte Genomics, Palo Alto CA.) which corresponding information is incorporated herein by reference.
  • LIFESEQ GOLDTM database a cDNA sequence is derived from a cDNA library constructed from a primate, (e.g., a human tissue).
  • Each tissue is generally classified into an organ/tissue category (such as, e.g., cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract).
  • organ/tissue category such as, e.g., cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of libraries in each category is counted and divided by the total
  • each LP sequence of the invention is also searched via BLAST against the UniGene database.
  • the UniGene database contains a non-redundant set of gene- oriented clusters.
  • Each UniGene cluster theoretically contains sequences that represent a unique gene, as well as related information such as the tissue types in which the gene has been expressed and map location.
  • Particularly interesting portions, segments, or fragments of LP's of the present invention are discovered based on an analysis of hydrophobicity plots calculated via the "GREASE” application, which is a computer program implementation based on the Kyte- Doolittle algorithm (J. Mol. Biol.
  • LP318a(cl6hDGL) & LP318b(c22hDGL) are novel human polypeptides.
  • LP318a(cl6hDGL) nucleic acid sequence was discovered using a normalized human brain cDNA Library whose construction is based generally on methods of Ko (1990) Nucleic Acids Res. 18(19): 5705-11, and Soares, et al (1994) Proc. Natl. Acad. Sci.91:9228-9232.
  • tissues from twelve brain subregions (Hypothalamus, Thalamus, Amygdyla, Sensory Cortex, Motor Cortex, Hippocampus, Cerebellum, Pons and Locus Coeruleus, Caudate/Putamen/Nucleus Accumbens, Entero-Cortex and Anterior Hippocampus, Prefrontal cortex, Anterior Cingulate Cortex) were obtained from the Harvard Medical School Tissue Bank and used to make mRNAs aliquots that were used to generate cDNAs.
  • the cDNAs were amplified using the polymerase chain reaction (PCR) and subsequently normalized by determining the ratio of high-, medium-, and low- abundance control genes.
  • PCR polymerase chain reaction
  • LP318a(cl6hDGL) sequence was discovered using this library.
  • LP318b(cl6hDGL) was discovered using nucleic acid sequence information obtained from LP318a(cl6hDGL). Sequence analysis of LP318a(cl6hDGL) amino acid structure demonstrates that
  • 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.
  • LP318b(cl6hDGL) nucleic acid sequence was localized to the C22qll region of human chromosome 22.
  • the following diseases, conditions, syndromes, disorders, and/or pathological states have also been mapped to this and surrounding regions of human chromosome 22, such as, for example: CATCH 22 syndrome, which is a spectrum of human conditions collectively referred using this medical acronym to refer to the cardiac anomalies, abnormal facial features, thymic hypoplasias, cleft palate, hypocalcemias, and chromosome 22 microdeletions that are associated with it (see, e.g., Krahn, et al., 1998 Mayo Clinic Proceedings.
  • CAFS conotruncal anomaly face syndrome
  • hypoparathyroidism Patients with DiGeorge syndrome may present with impaired immune function, heart failure, hypocalcemia, facial dysmorphism, impaired hearing, and mental retarda ⁇ on.
  • the syndrome which is a significant cause of heart and craniofacial defects as well as mental retardauon, is probably underdiagnosed and presents a large spectrum of presentation, from cases where the most prominent feature of the syndrome is hypocalcemia with hypoparathyroidism, to cases with asymptomatic, latent or late-onset hypocalcemia.
  • a recommenda ⁇ on to clinicians whom have patients presenting with late-onset or recurrent hypoparathyroidism is to perform a geneuc analysis of the human 22ql 1 region to determine if the individual has a feature of DiGeorge syndrome (see, e.g., Hong R.
  • an isolated and/or recombinant DNA molecule comprising LP318b(cl6hDGL) nucleic acid sequence meets the statutory utility requirement of 35 U.S.C. ⁇ 101 since it can be used to hybridize near sequence associated with one or more of the above stated diseases, conditions, syndromes, disorders, and/or pathological states and thus, LP22hDGL would serve as a marker for a such a disease, condiuon, syndrome, disorder, and/or pathological state.
  • compositions comprising LP318b(cl6hDGL) polypeptides or polynucleotides (fragments thereof), LP318b(cl6hDGL) agonists or antagonists, and/or binding compositions (e.g, LP318b(cl6hDGL) antibodies) will also be useful for diagnosis, prognosis, treatment, amelioration, and/or intervention of such an above referenced disease, condition, or state.
  • LP318a(cl6hDGL) and LP318b(cl6hDGL) are encoded by paralogous genes, which arose during some gene duplication event.
  • LP318a(cl6hDGL) nucleic acid sequence is not located on human chromosome 22, it also will be useful as a distinct marker for detecting, marking, associating with, and/or diagnosing individuals present with CTAFS, VCFS, DiGeorge, CATCH 22 or CTAFS-, VCFS-, DiGeorge-, or CATCH 22-l ⁇ ke phenotypes. Such situations are not uncommon.
  • LP318a(cl6hDGL) does not map to the chromosomal region deleted in, for example, CATCH 22 patients an LP318a(cl6hDGL) mutein or variant could have evolved a degree of independence to modify such conditions or LP318a(cl6hDGL) could affect other components of the same signaling pathway (see, e g, a similar situation described by Clouthier, et al, 1998 Development 125: 813-824, where endothelin receptor dysfunction contributes to cranial and cardiac defects that mimic CATCH 22 phenotypes).
  • VCFS velocardiofacial syndrome
  • an isolated and/or recombinant DNA molecule comprising LP318a(cl6hDGL) nucleic acid sequence also meets the statutory utility requirement of 35 U S C. ⁇ 101 since it can be used to hybridize near sequence associated with one or more of the above stated diseases, conditions, syndromes, disorders, and/or pathological states and thus serve as a marker for a such a disease, condition, syndrome, disorder, and/or pathological state.
  • 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).
  • Such behavioral abnormalities have been associated with defects in brain morphology (such as, e.g, defects in the midline structures of the brain) (see, e.g, Vataja & Elomaa 1998 Brit J of Psychiatry 172: 518-520).
  • genes encoding components of the nodal signaling pathway must be expressed only on one side of structures in the developing embryo to ensure correct placement and patterning. Errors in the nodal signaling pathway, for example, randomize the sidedness and morphology of the heart and other organs.
  • 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.
  • midline tissues in the developing vertebrate brain repress genes such as, e.g, cyclops and pitx2 on the right side of the developing diencephalon thus leading to morphological asymmetries of the nervous system.
  • LP318a(cl6hDGL) may play a role in this system, for example, by having an effect on the nodal signaling system or by, e.g, modulating the pitx2 or cyclops effector portion of the system.
  • the Drosophila protein with sequence similarity to LP318a(cl6hDGL) and LP318b(cl6hDGL) (see Table 1 below) is proposed to be a ligand protein further supporting such a ligand like function here for LP318a(cl6hDGL) or LP318b(cl6hDGL).
  • a two-hybrid type of system for identifying protein-protein interactions is encompassed herein to determine potential interactions of LP318b(cl6hDGL) and/or LP318a(cl6hDGL) with any of the currently described proteins known to influence morphological asymmetries (e.g, such as those described for the brain in Concha, et al, 2000 Neuron 28: 399-409; and Liang, et al, 2000 Development 127:5101-112).
  • morphological asymmetries e.g, such as those described for the brain in Concha, et al, 2000 Neuron 28: 399-409; and Liang, et al, 2000 Development 127:5101-112).
  • Such methods of determining protein-protein interactions are well known in the art (see, e.g. Fields and Song, 1989 Nature 340:245-6 for descriptions of the original yeast two-hybrid system design.
  • 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.
  • LP318a(cl6hDGL) sequence (SEQ ID NO: 1) is expressed in the following number of LIFESEQ GOLDTM database tissue and cDNA libraries- Genitalia, Female 1/106; Genitalia, Male 4/114,Germ Cells 1/5, Hemic and
  • compositions comprising
  • LP318a(cl6hDGL) polypeptides (or fragments thereof), polynucleotides (or fragments thereof), and/or LP318a(cl6hDGL) antibodies (or LP318a(cl6hDGL) binding compositions), and related reagents are also useful for the diagnosis, prognosis, treatment, amelioration, and/or intervention of a disease, condition, or state including, but not limited to, e.g, cell proliferative, autoimmune /inflammatory, coagulative, cardiovascular, neurological, and developmental disorders.
  • LP318a(cl6hDGL) (start (atg) and stop (tga) codons are indicated in bold typeface and underlined) .
  • the underlined portion is a predicted signal sequence (Met-1 to Lys-40)
  • a predicted SP cleavage site is between Lys-40 and Ala-41 indicated as follows 1
  • An optional predicted signal sequence MRLPPKVIFLLRSISKAVA (Met-1 to Ala-19) based on a different signal peptide analysis allocates an alternative cleavage site between Ala-19 and Ala-20 indicated as follows 1
  • MRLPPKVIFLLRSISKA VA ⁇ ATDWA 24 Alternative cleavage points may represent alternative mature LP318a(cl6hDGL) variants (all of which are encompassed herein)
  • MRLPPKVIF LRSISKAVAATD AHSGHRWVTGSRTFDRKAMGCQWP C
  • VSPGVQVT NLHGEASYLLQA LGSLCSPWAAPRVGP
  • An LP318a(cl6hDGL) Mature Sequence (172aa) Mature Sequence (172aa) :
  • a predicted mature LP318a(cl6hDG ) sequence is as follows:
  • LP318a(cl6hDGL) Additional interesting sections of LP318a(cl6hDGL) are the discovered portions of LP318a(cl6hDGL) from Pro-4 to Arg-12; Ser-13 to His-28; Trp-30 to Thr-36; Arg-39 to Leu-50; Trp-51 to Thr-59; Leu-60 to Leu-73; T -106 to Cys-116; Ala-117 to Ile-130; Ser- 131 to Gly-139; Phe-144 to Glu-156; Leu-161 to Asp-169; and Gly-196 to Asn-209. These fragments were discovered based on analysis of antigenicity plots.
  • LP318a(cl6hDGL) structures e.g., such as a helix, a strand, or a coil
  • LP318a(cl6hDGL) helix structures Leu-11 to Val-18; Ser- 67 to Ala-72; and Glu-184 to Phe-187.
  • coil structures are Met-1 to Pro-4; His-25 to His-28; Gly-33 to Asp-38; Ala-41 to Trp-46; Ser-53 to Gly-55; Leu-62 to Glu-65; Gly-74 to Ala-93; Ser-98 to Pro-100; Cys-110 to Cys-110; His-126 to His- 126; Phe-134 to Gly-139; Val-150 to Ala-155; Glu-173 to Lys-182; Glu-195 to Gly-196; and Asp-208 to Asp-212.
  • strand structures are Leu-48 to Trp- 51; Gln-57 to Leu-60; Met-94 to Arg-96; Ala-141 to Leu-143; and Tyr-197 to Glu-201. Further encompassed by the invention are contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one coil-strand- coil-helix-coil-strand-coil motif of LP295 combines the Ser-53 to Gly-55 coil, with the Gln- 57 to Leu-60 strand, the Leu-62 to Glu-65 coil, the Ser-67 to Ala-72 helix, the Gly-74 to Ala- 93 coil, the Met-94 to Arg-96 strand, and the Ser-98 to Pro-100 to form an interesting fragment of contiguous amino acid residues from Ser-53 to Pro-100.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • 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.
  • LP318b(cl6hDGL) structures e.g., such as a helix, a strand, or a coil
  • LP318b(cl6hDGL) helix structures Ile-4 to Leu-9; Phe-52 to Ala-61; Thr-91 to Gly-98; Thr-107 to Phe-115; His-117 to Arg-121; Ile-145 to Ala-164; Glu-171 to Asp-178; Ile-188 to Glu-196; Asn-205 to Leu-208; Gln-281 to Ser- 285; Glu-290 to Lys-296; Pro-301 to Ile-304; Lys-330 to Ser-334; and Gln-352 to Lys-356.
  • 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.
  • strand structures are Ala-17 to Leu-19; Val-129 to Ile-131; Ala-270 to Ile-273; and Met-392 to Ile-395.
  • contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one helix-coil-coil-strand-coil motif of LP318b(cl6hDGL) combines the Gln-352 to Lys-356 helix, with the Val-361 to Thr-366 and Gln-381 to Pro-390 coils, and the Met-392 to Ile-395 strand to form an interesting fragment of contiguous amino acid residues from Gln-352 to Ile-395.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP288 is a novel primate (e.g., human) polypeptide (SEQ ID NO: 6), which is a newly discovered member of the LDL receptor family of proteins. Specifically, LP288 appears to be a member of the LDL receptor-related group of proteins (LRPs), which are found throughout the animal phyla ranging, for example, from invertebrates (such as, e.g., worms such as, e.g., Caenorhabditis elegans), to insects (such as, e.g., Drosophila melanogaster), to birds (e.g., chickens), to mammals (e.g., rodents), and to primates (e.g., humans).
  • LRPs LDL receptor-related group of proteins
  • LRPs LDL receptor-related proteins
  • LRPs exhibit typical ligand binding characteristics — high affinity and broad specificity or a "one-receptor-many-ligand" profile.
  • LRPs were considered simply as cellular transporters for cholesterol and other lipids, however, this view has changed and other important functions exist for these proteins (e.g., such as regulators of developmental processes and participants in synaptic transmission) (see, e.g., Gotthardt, et al. 2000 J. Biol. Chem.
  • LP288 like other LRPs, will also recognize non-lipoprotein ligands and function in a wide variety of biological processes.
  • an insect LRP Drosophila arrow
  • LP288 exhibits sequence similarity to both Arrow and another recently described insect LRP (designated CG8909) supporting the view that LP288 may also function in the vertebrate Wnt/Wng signaling pathway
  • LRPs share common structures (such as e.g., amino acid motifs, modules, and/or domains) that are arranged in characteristic locations within an LRP (e.g., most easily visualized with respect to their positioning in a primary LRP amino acid sequence). For example, the following amino acid motifs, modules, and/or domains are routinely found in characteristic locations in LRPs.
  • LRP ligand-binding-like domains such as, e.g., the low-density lipoprotein receptor class A module
  • LRP-A module or domain contains between 2-12 complement-type cysteine rich repeats.
  • LP288 contains approximately eight LRP-A-like domains at its N-terminad most location.
  • one or several epidermal growth factor (EGF)-l ⁇ ke domains are characteristically positioned LP288 contains two such EGF-like domains at this location.
  • EGF epidermal growth factor
  • LP288 contains two such EGF-like domains at this location.
  • N-terminal- most EGF-like domains is a region characterized by the presence of multiple YWTD-like motifs that is flanked C-terminad by one or several EGF- like domains Characteristically, in all LRPs reported to date, it has been shown (Springer 1998 J. Mol Biol.
  • YWTD containing regions are never contiguous but are always separated by EGF- or FN3-l ⁇ ke domains.
  • some YWTD regions are flanked by single EGF-like domains while other YWTD regions are flanked by multiple (e.g., up to 11 EGF-like domains in the C. elegans LRP1), however two YWTD regions are never adjacent).
  • LP288 exhibits such an ordering so that each LP288 YWTD region is made up of multiple YWTD-like motifs that are not contiguous but are separated by one or more flanking EGF-like domains
  • flanking EGF-domains As models, Springer demarcated the boundaries of LDR EGF-like domains as being approximately two or three amino acid residues before the first cysteine of the EGF-like domain and two or three residues after the last cysteme of the EGF-like domain.
  • Every LRP YWTD containing region is made up of six separate YWTD repeat sequences (each repeat sequence, designated as YWTD repeat Nos. 1-6, is approximately 40-44 residues in length).
  • a set of six YWTD repeats make up a structural unit that Springer defines as a single YWTD domain (to avoid confusion, Appbcants refer to similar LP288 regions as YWTD islands).
  • the sequence and structure of the LP288 YWTD regions conform to such a model.
  • Each YWTD region in LP288 can be further subdivided into a set of six individual YWTD-like repeats, which possess specific characteristic features. Every LP288 YWTD island is bounded by at least one EGF-like domain.
  • 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.
  • each YWTD island is characteristically made up of six individual YWTD repeats (designated, from the N- to C-terminad direction, as YWTD repeats Nos. 1- 6). Each YWTD island is flanked by one or more EGF-like domains. After analyzing YWTD repeats from 89 such YWTD islands, Springer demonstrated that each YWTD repeat has within it a characteristic motif sequence (comprising about five contiguous amino acid residues), which is located at the beginning of the repeat (e.g., within the first 5-7 amino acids of the repeat sequence). For example, YWTD repeats Nos.
  • YWTD-like consensus motif e.g., Tyr-Trp-Thr-Asp
  • a different (though similar) motif is characteristically found.
  • similar residues such as, LFAN (Leu-Phe-Ala-Asn)
  • the repeat sequences found at YWTD repeat position No. 1 are similar in length to other YWTD repeat sequences and, except for the YWTD-like motif itself, sequences at the No. 1 repeat positions are as homologous to other repeats (e.g., those at positions Nos.
  • LFAN-like motifs have also been shown to be approximately equivalent to YWTD-like motifs (see, e.g., J. Mol. Biol (98) 283: 837-62), further supporting the idea that a repeat sequence at position No. 1 is, in fact, a YWTD repeat sequence like the repeat sequences at positions Nos. 2-6.
  • Springer also suggests that another characteristic feature of YWTD repeat motifs is that the amino acid residue positioned before each YWTD- like or LFAN-like motif is occupied by a hydrophobic amino acid residue. This feature is typically found in every repeat throughout every YWTD island.
  • LP288 has four YWTD islands, each of which is flanked by at least one EGF-like domain. Every LP288 YWTD island is made up of six individual YWTD repeats, which are approximately 40-43 contiguous amino acids in length. Moreover, each LP288 YWTD repeat contains an amino acid motif sequence, located at the beginning of the repeat,that has either a YWTD-like or LFAN-like motif depending upon which position the motif sequence occupies in the YWTD island (e g., position No. 1, 2, 3, 4, 5, or 6). For instance, as predicted, all LP288 position No. 1 repeats contain an LFAN-like motif at the beginning of the repeat sequence.
  • LP288 hLFAN-like motifs are found at the No. 1 repeat positions of each of the four LP288 YWTD domains: LLFAN (for LP288 YWTD domain 1), LLFAR (for LP288 YWTD domain 2), LIFAR (for LP288 YWTD domain 3), and LLFSS (for LP288 YWTD domain 4). Comparison of these four motifs results in a consensus LFAN-like motif for LP288 of "lFup.”
  • all LP288 repeat sequences at positions Nos. 2-6 in the LP288 YWTD islands contain a YWTD-like motif preceded by a hydrophobic amino acid residue.
  • a hydrophobic amino acid residue For example, the following hYWTD-like motifs (where h is any hydrophobic amino acid residue) are found at the beginning of LP288 YWTD repeat positions Nos.
  • VFWSD VFWSD, LYWTD, IYWTD, MYWVD, and LYWTD (for LP288 YWTD domain 1); VYWTD, LYWTD, MYWTD, LYWAD, and IYWTD (for LP288 YWTD domain 2); VYWSD, VYWTD, MYWTD, LYWAD, and IYWTD (for LP288 YWTD domain 3); and VYYTD LYWTD, LFWTD, IYWVD, and IYWTD (for LP288 YWTD domain 4).
  • a cladistic-like analysis comparing the sequences of every YWTD repeat in LP288 shows that a positional effect exists for the sequence of any particular YWTD repeat within any LP288 YWTD domain. So that, for instance, a repeat sequence at position No. 3 is more similar to any other repeat sequence at that same position (i.e. No. 3) in any other island than it is to other YWTD repeat sequences. For example, all four position No. 1 YWTD repeat sequences in LP288 are more closely similar (using sequence identity as a measure) to each other than to repeat sequences at other positions in the LP288 YWTD islands.
  • LP288 YWTD islands' 1 and 2 are ad j acently ordered followed by the subsequent adjacent ordering of islands 3 and 4.
  • sequence similarity combined with such ordering suggests that the conservation of sequences within a particular positional YWTD repeat is maintained to functionally conserve the resulting higher order structures that make up the blades of the beta-propeller encoded by a YWTD island as each repeat in a YWTD island is used to construct subunits of the beta- propeller structure.
  • adjacent blades of separate beta-propeller structures have been proposed to interact to form higher order structures. Accordingly, it is possible that repeat position No. 5 in LP288 YWTD islands' 1 and 2 and/or repeat position sequences No.
  • LP288 islands' 3 and 4 may interact to form higher order multi-beta-propeller structure/ s (see, e.g., Fulop and Jones 1999, Curr. Opin. Struct. Biol. 9:715-721). Furthermore, it is possible that a particular position of an LP288 YWTD repeat within a YWTD island has a similar structural and/or functional role from one island to another thus compelbng evolutionary selection to maintain sequence similarity at specific repeat positions between different islands.
  • LRPs e.g., vertebrate LRP1, and LRP2
  • LRPs also exhibit groupings of two or four YWTD islands that are separated from other such groups by only single EGF-bke domains between the adjacent YWTD islands of a group.
  • Springer suggests that the proximity of these LRP YWTD islands imply that they act in concert, for example, in binding a single bgand.
  • the groupings in LP288 add further weight to the evidence that LP288 is an LRP.
  • the groupings of YWTD islands in LP288 may convey a similar function by promoting mutual interactions (e.g., to bind a single bgand).
  • LDLRs low-density bpoprotein receptors
  • LRPs beta-propeller structures proposed to form from YWTD islands (see, e.g., Springer 1998 J. Mol. Biol. 283:837-62 and references cited therein).
  • An additional characteristic feature of LRPs are that these receptors typically have a single transmembrane segment which is followed by a cytoplasmic tail containing characteristic intracellular binding motifs.
  • LP288 possesses both a single transmembrane segment and intracellular binding motifs that have been described as being characteristic for LRPs.
  • LDLRs characteristically have at least one NPxY motif in their cytoplasmic tail (where N is asparagine; P is probne; Y is tyrosine; and x is any amino acid residue).
  • NPxY motifs have been reported to function as internabzation signals (e.g., required for clustering of LRPs into coated pits).
  • others have recently proposed that a cytoplasmic YxxL-bke motif and di-leucine repeats (LL) rather than NpxY motif, serve as the dominant agents for LRP endocytosis (see, e.g., Li, et al. 2000 J. Biol.
  • LP288 contains both NPxY-bke (e.g., NPSY), YxxL-bke (e.g, YNQL, and WDDL), YxxP-bke (e.g, YSNP), and di-leucine repeat (LL) (e.g, GLLR, and QLLQ) sequence motifs (among others) in its cytoplasmic portion.
  • NPxY-bke e.g., NPSY
  • YxxL-bke e.g, YNQL, and WDDL
  • YxxP-bke e.g, YSNP
  • LL di-leucine repeat
  • LRPs may both endocytosis (e.g, through coated pits) and also function via non-internabzed intracellular signabng pathways (e.g, MAP kinase pathways) (see, e.g, Gotthardt, et al. 2000 J. Biol. Chem 275 No. 33: 25616-25624, and Pandur & Kuhl, 2001 BioEssays 23:201-210).
  • the abibty to direct an LRP toward one or another pathway e.g.
  • internatibzed or non-internabzed may be mediated depending upon what particular binding partners are complexed with the cytoplasmic portion of an LRP.
  • DABl binding to LRP tails competes and prevents bound LRP from clustering into coated pits and thus, DABl prevents subsequent LRP endocytosis. This data suggests a mechanism by which alternative signaling through LDL receptor family members is accompbshed.
  • LRP cytoplasmic tail e.g, via scaffold and adaptor proteins that may be engaged in intracellular signabng pathways
  • LRP system e.g., via scaffold and adaptor proteins that may be engaged in intracellular signabng pathways
  • Such a multi-functional LRP system would be dynamic depending on various factors controbng it (such as, e.g, the number and kind of binding partners, the on-off rates of binding, binding partner local concentrations, the timing, and/or seeding of initial complex binding partners, binding constants and/or specificities or affinities to particular LRP cytoplasmic motifs, etc.).
  • the NPxY motif is also a substrate for various intraceUular binding partners (such as, e.g, the She adaptor protein, which has the abibty to interact with a large number of tyrosine-phosphorylated proteins), and that the NPxY motif also binds a PI/PTB domain, which itself has been identified as a binding motif in the She adaptor protein (Margobs 1996 J Lab Cbn Med 128(3): 235-41), the NPxY motif may be a context-dependent motif that functions both in endocytosis or via an intraceUular signabng pathway depending upon the interaction of other members of a binding complex on an LRP cytoplasmic tail. Such a system would resemble the combinatorial-bke functioning features of DNA transcriptional regulator complexes (e.g, bke those seen in "turning on” HOX genes).
  • 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.
  • a given LRP receptor may bind multiple signabng molecules, yet activation of the appropriate intraceUular signabng pathway would depend on the particular binding partner members of a complex, e.g. such as co-receptors (e.g, Wnt) for the extraceUular portion of a complex and/or adaptor or scaffold proteins for the intraceUular portion of a complex.
  • co-receptors e.g, Wnt
  • Wnt co-receptors
  • Appbcants invention encompasses such complexes that are formed with LP288 (e.g, both extraceUular, intraceUular, and complexes including both intra- and extra-ceUular complexes).
  • Appbcants invention encompasses LP288 variants whose bias drive pathway specificity for LP288 binding complexes, for example, by biasing the formation of particular LP288 complexes (both intra- and extra-ceUularly).
  • LP288 variants are encompassed herein in which, for example, cytoplasmic-bke motifs are removed, added, and/or mutated.
  • LP288 variants are encompassed herein that, e.g, bias which pathway an LP288 would take (e.g, endocytosis, or intraceUular signabng).
  • Non-bmiting examples of such LP288 variants include those in which, for example, YxxL, NPxY, tSxV, or YxxP LP288 motifs have been removed, added, relocated, and/or mutated (e.g, such as by substituting a tyrosine residue (Y) for an X in an YxxP motif; or by adding a second terminal SxV motif to the end of an LP288 cytoplasmic tail to, e.g, increase the binding affinity for, e.g, PDZ domains).
  • bias which pathway an LP288 would take e.g, endocytosis, or intraceUular signabng.
  • Non-bmiting examples of such LP288 variants include those in which, for example
  • LRPs interact (e.g, through proteins having either PID or PDZ domains, with a much broader range of proteins than had previously been recognized (such as, e.g, cytoplasmic adaptor and scaffold proteins such as, e.g, SEMCAP-1, JIP-1, PSD-95, JIP-2, Tabn homologue, OMP25, CAPON, DABl, ICAP-1, MINT2, PIP4, 5-Kinase homologue, Sodium channel brain 3, and APC subunit 10).
  • proteins having either PID or PDZ domains with a much broader range of proteins than had previously been recognized (such as, e.g, cytoplasmic adaptor and scaffold proteins such as, e.g, SEMCAP-1, JIP-1, PSD-95, JIP-2, Tabn homologue, OMP25, CAPON, DABl, ICAP-1, MINT2, PIP4, 5-Kinase homologue, Sodium channel brain 3, and APC subunit 10).
  • binding partners of LP288 signabng complexes that form with a cytoplasmic portion of LP288 such as, e.g, the binding partners SEMCAP-1, JIP-1, PSD-95, JIP-2, Tabn homologue, OMP25, CAPON, DABl, ICAP-1,
  • an insect LDLR protein (Drosophila arrow, which is homologous to vertebrate LRP5 and LRP6) forms extraceUular and intraceUular heteromeric complexes with members of the Wnt/Wg signabng pathway, such as, for example the transmembrane proteins Frizzled (Fz) and Dfrizzled (DFz2) (see, e.g, Pandur & Kuhl, 2001 BioEssays 23:201-210).
  • Frizzled Frizzled
  • DFz2 Dfrizzled
  • LP288 shows sequence similarity to both the Drosophila arrow protein and to another Drosophila LDRL-bke protein related to arrow (designated CG8909). Both arrow and CG8909 are reported to be LDRL-bke receptors exhibiting sequence similarity to primate, rodent (LRP5 and LRP6), fish, and worm LDRL-bke receptor proteins (see, e.g, the report on CG8909 in the Drosophila Flybase located at http: //flybase.bio.indiana.edu).
  • LP288 may also play a role in developmental events mediated by vertebrate homologs of the Wnt/Wg cascade of proteins (e.g, such as vertebrate homologs of DrosopHla genes known to act downstream of wingless, such as, e.g, Adenomatous polypopsis cob tumor suppressor homolog 2, Ape, armadiUo, arrow, Axin, decapentaplegic, disheveUed, engrailed, eyebd (antagonizes Wingless signabng), frizzled (receptor for Wingless), frizzled2 (receptor for
  • 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
  • a Xenopus dsh protein when co-expressed with rat frizzled family members in a Xenopus blastomer, translocates from a cytoplasmic pool to a membrane location, indicating both the conserved functional association of these proteins and their sequence conservation (since one protein is from an amphibian and the other is from a rodent).
  • phenotypes resulting from mutations in a mouse homolog of arrow (LRP6) display a variety of defects such as, for example, midbrain/hindbrain morphogenetic defects, axis truncations, and bmb patterning defects.
  • LP288 has five PxxP-bke motif sequences (PDEP, PPAP, PVLP, PNTP, and PAAP), where "P" is Probne, and x is any amino acid residue.
  • PxxP motifs are found intraceUularly in probne rich regions, for example, such as in the cytoplasmic tails of receptors. The PxxP motif is known to bind with SH3 domains of various intraceUular proteins (see, e.g, Kay, et al. 2000 FASEB J. 14:231-241).
  • the intraceUular binding sites in an LP288 would control the system (e.g, leading to activation of a different signabng cascade, such as, for example, a JAK/STAT pathway).
  • LP288 did become internabzed (e.g, via binding of unique extraceUular bgand/s or, e.g, by formation of a specific extraceUular binding complex)
  • the previously sequestered PxxP motifs would now, in effect, become "unmasked" via internabzation and be made available for interaction with, for example, SH3-bke binding partners.
  • LP288 is a novel LRP member of the low-density bpoprotein receptor family.
  • LP288's possession of a repertoire of domains and modules, that have been shown to characteristic for the LDLR family e.g, having multiple bgand binding domains, EGF modules, YWTD doma ⁇ n(s), a single transmembrane 'domain', and a cytoplasmic tail with characteristic sequence motifs.
  • LP288 nucleic acid sequence (SEQ ID NO: 5) is expressed in the foUowing number of LIFESEQ GOLDTM database tissue and cDNA bbranes: Cardiovascular System 5/68; Connective Tissue 2/47; Digestive System 16/148; Embryonic Structures 3/21; Endocrine System 6/53; Exocnne Glands 5/64; Genitaba, Female 11/106; Genitaba, Male 13/114; Hemic and Immune System 19/159; Liver 5/35; Musculoskeletal System 3/47; Nervous System 65/198; Pancreas 2/24; Respiratory System 6/93; Skin 2/15; and Urinary Tract 7/64.
  • compositions comprising LP288 polypeptides (or fragments thereof), polynucleotides (or fragments thereof), and/or LP288 antibodies (or LP288 binding compositions), and related reagents are also useful for the diagnosis, prognosis, treatment, ameboration, and/or intervention of a disease, condition, or state including, but not bmited to, e.g, ceU probferative, autoimmune/inflammatory, coagulative, cardiovascular, neurological, and developmental disorders.
  • the underlined portion is a predicted signal sequence (Met-1 to Ala-20).
  • a predicted SP cleavage site is between Ala-20 and Ser-21 indicated as follows: 1 MRRQWGALLLGALLCAHGLA ⁇ SSPE 24.
  • LP288 variant encompassed by the present invention (comprising generally the extracellular portion of a mature LP288). Such an LP288 variant could be used as a competitive binding agent for various LP288 ligands.
  • An LP288 Variant ( 154aa) : The following is an LP288 variant encompassed by the present invention (comprising generally the intracellular portion of LP288) Such an LP288 variant could be used as an agonist or antagonist for LP288 intracellular signaling
  • LP288 interesting portions of LP288 are the segments: Cys-378 to Cys-393 (CTCHTGYRLTEDGHTC ) , Cys-418 to Cys-433 ( CWCETGYELRPDRRSC ) , and Cys-722 to Cys- 736 (CACPTGFRKISSHAC ) which have been discovered to be EGF-bke domain signatures.
  • an EGF domain includes six cysteine residues (here, they would be LP288 cysteines: C378, C393, C418, C433, C722, and C736).
  • An additionaUy interesting segment of LP281 is the segment Asp-395 to Cys-418 ( DVNECAEEGYCSQGCTNSEGAFQC ) , which has been discovered to be a calcium-binding EGF-bke domain.
  • An additionaUy interesting portion of LP288 is a segment identified as a potential aspartic acid and asparagine hydroxylation site (CTNSEGAFQCWC, from Cys-409 to Cys-420).
  • AdditionaUy, interesting portions of LP288 are segments: Cys-44 to Cys-66 ( CIPAQWQCDGDNDCGDHSDEDGC ) , Cys-83 to Cys- 105
  • LDLRA modules of LP288 are segments: Cys-42 to Glu-63 (CTCIPAQWQCDGDNDCGDHSDE) , Cys-83 to Cys-105 (CIRRSWVCDGDNDCEDDSDEQDC) , Cys-122 to Cys-143 (CIRSLWHCDGDNDCGDNSDEQC) , Cys-160 to Cys-182 (CIAEHWYCDGDTDCKDGSDEENC), Cys-203 to Cys-225 (CILDIYHCDGDDDCGDWSDESDC), Cys-243 to Cys-265 ( CINAGWRCDGDADCDDQSDERNC ) , Cys-282 to Cys-304
  • LDLRA modules have been found in other receptor related to the LDL receptor, for example: vertebrate low-density bpoprotein receptor-related protein 1 (LRPl) (reviewed in Krieger & Herz 1994 Annu. Rev. Biochem. 63:601 -637) and vertebrate low-density bpoprotein receptor-related protein 2 (LRP2) (also known as gp330 or megabn), contain LDLRA modules.
  • LRPl vertebrate low-density bpoprotein receptor-related protein 1
  • LRP2 vertebrate low-density bpoprotein receptor-related protein 2
  • AdditionaUy interesting portions of LP288 are respectively, LP288 YWTD islands 1-4 are approximately:
  • LP288 YWTD repeats (Nos 1-6) for LP288 YWTD island No 1 are approximately: (GPEPVLLFANRIDIRQVLPHRSEYTLLLNNLENAIALDFHH) , (ELVFWSDVTLDRILRANLNGSNVEEWSTGLESPGGLAVDWVH) , (DKLYWTDSGTSRIEVANLDGAHRKVLLWQNLEKPRAIALHP ) , (GTIYWTDWGNTPRIEASSMDGSGRRIIADTHLFWPNGLTIDYA) , (RR Y VDAKHHVIERANLDGSHRKAVISQGLPHPFAITVFED) , and
  • LP288 YWTD repeats Nos. 1-6
  • LP288 YWTD repeats are approximately: (KFLLFARRMDIRRISFDTEDLSDDVIPLADVRSAVALDWDSRD) , (DHVY TDVSTDTISRAKWDGTGQEVWDTSLESPAGLAIDWVTN) , (KLYWTDAGTDRIEVA TDGSMRTVLIWENLDRPRDIWEPMGGY) , (MYWTDWGASPKIERAGMDASGRQVIISSNLTWPNGLAIDYGSQR) , (LYWADAGMKTIEFAGLDGSKRKVLIGSQLPHPFGLTLYGERI) , and (YWTDWQTKSIQSADRLTGLDRETLQENLENLMDIHVFHRRRPPV) AdditionaUy, interesting portions of LP288 are LP288 YWTD repeats (Nos.
  • LP288 YWTD island No. 3 are approximately: ( FLIFARRIDIRWSLDIPYFADV PINITMKNTIAVGVDPQEG ) , ( KVYWSDSTLHRISRANLDGSQHEDIITTGLQTTDGLAVDAIGR) ,
  • LP288 ( PGLVPPAPRATGMSEKSPVLPNTPPTTLYSSTTRTRTSLEEVEGRCSERDARLGLCARSNDAVPAAP) from about Pro-1652 to about Pro-1718. AdditionaUy, interesting portions of LP288 are the LP288 cytoplasmic domain is approximately,
  • the seven-amino acid, C-terminal domain of LP288 contains a terminal tSxV motif (where S is serine, x is any amino acid, and V is vabne). This motif has been suggested to interact with PDZ domains of various intraceUular proteins.
  • LP288 Additional interesting sections of LP288 are the discovered portions of LP288 from Leu-9 to Ala-20; Gly-40 to Asp-56; Asp-64 to Pro-73; Gly-82 to Asp-95; Gln-103 to Leu-126; Asn-133 to Lys-147; Cys-155 to His-164; Pro-189 to Ile-207; Asp-213 to Asp-224; Arg-233 to Cys-250; Glu-262 to Gln-274; As ⁇ -300 to Gln-311; Phe-317 to Lys-329; Pro-351 to Gln-367; Asp-395 to Leu-426; Leu-473 to Leu-481 ; Asn-498 to Val-518; Thr-528 to Asn-539; Leu-540 to Trp-550; His-562 to Pro-577; Ile-579 to Asp- 594; Trp-599 to Val-615; Asn-700 to His-711; Ser-716 to Lys-730
  • LP288 structures e.g, such as a hebx, a strand, or a coil
  • the foUowing LP288 hebx structures Gln-4 to Leu-8; Leu-464 to Asn-466; Asn-469 to Ile-471; Leu-489 to Leu-493; Gln-738 to Arg-748; Arg-979 to Asn-988; Gln-1300 to Arg-1304; Leu- 1406 to Arg-1411; Ile-1493 to Arg-1498; Arg-1538 to Ser-1541; Arg-1700 to Arg-1703; Ala- 1742 to Tyr-1747; Trp-1823 to Ser-1831; and Thr-1860 to Glu-1868.
  • coil structures are Met-1 to Arg-2; Gly-18 to His-31; Leu-39 to Thr-43; Asp-52 to Gly-65; Thr-70 to Leu-74; Cys-78 to Lys-82; Asp-91 to Tyr-121; Cys-129 to Asp-140; Cys- 148 to Ser-159; Cys-167 to Asn-159; Cys-167 to Asn-192; Gly-201 to Gly-201; Cys-210 to Leu-242; Ala-246 to Met-269; Cys-277 to Arg-281; Cys-289 to Gln-316; Trp-320 to Glu-355; Asn-360 to Ala-365; His-380 to Gly-383; Glu-388 to Asp-395; Gly-403 to Ala-415; Cys-419 to Pro-440; Leu-454 to Ser-458; Ser-485 to Ser-485; Asn-496 to Ser-500; Gly-509 to Gly-515;
  • strand structures are Thr-33 to Cys-34; Arg-85 to Val-89; Leu-126 to Trp-127; Ile-161 to Tyr-166; Leu-205 to Tyr-208; Cys-243 to Ile-244; Val-283 to Trp-287; Val-375 to Cys-379; Arg-385 to Leu-386; Val-441 to Phe-444; Glu-503 to Ser-507; Ala-517 to Trp-520; Leu-525 to Trp-527; Arg-534 to Ala-538; Ala-558 to Leu-561; Ile-579 to Ala-581; Arg-590 to Ala-593; Thr-604 to Asp-606; Met-612 to Val-615; Val-621 to Arg-624; Lys-633 to lle-636; Ala-645 to Val-648; His-711 to Leu-712; Thr-721 to Cys-722; Tyr-788 to Thr-790; Glu-808 to Asp-812
  • one hebx-coil- strand-coil motif of LP288 combines the Leu- 1406 to Arg-1411 hebx, with the Asp-1413 to Asn-1418 coil, the Glu-1420 to Ile-1423 strand, and the Arg-1425 to Gly-1432 coil form an interesting fragment of contiguous amino acid residues from Leu-1406 to Gly-1432.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP288 variants such as, e.g, variants comprising only an extraceUular region of LP288.
  • Other LP288 variants encompassed by the present invention are LP288 variants that have mutated, truncated, and or missing cytoplasmic portion/s (e.g, a mutant LP288 lacking the carboxyl intraceUular domain).
  • an LP288 variant lacking a carboxyl intraceUular domain may act as an antagonist to vertebrate members of the Wnt signabng group.
  • the cytoplasmic portion of LP288 contains a tetra-amino- acid motif NPxY, which also plays a central role in mediating the interaction of receptor tails with endocytotic machinery, and also serves as a docking site for cytoplasmic adaptor and scaffold proteins. Mutations in this region of an LDL receptor result in impaired endocytosis of LDL by, e.g, the bver, which in turn leads to elevated plasma cholesterol concentrations and coronary artery disease. Accordingly, further LP288 variants encompassed herein include those involving modifications to LxxY, tSxV an NPxY sequence of LP288.
  • NPxY sequence for example, addition of an NPxY sequence to LP288 cytoplasmic portion is contemplated so that, e.g, increased internabzation of bpoprotein-bke molecules would occur thereby treating, e.g, an atherosclerotic-bke condition.
  • LP288 FUNCTIONS Given the teachings suppbed herein, for example, of: LP288 primary amino acid, LP288 higher order structures; the relationship of LP288 amino acid sequence to higher order structural features; the comparabibty of LP288 sequence and/or LP288 higher order structure with known LRPs (such as, e.g, members of the low density bpoprotein-related protein family, such as, e.g, LRPl, alpha-2-macroglobin receptor, LRP2, LRP5, and LRP6); and the relationship of higher order structural features of LRPs with their known functions; it is bkely that an LP288 or an LP288 variant plays similar roles in a variety of physiological processes.
  • LRPs such as, e.g, members of the low density bpoprotein-related protein family, such as, e.g, LRPl, alpha-2-macroglobin receptor, LRP2, LRP5, and LRP6
  • Some non-bmiting functions an LP288 or LP288 variant is bkely to participate in, modulate, maintain, effect, or regulate are those such as, for example: bgand endocytosis; maintenance of plasma cholesterol concentrations; modulation of bpases; bpoprotein processing; protein and/or remnant scavenging; mediation of the ceUular uptake of apoE-containing, remnant-bke bpoproteins; processing apobpoproteins (e.g, such as ApoE, ApoE-4, etc.); bpid metabobsm, catabobsm, clearance, and/or recycbng; amyloid clearance (e.g, from the nervous system, e.g, from the CNS, e.g, from the brain); bver function; plasma clearance of chylomicron remnants; plasma clearance of activated alpha 2- macroglobubn; local modulation of complexes between plasminogen activators and their endogenous inhibitors (e.g,
  • prevention of uptake of such entities may modulate tumorogenesis); prevention of atherosclerotic disease; maintenance of protease homeostasis; modulation of local serpin concentrations; regulation of protease-serpin complexes; coronary artery diseases, conditions, and/or syndromes; ceU adhesion; ceU migration (e.g, receptor (uPAR)-bound urokinase (uPA) binds its inhibitor PAI-1, which is locaUzed in an extraceUular matrix, and the resulting complex is internabzed by endocytotic receptor activity of LRPs.
  • uPAR receptor
  • uPA urokinase
  • LRP such as, e.g, in the hippocampus
  • brain development e.g, such as in defects of CNS development, such as, e.g. holoprosencephaly
  • embryogenesis e.g, such as axis formation
  • induction of receptor clustering such as, e.g, inducing clustering of NMD A receptors thereby stimulating local Ca +2 influx
  • LRP2 N-terminal portion of LRP2 is the autoantigenic target responsible for the generation of this rodent disease suggesting that a similar LRP2-bke region in other proteins (such as LP288) may also generate similar human conditions or disorders (see, e.g, Oleinikov, et al. 2000 J. Amer Soc Neph 11-57-64, inco ⁇ orated herein for the assay methods described therein).
  • LP288 Given LP288's similarity to LRP2, similar N-terminal cysteine-bke regions in LP288 may also be autoantigenic thus leading to similar autoimmune disorders, conditions, or syndromes that would affect e.g, the kidney); diabetic-bke conditions and/or obesity-bke conditions (recently, it has been shown that insuhn activity regulates LRP presentation in adipocytes and can be inhibited by phosphatidybnositide 3-k ⁇ nase treatment (see, e.g, Ko, et al, 2001 Biochemistry 40:752-759, inco ⁇ orated herein for the assay methods described therein)); aging and/or insubn resistance (e.g, insubn resistance and chylomicron clearance is significantly reduced in aged humans and rodents.
  • insubn activity regulates LRP presentation in adipocytes and can be inhibited by phosphatidybnositide 3-k ⁇ nase treatment (see, e.g
  • age related insubn resistance may lead to detrimental plasma bpoprotein profila due to the reduced expression of LRPs.
  • age related insubn resistance may account for aberrant LRP expression on adipocytes leading to disorders associated with obesity); vitamin metabobsm, organ development; neuronal degeneration; neuronal pathfinding; axon guidance; regulation of synaptic transmission; protein conformational disorders or diseases; Alzheimer or Alzheimer- bke conditions; activation and /or modulation of MAP kinase pathways; local organization of the cytoskeleton; ceU adhesion, and endocytosis of various LDL-bke bgands.
  • Non-bmiting examples of bgands for LP288 include, for instance, bpoproteins containing ApoE, bpases, proteases (such as, e.g, PA, alpha2M, or PAI-1) and/or protease/serpin inhibitor complexes, hepann-binding growth factors (e.g., midkine (MK)), and signabng proteins (such as, e.g. Rein or Reln-bke proteins, vertebrate Wnts or vertebrate Wnt-bke proteins, or TSP-1 or TSP-1-bke proteins).
  • proteases such as, e.g, PA, alpha2M, or PAI-1
  • MK midkine
  • signabng proteins such as, e.g. Rein or Reln-bke proteins, vertebrate Wnts or vertebrate Wnt-bke proteins, or TSP-1 or TSP-1-bke proteins.
  • Non-bmiting examples of cytoplasmic binding partners and/or effectors of bgand binding of LP288 are, e g. endocytotic machinery proteins; Dabl; XI 1; Fe65;J ⁇ pl; J ⁇ p2; PIP 4,5 bnase; PSD95; SEMCAPl, OMP25; ICAP-1; and Capon (among others).
  • LRPs serve as gateways for the entry ceUular of exotoxin A from Pseudomonas aeruginosa (PEA) (see, e.g, Kounnas et al. 1992a J. Biol. Chem. 267:12420-12423).
  • Pseudomonas aeruginosa an increasingly prevalent opportunistic human pathogen, is the most common gram-negative bacterium found in nosocomial infections.
  • An LP288 variant (such as, e.g, an LP288 variant comprising only an extraceUular portion of a mature complete LP288) may play a protective role by acting as a competitive "secreted" binding agent for released exotoxin A thus preventing it from being taken up into ceUs were its subsequent detrimental effects are reabzed. Consequently, LP288 would have a specific utibty in currently available form as a means to treat a known biological poison.
  • LP288 in toxin uptake (such as, e.g, exotoxin A from Pseudomonas aerugionosa (PEA))
  • PPA Pseudomonas aerugionosa
  • LRP deficient ceUs that can be subsequently transfected with various LP288 constructs (using ordinary genetic engineering techniques).
  • 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 is a novel primate (e.g, human) polypeptide (SEQ ID NO: 8), which is a newly discovered member of the immunoglobubn superfamily (IgSF) of proteins.
  • SEQ ID NO: 8 a novel primate polypeptide
  • IgSF immunoglobubn superfamily
  • LP288 is a novel member of the IgLON family, which includes, e.g, such proteins as LAMP, OBCAM, Neurotrimin (NtM), CEPU, GP50, KILON, and GP55. Characteristic features of members of the IgLON family include the presence of conserved cysteines as weU as a number of Asn-bnked potential glycosylation sites. Sequence similarity (at the amino acid level) with other known IgLON proteins suggests that LP289 is involved in ceU recognition, ceU adhesion, and/or opiod-type receptor functioning.
  • IgLONs among the IgLONs described to date, common structures (such as e.g, amino acid motifs, modules, and/or domains), arranged in characteristic locations within an IgLON protein are found.
  • the foUowing amino acid motifs, modules, and/or domains are routinely found in characteristic locations in proteins that are members of the IgLON grouping.
  • a series e.g, typicaUy three
  • immunoglobubn-bke C2-type, domains IgC2
  • Each lmmunoglobuhn-hke C2-type domain characteristicaUy contains two conserved cysteine residues in the domain sequence that form disulfide bonds with each other (when drawn in cartoon form the Ig-C2-bke domains resemble a series of loops, in which each loop is closed by disulfide bridges formed between the two conserved cysteine residues of the Ig-bke-C2- type domain; see, e.g. Fig. 2C in Funatsu, et al. 1999 J. Bio. Chem. 274:8224-8230).
  • LP289 foUows this pattern by possessing three immunoglobubn-bke C2-type domains, each of which has two conserved cysteine residues moreover, LP289 has a number of putative N-bnked glycosylation sites. FoUowing the immunoglobubn-bke C2-type domains (i.e., moving C-terminad along the primary amino acid structure of a typical IgLON), most IgLONs possess a GPI-anchor bke motif foUowed by a hydrophibc spacer region and a C-terminal signal sequence of predominately hydrophobic amino acids. LP289 also exhibits this pattern since it does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of a GPI anchor site membrane by attachment via a phosphatidybnositol bnkage.
  • LP289 exhibits sequence identity/ similarity at the amino acid level to members of the immunoglobubn protein superfamily, most notably with the various IgLON cell-adhesion molecules known as (see, e.g, Schofield, et al. 1989 EMBO J 8:489-495, Hachisuka, et al, 2000 Dev. Brain Res. 122:183-191, Funatsu et al, 2000 J Comp Neurology 424:74-85). LP289 shares sequence homology with: (1) opiod-binding protein ceU adhesion molecule
  • OBCAM also designated OPCML, a protein that binds opioid alkaloids in the presence of acidic bpids, exhibiting selectivity for mu bgands.
  • OPCML a protein that binds opioid alkaloids in the presence of acidic bpids, exhibiting selectivity for mu bgands.
  • OBCAM is pnncipaUy expressed in the gray matter in a pattern that suggests OBCAM plays a role in the synaptic machinery of the nervous system (such as, e.g, modulating opiod receptor functioning) (see, e.g, Loh and Smith 1996 "Regulation of Acute and Chronic Opiod Receptor Functions by OBCAM a ceU Adhesion-bke Molecule" in NIDA Research Monograph 161 titled, "Molecular Approaches to Drug Abuse Research: Vol.
  • OBCAM expression is observed primarily on dendntes of AVP-secreting magnoceUular neurons while KILON is expressed mainly on dendntes of AVP secreting neurons and occasionaUy on OXT-secreting neurons suggesting that KILON and OBCAM confer the abibty of magnoceUular neurons of the hypothalamus to rearrange synaptic connectivity (see, e.g, Miyata, et al, 2000 Jour. Comp.
  • Neurot ⁇ min a subfamily of differentiaUy expressed neural ceU adhesion molecules that have been shown to regulate the development of neuronal projections via attractive and repulsive mechanisms that are ceU type specific and are mediated by homophibc and heterophibc interactions (see, e.g, Struyk, et al. 1995 J. Neurosci. 15:2141-2156, Gil, et al , 1999 J.
  • CEPUS a molecule that provides a favorable route for migrating neurons to generate a neuron-specific guidance in developing neurons in vivo (see, e.g, Kim, et al.1999 Mol CeUs 9(3):270-276); (4) KILON, an IgLON member specificaUy expressed in the dentate gyrus (DG) of the adult rat that is involved in neurite outgrowth and capable of interacting with LAMP (see, e.g, Funatsu, et al. 1999 J. Bio. Chem.
  • DG dentate gyrus
  • LAMP bmbic associated membrane protein
  • LAMP protein may be important in nerve growth and differentiation, epilepsy, Alzheimer's disease, and schizophrenia or schizophrenic-bke conditions. LAMP contributes to the guidance of developing axons and remodebng of mature circuits in the bmbic system.
  • the LAMP protein is essential for normal growth of the hippocampal mossy fiber projection. LAMP is attached to the membrane by a GPI-Anchor. It is expressed on bmbic neurons and fiber tracts as weU as in single layers of the superior colhculus, spinal chord, and cerebeUum.
  • LP289 nucleic acid also has similarities to additional nucleic acids, described as having similar or analogous properties, including (1) chicken mRNA for CEPU-1, an immunoglobubn superfamily molecule expressed by developing cerebeUar Purkinje ceUs (Spaltmann and Brummendor Neurosci. 16 (5), 1770-1779 (1996)); (2) chicken CEPU gene identified as a neural secreted glycoprotein belonging to the immunoglobubn-bke opioid binding ceU adhesion molecule (OBCAM) subfamily, (Kim et al, 1999 Mol. CeUs 9 (3), 270- 276); and (3) Bovine mRNA for opioid binding protein/ceU adhesion molecule OBCAM.
  • OBCAM immunoglobubn-bke opioid binding ceU adhesion molecule
  • PI phosphatidyl inositol
  • LP289 is developmentaUy regulated, for example, other IgLON members show such regulation, e.g, during early development (El 6, embryonic day 16) OBCAM is found on post mitotic neurons and in fiber tracts in the CNS that contain expanding axons suggesting the OBCAM functions in axonal outgrowth.
  • 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.
  • LP289 has a hydrophobic C-terminal sequence consistent with that found on other GPI-linked proteins, typically, this type of sequence is cleaved during post translational processing, such as, e g , when a protein is inserted into a membrane via a GPI linkage (see, e g , Cross, Ann Rev Cell Biol 6 1 -39, 1990, Ferguson and Williams, Ann Rev Biochem 57 285-320, 1988, Gerber et al , J Biol Chem 267 18168-12173, 1992)
  • a putative GPI anchor attachment point in LP289 is indicated (by double underlining) at or near (e g , within 1, 2, 3, 4, or 5, amino acid residues either C-terminad or N terminad to the indicated asparagine), the asparagine (N) in the following LP289 sequence (SLENSAP)
  • the putative GPI anchor attachment point in LP289 is indicated (by double underlining) at or near (e g , within 1, 2,
  • Applicants invention encompasses 1, 2, 3, 4, or 5, amino acid residues either and/or both C terminad and/or N terminad to a predicted GPI site described herein Furthermore, Applicants invention encompasses LP variants in which LP289 alterations prevent a typical GPI modification thereby resulting in a soluble/ secreted LP289 variant Sequence C-terminad to such an LP289 putative GPI anchor site is typically removed during processing to a mature LP289 form LP289 sequence that is typically removed during process of LP289 is indicated below by waved underling (SAPRPPGLLALLSALGWLWWRM) LP289 polypeptides encompassed herein include full-length forms encoded by an ORF disclosed herein, as well as any mature forms therefrom Such a mature LP289 could be formed, for example, by the removal of a signal peptide Further as used herein, a "mature" LP encompass, e g , post-translational modifications other than proteolytic
  • 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 further alternate predicted mature LP289 sequence is as follows:
  • 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.
  • a non-limiting example of such a variant LP289 sequence encompassed herein is as follows: VISRGLLSQSLEFNSPADNYTVCEGDNATLSCFIDEHVTRVAWLNRSNILYAGNDRWTSDPRVRLLINTPEE FSILITEVGLGDEGLYTCSFQTRHQPYTTQVYLIVHVPARI TSriSSPVTVNEGGNVNLLCLAVGRPEPTVT RQLRDGFTSEGEILEISDIQRGQAGEYECVTHNGV SAPDSRRVLVTVNYPPTITDVTSARTALGRAALLRC EAMAVPPADFQ YKDDRLLSSGTAEGLKVQTERTRSMLLFANVSARHYGNYTCRAANRLGASSASMRLLRPG SLE* Analysis of the primary amino acid structure of LP289 demonstrates that LP289 possesses typical IgLON characteristics, including homology to known IgLON members and IgLON-bke motifs.
  • Particularly interesting portions or fragments of a fuU length LP289 polypeptide include, e.g, a discovered putative signal peptide-bke sequence from Met-1 to Ser-30 (MPPPAPGARLRLLAAAALAGLAVISRGLLS ) .
  • An alternative predicted cleavage site results in an alternate putative signal peptide-bke sequence from Met-1 to Ala-22 (MPPPAPGARLRLLAAAALAGLA) .
  • GDNATLSCFIDEHVTRVAWLNRSNILYAG DR TSDPRVRLLINTPEEFSILITEVGLGDEGLYTCSF from about Gly-147 to about Thr-197: (GGNV LLCLAVGRPEPTVTWRQLRDGFTSEGEILEISDIQRGQAGEYECVT) ; and from about Gly-
  • LP289 Additional interesting sections of LP289 are the discovered portions of LP289 from about Ala-8 to about Ala-22 from about Phe-35 to about Asp-48 from about Asn-49 to about Thr- 61 from about Leu-66 to about Asp-77; Pro-83 to about Phe-95 from about Ser-96 to about Glu-107 from about Gly-119 to about Gln-119 from about Val-129 to about Ser-140 from about Pro-141 to about Asn-152 from about Leu-152 to about Val-164 from about Trp-166 to about Glu-176 from about Gly-177 to about Gly-188 from about Gln-189 to about Val- 201 from about Thr-213 to about Ala-226 from about Arg-227 to about Leu-236 from about Phe-248 to about Ser-259 from about Thr-269 to about Phe-278 from about Ser-303 to about Leu-3 2.
  • LP 289 secondary structures are the foUowing LP289 coil structures: from about Met-1 to about Pro-6; from about Gly-27 to about Asp-40; from about Glu-46 to about Asn-49; from about Asn- 67 to about Ser-69; from about Ala-74 to about Arg-84; from about Asn-90 to about Glu-94; from about Gly-103 to about Gly-108; from about Thr-116 to about Thr-122; from about Pro-132 to about Ala-133; from about Ile-138 to about Pro-141 ; from about Glu-146 to about Asn-149; from about Val-157 to about Thr-163; from about Asp-171 to about Gly- 177; from about Gly-188 to about Glu-192; from about His-198 to about Ser-207; from about
  • Particularly interesting LP289 hebx structures are: from about Leu- 12- to about Gly-20; from about Arg-227 to about Glu-239; from about Ala-302 to about Arg-305; and from about Leu-326 to about Ala-328.
  • Particularly interesting strands are from about Tyr-42 to about Cys-45; from about Leu-52 to about Phe-55; from about Ala-64 to about Ala-64; from about Ile-71 to about Tyr-73; from about Val-85 to about Leu-87; from about Ser-96 to about Glu-101; from about Tyr-110 to about Cys-112; from about Gln-124 to about Val-129; from about Ile-135 to about Asn-137; from about Thr-143 to about Nal- 144; from about Asn-151 to about Leu-155; from about Nal-164 to about Trp-166; from about Tyr-193 to about Thr-197; and from about Arg-209 to about Nal-214.
  • one strand-coil-hebx-coil motif of LP289 combines the Arg-209 to Val-214 strand; and the Asn-215 to Val-223 coil; and the Arg-227 to Glu-239 hebx; with the Ala-242 to Phe-248 coU to form an interesting fragment of contiguous amino acid residues from about Arg-209 to about Phe-248.
  • Other combinations of contiguous amino acids contemplated are also encompassed as can be easily determined.
  • LP289 splice variants such as, e.g, spbce variants in which alterations in the processing of an LP289 mR ⁇ A results in a soluble/secreted LP289 that is not anchored to a ceU membrane.
  • a fuU-length LP289 spbce variant is from about Met-1 to about Thr-33
  • MPPPAPGARLRLLAAAALAGLAVISRAASSTST ( MPPPAPGARLRLLAAAALAGLAVISRAASSTST ) , with a predicted cleavage site as indicated as foUows: MPPPAPGARLRLLAAAALAGLA ⁇ VISRAASSTST.
  • the resulting mature LP289 spbce variant sequence (also known as LP343; see below) is from about Val-23 to about Thr-33 (VISRAASSTST).
  • This LP289 spbce variant sequence was discovered from a brain cD ⁇ A bbrary further supporting the role of this LP289 variant in neural functions described herein.
  • LP289 nucleic acid and the splice variant nucleic acid (whose polypeptide product is designated as LP343 (SEQ ID NO: 9)), indicates that during mRNA processing the parent of LP289 nucleic acid sequence the 2nd exon is skipped in the LP289 splice variant (see below).
  • the resulting LP289 splice variant created is a small peptide (approximately 11 amino acid residues in length after removal of a signal sequence).
  • exon 2 LP289 GCCGCCCTGGCCGGCTTGGCCGTCATCAGCCGAGGGCTGCTCTCCCAGAGCCTGGAGTTC LP343 GCCGCCCTGGCCGGCTTGGCCGTCATCAGCCGAG
  • LP289 Splice Variant Nucleic Acid Sequence (754 bp) (ORF 19-120) : LP289 splice variant (LP343) start (atg) and stop (tga) codons are indicated in bold typeface and underlined. >ds42802 Nucleic acid sequence is:
  • LP289 SASMRLLRPGSLENSAPRPPGLLALLSALGWLW RM This LP289 splice variant (LP343; SEQ ID NO: 10) may be useful as a therapeutic peptide to treat nervous system diseases.
  • LP343 (LP289 spbce variant) may cross the blood-brain barrier since it is a smaU amino-acid peptide after cleavage of its predicted signal peptide.
  • the LP289 splice variant (LP343) may also self- interact with the parent LP289 to modulate for example, parent LP289 expression, LP289 binding characteristics, LP2289 placement in the ceU.
  • LP343 may act as a secreted factor that can function as, e.g, a growth factor in the estabbshment of neural circuitry that has been estabbshed, maintained, or remodeUed by, e.g, parent LP289 or another IgLON (as described in detail herein). Furthermore, LP343 may act as a bgand for the parent LP289 or other IgLON member. Such spbcing interactions are not unheard of, for example, Karpa, et al.
  • LP289 primary amino acid and higher order structures Given the teachings suppbed herein of: LP289 primary amino acid and higher order structures, the relationship of the LP289 amino acid sequence and higher order structural features compared with known IgSF members (e.g, such as IgLON members (such as, e.g, LAMP, OBCAM, Ntm, CEPU, GP50, KILON, and GP55) and their higher order structural features (including the known functions of these IgLON members and their higher order structures), it is bkely that an LP289 or an LP289 variant play similar roles in a variety of physiological processes.
  • IgSF members e.g, such as IgLON members (such as, e.g, LAMP, OBCAM, Ntm, CEPU, GP50, KILON, and GP55) and their higher order structural features (including the known functions of these IgLON members and their higher order structures).
  • Some non-bmiting examples of functions an LP289, LP289 variant, or an LP289 binding agent is bkely to participate in are, for example, those such as: neuorogenesis; the formation, development, and/or modification of regional centers in the brain such as, for example, nuclei of, for example, the forebrain: such as, for example, the olfactory bulb and cortex; the neocortex; the stnatum, the nucleus accumbens; the basal forebrain; the bmbic circuit; the thalamus
  • the hypothalamus (including, for example, anterior lobe of the pituitary (adenohypophysis), posterior lobe of the pituitary (neurohypophysis), optic chiasm, preoptic nucleus, anterior nucleus, dorsomedial nucleus, ventromedial nucleus, posterior nucleus, mammiUary body, hypothalamic supraoptic nuclei (SON), and paraventncular nuclei (PVN)); the Midbrain, such as, for example, the tectum (
  • LP289 nucleic acids of the invention can be used to create LP289-denved polypeptides that interact with native LP289 located, for example, at a neural ceU surface either to stimulate the growth and differentiation activities of LP289 or to inhibit those activities.
  • Particularly preferred are such polypeptides that are soluble LP289 analogs having binding domains effective to bind LP289.
  • the LP289- ⁇ nh ⁇ b ⁇ tory polypeptides that are encoded by these nucleic acids can be used to treat diseases characterized by abnormal growth and functioning of neurons, such as, for example, neurons of the central nervous system, such as those involved in conditions of epilepsy, Alzheimer's disease, and schizophrema-bke diseases states or conditions.
  • LP289 nucleic acids of the invention are useful targeting agents because they bind LP289 found at the cell surfaces in the nervous system, for example, such as ceUs in the bmbic system.
  • targeting agents encompassed herein are bound covalently or noncovalently to a biological agent or a vehicle for debvenng biological agents such as, for example, debvery methods described herein or otherwise art known.
  • 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).
  • LP289 nucleic acids can also be used to transform stem ceUs to program their development as neural system neurons. These replacement neurons can be implanted to treat neuropathologies by reconnecting circuits involved in cognition, mood, memory and learning, and cardiovascular regulation, providing therapies for diseases, conditions, syndromes, etc , such as, for example, dementia (including without bmitation Alzheimer's disease, multi-infarct dementia, dementia associated with Parkinson's disease), aU forms of epilepsy, major depression, anxiety (including, without bmitation, manic-depressive iUness, generabzed anxiety, obsessive-compulsive disorders, panic disorder and others), schizophrenia, and schizophrenaform disorders (including without bmitation schizoaffecto disorder), cerebral palsy and hypertension.
  • dementia including without bmitation Alzheimer's disease, multi-infarct dementia, dementia associated with Parkinson's disease
  • aU forms of epilepsy major depression
  • anxiety including, without bmitation, manic-
  • Non-bmiting examples of stem cells that are useful in neural stem ceU replacement therapy include human cortical and subcortical fetal brain ceUs, porcine fetal brain ceUs, human subventricular zone ceUs, and gbal progenitor ceUs, including 02A ceUs (which are progenitors for aU gbal ceU types, including astrocytes and obgodendrocytes).
  • the LP289 nucleic acids of the invention can be used to create LP289-derived polypeptides that interact with LP289 located at a neuron ceU surface either to stimulate the growth and differentiation activities of LP289 or to inhibit those activities. Particularly preferred are such polypeptides that are soluble LP289 analogs having binding domains effective to bind LP289.
  • An LP289-inhibitory polypeptide can be used to treat diseases characterized by abnormal growth and functioning of neurons, such as epilepsy, Alzheimer's disease, and schizophrenia. Antisense strategies to inhibit the expression of LP289 can also be used to treat these diseases.
  • nucleic acids of the invention are to create targeting polypeptides for directing the debvery of biological agents to the nervous system where LP289 is expressed (for example, such as, the bmbic system, the dentate gyrus, the forebrain, the thalamus, the midbrain, etc.).
  • the LP289 polypeptides are useful targeting agents because they bind to LP289 found at the ceU surface of neuronal ceUs such as, for example, in the CNS, such as, for example, in the bmbic system.
  • 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
  • Further agents include, without bmitation, antidepressants, neuroleptics, anti- epileptics and antagonists of neurotransmitter receptors (such as type la, 1 b, II or III dopamine receptors, A and B adrenergic receptors and 5-HT receptors).
  • neurotransmitter receptors such as type la, 1 b, II or III dopamine receptors, A and B adrenergic receptors and 5-HT receptors.
  • the abibty of LP289 expression to modulate growth and/or differentiation of various embryonic ceU populations can be tested using a substrata of CHO ceUs, which have been transfected and/or transformed by LP289 (with controls of CHO ceUs transfected and/or transformed with a vector only).
  • ceUs from various CNS areas are tested such as, for example, ceUs from hippocampal and penrhinal cortex in comparison to ceUs from non-LP289 expressing areas, such as, for example, cardiovascular or connective tissue system ceUs.
  • Primary neurons from rodent El 6 embryos are prepared as outbned by Fern and Levitt, 1993 Cerebral Cortex 3; 187-198.
  • ceUs are marked by adding bpophibc dye PKH26 (Sigma Chemical Co, St. Louis, MO); if they are not so marked, an antibody stain is used later in the experiment to identify neural cells.
  • the ceUs are plated in DMEM/ 10% FCS at a density of 5 x 103 ceUs/ml per cm2, onto coversbps on which there are monolayers of transformed CHO ceUs. After 48 hours in culture, the ceUs attached to the coversbps are fixed with formaldehyde and, if the neural ceUs are not dye-marked, stained for neural ceUs with anti- MAP2, as described in Fern and Levitt, 1993 Cerebral Cortex 3: 187-198. For each experiment, six coversbps are examined and the longest neuron in a randomly selected field of 10- 5 process-bearing ceUs is measured.
  • 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.
  • LP289 and Neural Development and Neural Circuits Based on an analysis of LP289, such as, e.g, its homology to other IgLON members, LP289 is bkely to be involved in the regional specification of the central nervous system, e.g, regional specification of the brain, such as, e.g, the formation, maintenance, and/or modulation of neural circuits in the brain. For example, individual members of the IgLON family are expressed on distinct populations of neurons that, for the most part, form functional circuits in the nervous system, such as, e.g, in the brain.
  • the IgLON member Neurotnmin has an expression pattern that is largely complementary to that of LAMP, with the highest expression of Ntm in the sensonmotor cortex, Neurotnmin's expression in layers IV, V, and VI of the cortex, the subplate, and the rostral lateral thalamus as weU as in the pontine nucleus and cerebeUum suggests a potential role in the development of thalamocortical and pontocerebeUar projections, respectively (Struyk et al, 1995)
  • the IgLON member OBCAM has a more restricted distribution, with highest expression in the cortical plate and hippocampus (Struyk et al, 1995 J Neurosci 15:2141-2156) another member of the IgLON family of proteins.
  • the IgLON member LAMP is expressed by cortical and subcortical neurons of the bmbic system (Levitt, 1984 Science 223:299-301) and has been strongly impbcated in the development of pro j ections in this system (Pimenta et al, 1995 Neuron 15:287-297; Zhukareva and Levitt, 1995 Mol CeU Neurosci 10:43-55).
  • LAMP has been shown to play a role in specifying a subset of thalamocortical projections, which at early developmental stages are selectively expressed in the penrhinal and frontal bmbic cortex and medial bmbic thalamic nuclei (Levitt, 1984 Science 223:299-301; Horton and Levitt, 1988; Pimenta et al, 1996).
  • LAMP acts homophibcaly to promote adhesion and growth of bmbic axons (Pimenta et al, 1995 Neuron 15:287-297; Zhukareva and Levitt, 1995 Mol CeU Neurosci 10:43-55), and antibody perturbation studies show that LAMP can regulate the formation of septohippocampal and lntrahippocampal circuits (KeUer and Levitt, Neuroscience 28: 455- 474, 1989; Pimenta et al, 1995 Neuron 15:287-297).
  • LAMP acts as an attractive guidance signal that also induces branch formation while nonbmbic thalamic fibers are deflected and axonal branching is inhibited by LAMP.
  • neocortical and bmbic stnatal dopamine circuitry such as, e.g, circuitry of dopamine receptor neurons in the substantia mgra (SN) whose axons connect to neostriatal (caudate -putamen) nucleu or dopamine receptor neurons in the ventral tegmental area (VTA) whose axons connect with bmbic or ventral stnatum, including nucleus accumbens nucleu,
  • VTA ventral tegmental area
  • LP289 plays a role in the formation, maintenance, and/or such remodebng of CNS neural circuits by e.g. promoting the adhesion and/or growth of developing neural extensions (such as, for example, developing neu tes).
  • LP289 expression in the formation, maintenance, and/or modulation of such circuits would have important consequences for diseases, syndromes, or conditions of mood, thought, appetite, addiction, and/or emotion. Consequently, it would be useful to examine the role of LP289 in such neural circuits.
  • LP298's role in the nervous system is to determine if an LP289 binding agent, such as, for example, an antibody or antibody binding fragment directed against an LP289 polypeptide (or fragment thereof) interferes in the post natal development of a neural circuit of the central nervous system, e.g, such as an hippocampal circuit, the foUowing or similar experiments can be carried out (other known neural circuits and their corresponding neuronal architecture can be examined in a similar fashion using similar methods): Newborn Sprague-Dawley rats are in j ected lntraventncularly with Fab fragments of anti-LP289, control anti-paramyosin IgG, and anti-Ll.
  • an LP289 binding agent such as, for example, an antibody or antibody binding fragment directed against an LP289 polypeptide (or fragment thereof) interferes in the post natal development of a neural circuit of the central nervous system, e.g, such as an hippocampal circuit, the foUowing or similar experiments
  • Anti-Ll which binds to developing axons, is as described by Sweadner, J. Neurosci. 3: 2504-2517, 1983.
  • AU antisera are purified on a protein A column using a protein A affinity enhancement buffer (the
  • Fab fragments are prepared from the antisera by digestion with immobihzed papain (Pierce, Rockford, IL) and purified by protein-A affinity chromatography.
  • the Fab fragments (10 /eg in 10 ul of sabne) are injected on postnatal day 0, 2, 4, and 6 into the cisterna magna using a 35-32 gauge needle.
  • the animals are sacrificed by transcardial perfusion with 4 9 % sodium sulfide in 0.1 M phosphate buffer (pH 7 4). Brains are fixed in Carnoy's solution together with 1.2 % sodium sulfide.
  • Paraffin sections of the brains are prepared (in this instance) for mossy fiber staining using the Timm method (see, Haug, Adv. Anat. Embryol CeU Biol 47- 1-71, 1973) although other methods of examining neural arthitecture in other region of the brain can also be used.
  • Subfields are analyzed for density of innervation using the Bioquant OS/2 image analysis system (R & M Biometrics, NashviUe, TN) to examine neural architecture, for example, such as the mossy fiber projection of granule ceUs to pyramidal neurons of the hippocampus express.
  • Results are examined to determine if anti-LP289 treatment, but not the other antibody treatments, results in an uncharacteristic neuronal architecture, such as, for example, a diffuse pattern of mossy fiber projections indicating misdirected fibers
  • Quantitative effects can also be determined, for example, a positive effect of LP289 treatment should result in a statisticaUy significant increase in the area occupied by, for example, mossy fiber projections.
  • Spinal Cord Regeneration Model To evaluate the role LP289 in a spinal cord regeneration response (based on the methods of O'Hara, and Chernoff 1994 Tissue and CeU, 26: 599-611; Chernoff, et al. 1998 Wound Rep. Reg.
  • LP319a & LP319b are a novel primate (e.g, human) polypeptides (SEQ ID NO: 12 & 14), which are a newly discovered variant members of the IgLON family, which includes, e.g, such proteins as LAMP, OBCAM, Ntm (neurotrimin), CEPU, GP50, KILON, and GP55. See above for a description of IgLON sequence and structures.
  • LP319a & LP319b as opposed to LP289, however, are not canonical IgLON members since they display features at the amino acid level that are different from a typical IgLON, e.g, LP319a has one, not two, conserved cysteines in the most N-terminad Ig-bke C2-type domain; however, the second LP319a Ig-bke C2-type domain (moving C-terminad) resembles a typical IgLON Ig- bke C2-type domain, while the third Ig-bke C2-type domain contains only one cysteine and appears truncated (in comparison to other IgLONs).
  • LP319a contains no GPI- anchor-bke motif foUowed by a hydrophibc spacer region and a C-terminal signal sequence of predominately hydrophobic amino acids. Accordingly, native LP319a is bkely to be a secreted IgLON similar to CEPUS, a soluble counterpart to the cerebeUar Purkinje ceU specific antigen, CEPU-1, which is a secreted IgLON member that is bebeved to provide a favorable route for migrating CEPU-positive population of neurons to generate a neuron- specific guidance in developing neurons in vivo. It is bkely that native LP319a performs a similar role as CEPUS.
  • Appbcants invention encompasses, however, variant LP319s such as, e.g, variants in which a C-terminad GPI anchor sequence and/or an Ig-C2 bke domain is fused to a native LP319a sequence.
  • LP319b is also not a typical IgLON in that it displays features at the amino acid level which differ from other IgLONs SpecificaUy, LP319b does not exhibit three typical IgLON Ig-bke-C2-type domains.
  • the carboxy-most Ig-hke-C2-type domain of LP319b contains only one conserved cysteine and appears truncated in comparison to other IgLONs.
  • LP319b contains no GPI-anchor-bke motif foUowed by a hydrophibc spacer region and a C-terminal signal sequence of predominately hydrophobic amino acids Accordingly, native LP319b is bkely to be a secreted IgLON similar to CEPUS, a soluble counterpart to the cerebeUar Purkinje ceU specific antigen, CEPU-1.
  • LP319a sequence (SEQ ID NO: 11) is expressed in the foUowing number of LIFESEQ GOLDTM database tissue and cDNA bbraries: Digestive System 1/151; Embryonic Structures 3/23; 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.
  • LP319b nucleic acid sequence (SEQ ID NO: 13) is expressed in the foUowing number of LIFESEQ GOLDTM database tissue and cDNA bbraries:
  • LP319a start (atg) and stop (tga) codons are indicated in bold typeface and underlined.
  • a predicted mature LP319a sequence is as follows:
  • 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
  • An LP319b Mature Sequence (226aa) A predicted mature LP319b sequence is as follows:
  • An Alternate P319b Mature Sequence (234aa) An alternate predicted mature LP319b sequence is as follows: VISRGLLSQRLEFNSPADNYTVCEGDNATLSCFMDEHVTRVAWLNRSNILYAGNDRRTRDPRVRLLINTSEE FSILVTEVGLGDEGLYTCSFQTRHQPYTTQV ⁇ LIV ⁇ VPARVVT ⁇ ISSPVMV EGGNVNLLCLAVGRPEPTVT RQLRDGFTSEGEILEISDILRGQAGEYECVTHNGVNSAPDSRRVLVTVNYPPTITDVTSARTALGRAAYCAA KPWRFPPRISSGIRMTDY
  • An P319b Variant Sequence (286aa) A variant LP319b sequence with a fusion of LP289 carboxy amino acid sequence resulting in a complete third Ig-like C2 domain and a GPI anchor sequence. The added sequence is indicated by underlining.
  • the added sequence is indicated by underlining.
  • LP319a & LP319b Analysis of the primary amino acid structure of LP319a & LP319b demonstrates that they possess typical IgLON characteristics, including homology to known IgLON members and IgLON-bke motifs. Based on the teachings supplied herein (e.g, the LP319a & LP319b sequence and their relationship with the domains, motifs, and signatures of other known
  • 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).
  • Some non-bmiting examples of functions an LP319a/b, LP319a/b variant, or an LP319a/b binding agent is bkely to participate in are, for example, those such as: neuorogenesis; the formation, development, and/or modification of regional centers in the brain such as, for example, nuclei of, for example, the forebrain: such as, for example, the olfactory bulb and cortex; the neocortex; the striatum, the nucleus accumbens; the basal forebrain; the bmbic circuit; the thalamus (including, for example, reticular thalamic nucleus, dorso-caudal nucleus, dorso-intermedial nucleus, dorso-orales nucleus, ventral-caudal nucleus, ventral- intermediate nucleus, ventral-orabs posterior
  • Particularly interesting portions or fragments of the fuU length LP319a polypeptide include, e.g, are two immunoglobubn-bke domains: the first from about Ser-30 to about Phe-99:
  • the LP319b immunoglobubn-bke domains are from about Gly-47 to about Phe-114: ( GDNATLSCFMDEHVTRVAWLNRSNILYAGNDRRTRDPRVRLLINTSEEFSILVTEVGLGDEGLYTCSF ) ; and from about Gly-147 to about Val-197
  • LP319a is the discovered heavy metal-associated-bke domain at the C-terminus of LP319a from about Val-197 to about Lys-224 ( VTVNYPPTITDVTSARTALGRAAYCAAK ) .
  • the heavy metal associated domain of LP319b is from about Nal-212 to about Lys-239 ( VTV ⁇ YPPTITDVTSARTALGRAAYCAAK ) .
  • AdditionaUy interesting segments of LP319a are discovered fragments from about Arg-11 to about Leu-29; from about Gln-31 to about Trp- 50; from about Asn-52 to about Ile-74; from about Thr-76 to about Asp-91; from about Glu-92 to about Gln-109, from about Val-110 to about Val-129; from about Gly-132 to about Arg-144; from about Pro-145 to about Glu-163; from about Leu-171 to about Val- 181; from about Thr-182 to about Val-195; from about Ala-222 to about Arg-231; from about Arg-11 to about Leu-28; from about Ser-30 to about Ala-49; from about Arg-53 to about Leu-73; from about Val-84 to about Tyr-95; from about Thr-96 to about Gln-109; from about Val-110 to about Val-120; from about Val-121- Asn-136; from about Val-142 to about Ile-164; from about
  • 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.
  • LP319a secondary structures e.g, such as a hebx, a strand, or a coil
  • the foUowing LP319a hebx structures from about Leu-13 to about Ala-17, and from about Arg-212 to about Leu-215.
  • Particularly interesting discovered coil structures are from about Met-1 to about Pro-6; from about Asn-36 to about Tyr-42; from about Asn-52 to about Ser-54; from about Ala-59 to about Asp-67; from about Asn-75 to about Glu-79; from about Gly-88 to about Gly-93; from about Arg-102 to about Thr-107 ' ; from about Pro-117 to about Ala-118; from about Ile-123 to about Ser-125; from about Glu-131 to about Asn-134; from aboutVal-142 to about Thr-148; from about Leu-154 to about Gly-162; from about Arg-172 to about Glu- 177; from about Asn-184 to about Ser-192; from about Asn-200 to about Ile-205; from about Ala-223 to about Gly-235; from about and Thr-239 to about Tyr-241.
  • Particularly interesting discovered strand structures are from about Ala-49 to about Trp-50; from about Ile-56 to about Tyr-58; from about Ser-81 to about Glu-86; from about Tyr-95 to about Cys- 97; from about Gln-109 to about Nal-114; from about Val-120 to about Asn-122; from about Asn-136 to about Leu-140; from about Val-149 to about Arg-152; from about Ile-164 to about Leu-165; from about Tyr-178 to about Thr-182; and from about Arg-194 to about Val-199.
  • Further encompassed by the invention are contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one coil-strand-coil-strand-coil motif of LP319a combines an Arg-102 to Thr-107 coil, with an Gln-109 to Val-114 strand, with an Pro-117 to Ala-118 coU, with an Val-120 to Asn-122 strand, and an Ile-123 to Ser-125 coU to form an interesting fragment of contiguous amino acid residues from about Arg-102 to about Ser-125.
  • Other such combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP319b interesting segments of LP319b are discovered fragments from about Leu-18 to about Phe-35; from about Asn-36 to about Thr-51; from about Arg-62 to about Ala-74; from about Arg-84 to about Glu-93; from about Glu-94 to about Gly-103; from about Glu- 122 to about Ala-133; from about Arg-134 to about Met-143; from about Gly-177 to about Leu-186; from about Gly-187 to about Val-196; from about Thr-197 to about Gly-208; from about Arg-209 to about Thr-221; from about Asp-222 to about Ala-238; from about Pro 240 to about Ser-249; from about Arg-11 to about Leu-29; from about Ser-30 to about Asp-40; from about Asn-41 to about Ala-50; from about Glu-58 to about Asp-77; from about Arg-78 to about Ile-89; from about Arg-91 to about Asp-106
  • LP319b Additional interesting sections of LP319b are the discovered portions of LP319b from about Arg-8 to about Val-23; from about Phe-35 to about Cys-45; from about Glu-46 to about Arg-62; from about Trp-65 to about Asp-77, from about Val-85 to about Phe-95; from about Ser-96 to about Thr-111; from about Ser- 137 to about Thr-150 from about Asn-151 to about Val-164 from about Thr-165 to about Glu-181 from about Ile-182 to about Gly-191 from about Glu-192 to about Val-201 from about Nal-212 to about Ala-226 from about Arg-227 to about Pro-240; and from about Trp- 241 to about Gly-250.
  • LP319b secondary structures e.g, such as a hebx, a strand, or a coil
  • the foUowing LP319b hebx structures from about Leu-13 to about Ala-17; and from about Arg-227 to about Leu-230.
  • Particularly interesting discovered coil structures are from about Met-1 to about Pro-6; from about Gly- 27 to about Gly-27; from about Asn-36 to about Asp-39; from about Glu-46 to about Cys- 49; from about Asn-67 to about Ser-69; from about Ala-74 to about Asp-82; from about Asn-90; from about Glu-94 to about Gly-103 to about Gly-108; from about Thr-116 to about Thr-122; from about Pro-132 to about Ala-133, from about Ile-138 to about Ser-140, from about Glu-146 to about Asn-149; from about Nal-157 to about Thr-163; from about Leu-169 to about Gly-177; from about Arg-187 to about Glu-192, from about Asn-199 to about Ser-207; from about Asn-215 to about Ile-220, from about Ala-238 to about Gly-25, and from about Thr-254 to about Tyr-256.
  • Particularly interesting discovered strand structures are from about Try-42 to about Cys-45; from about Ala-64 to about Trp-65; from about Ile-71 to about Tyr-73; from about Ser-96 to about Glu-101; from about Tyr-110 to about Cys-112; from about Gln-124 to about Val-129; from about Val-164 to about Arg-167; from about Ile-179 to about Leu-180; from about Tyr-193 to about Thr-197; and from about Arg-209 to about Val-214.
  • LP321 is a novel primate (e.g, human) polypeptide (SEQ ID NO: 16) that is a newly discovered member of the defensin family of antimicrobial peptides, which are generaUy recognized a having antibiotic, antifungal, and antiviral activities. SpecificaUy, LP321 exhibits sequence homology to enteric alpha defensins known as cryptdins. Defensins are a family of structuraUy related cysteine-nch peptides active against many Gram-negative and Gram- positive bacteria, fungi, and viruses (see, e.g, Lehrer, et al. CeU 64-229-230; Kagan, et al.
  • defensins are also caUed corticostatins (CS) because they inhibit corticotropin-stimulated corticosteroid production.
  • CS corticostatins
  • Defensins can kiU ceUs by forming voltage-regulated multimeric channels in the membrane of the susceptible ceU. Defensins play a significant role in innate immunity to infection and neoplasia.
  • Antimicrobial peptides are a prevalent mechanism of host defense found throughout nature (Kaiser & Diamond 2000 J Leukoc Biol 6:779-84). In mammals, defensins are among the most abundant of these broad-spectrum antibiotics, and are expressed in epithebal and hematopoietic ceUs among others. The defensin peptides are especiaUy abundant in neutrophils. In epithebal ceUs, defensins are found both as constitutively expressed and inducible genes. Induction has been observed in vitro by stimulation with bacterial bpopolysacchande (LPs) as weU as inflammatory mediators.
  • LPs bacterial bpopolysacchande
  • 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 NP-4.
  • 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
  • these peptides range in length from approximately 29 to about 35 amino acids and typicaUy, at the primary amino acid sequence level, they possess invariant cysteine residues that are involved in intrachain disulfide bonding.
  • expression of defensins was thought to be bmited to professional phagocytes, such as, for example, neutrophils and macrophages.
  • LP321 nucleic acid sequence (SEQ ID NO: 15) is expressed in the foUowing LIFESEQ GOLDTM database tissue and cDNA bbraries: Respiratory System 1/95. This LP321 expression pattern is commensurate with reports indicating that cryptdins are also found in lung marcrophages (see, e.g, Shirafuji, et al. 1999 Cbn. & Diagnos. Lab. Immun 6:336-340).
  • LP321 (SEQ ID NO 16) The underlined portion is a predicted signal sequence (Met 1 to Ala-19) A predicted SP cleavage site is between Ala-20 and Asp-21 indicated as follows 1 MKTLVLLSALVLLALQVQA'OP 21 An alternate predicted SP cleavage site is between Gln-16 and Val 17 indicated as follows 1 MKTLVLLSALVLLALQ ⁇ VQ 18 Both mature LP321 versions are encompassed herein LP321 polypeptides encompassed herein include full length forms encoded by an ORF disclosed herein, as well as any mature forms therefrom Such a mature LP321 could be formed, for example, by aminopeptidase modification, or by the removal of a signal peptide Further as used herein, a "mature" LP encompass, e g , post-translational modifications other than proteolytic cleavages (such as, e g , by way of a non-limiting example, glycosylations, mynstylations
  • a predicted mature LP321 sequence is as follows: DPIQEAEEETKTEEQPADEDQDVSVSFEGPEASAVQDLRVRRTLQCSCRRVCRNTCSCIRLSRSTYAS An LP321 variant Sequence ( 71aa ) :
  • An LP321 Variant Sequence ( 87aa ) : An al ternate LP321 .
  • the underlined portion is a predicted propeptide segment from about Met -1 to about Ser-52
  • MKTLVLLSALVLLALQVQADPIQEAEEETKTEEQPADEDQDVSVSFEGPEAS A predicted cleavage si te is between Ser- 52 and Ala-53 indicated as f ol lows : 1 MKTLVLLSALVLLALQVQADPIQEAEEETKTEEQPADEDQDVSVSFEGPEAS ⁇ AVQD 56 .
  • An LP321 Variant Sequence ( 28aa ) : A further alternate LP321 mature sequence comprising an alternate N terminal portion before the f irst cysteine is : LRDLVCSCRRVCRNTCSCIRLSRSTYAS
  • Stil l another alternate LP321 mature sequence comprising an alternate N terminal portion before the f irst cysteine is : GLLCSCRRVCRNTCSCIRLSRSTYAS
  • LP321 is the segment from about Met-1 to about Ser-52, which has been discovered to be a defensin propeptide-bke domain.
  • Other interesting segments of LP321 are the discovered portions of LP321 from about Ser-8 to about Ala-25; from about Asp-20 to about Phe-46; from about Ala-51 to about Arg-60; from about Arg-61 to about Cys-71 ; from about Ser-8 to about Ile-22; from about Gln-23 to about Glu-38, from about Asp-41 to about Ala-53; from about Arg-61 to about Arg-72, from about Cys-71 to about Leu-80; from about Val-17 to about Glu-27; from about Ile-22 to about Pro-35; from about Pro-35 to about Val-44; from about Glu-47 to about Asp-56; from about Val-59 to about Val-70; and from about Gln-64 to about Cys-77; whose discoveries were based on an analysis of hydrophobic
  • LP321 Additional interesting sections of LP321 are the discovered portions of LP321 from about Val-5 to about Gln-18; from about Ala-19 to about Glu-27; from about Tyr-29 to about Pro-35; from about Ser-43 to about Ala-51 ; from about Gln-64 to about Ser-81 ; and from about Ser-52 to about Arg- 61. These fragments were discovered based on analysis of antigenicity plots.
  • particularly interesting LP321 structures that have been discovered are the foUowing LP321 hebx structures: from about Leu-6 to about Leu-14, from about Ile-22 to about Glu-28; and from about Ala-53 to about Leu-57.
  • Particularly interesting discovered coil structures are from about Glu-6 to about Gln-14; from about Glu-47 to about Glu-50; from about Cys-71 to about Cys-75; and from about Leu-53 to about Ser-57.
  • Particularly interesting discovered strand structures are from about Val-42 to about Ser-45; and from about Cys-77 to about Arg-79.
  • one hebx-coil- strand-coil motif of LP321 combines the hebx from about Ile-22 to about Glu-28, with the coil about Glu-6 to about Gln-14, the strand from about Val-42 to about Ser-45, and the coil from about Glu-47 to about Glu-50 to form an interesting fragment of contiguous amino acid residues from about Ile-22 to about Glu-50.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP321 Functions Given the teachings suppbed herein, for example, of: an LP321 primary amino acid, LP321 higher order structures, the relationship of an LP321 amino acid sequence to higher order structural features, the comparabibty of an LP321 sequence and/or an LP321 higher order structure with a known defensin (such as, e g, members of the cryptdin protein family, such as, e g, mouse cryptdins 1-6), and the relationship of higher order structural features of a cryptdin with their known functions, it is bkely that an LP321, an LP321 variant, an LP321 hetero-or an LP321 homomultimer, and/or an LP321 binding agent (e.g, such as an LP321 antibody (or fragment thereof)) plays a similar role/s in a variety of physiological processes.
  • a known defensin such as, e g, members of the cryptdin protein family, such as, e
  • some non-hmiting examples of functions an LP321, an LP321 variant, an LP321 hetero- or an LP321 homomultimer, or an LP321 binding agent is bkely to have and/or participate in are, for example, those such as- mucosal immunity (such as, e.g, mucosal surfaces in, e.g, epitheba in an airway, sk n, oropharynx, a gingival crevice, and an urogenital surface); host defense; anti-microbial activity against (e.g, bacteria such as, e.g. Gram-positive bacteria, Gram-negative bacteria, S. typhimunm, S. typhimurium, S.
  • bacterial such as, e.g. Gram-positive bacteria, Gram-negative bacteria, S. typhimunm, S. typhimurium, S.
  • protists such as, e.g, G.
  • 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.
  • the invention also provides a method for treating a biological surface with an LP321, an LP321 variant, an LP321 hetero-or an LP321 homomultimer and/or an LP321 binding agent, wherein said surface is a mucosal surface; wherein said surface an epitheba in an airway; wherein said surface is skin, wherein said surface is a surface in the oropharynx; wherein said surface is a gingival crevice; and wherein said surface is a urogenital surface.
  • Such treatment is particularly advantageous in subjects that are immunocompromised due, such as, for example, to: malnutrition, radiation, burns, immunosuppressive infections or conditions, autoimmune disease, neonatabty, bone marrow transplantation, and/or chemotherapy.
  • Non- bmiting examples of how an LP321 of the invention can be administered are: oraUy, by nasogastric intubation, by transabdominal catheter, intravenously, or by aerosol inhalation.
  • an LP composition of the invention When administered orally, an LP composition of the invention is preferably in a delayed release formulation designed to permit release in, e.g, the intestinum carcum, the intestinum crassum, the intestinum ileum, the intestinum jejunum, the intestinum rectum, the intestinum ***, or the intestinum mesenteriale.
  • An LP321 of the invention can be administered as a composition with a physiologicaUy acceptable medium, and can be administered in combination with other agents such as, for example, a cryptdin, a defensin, a thionin, or it can be administered simultaneously, or sequentiaUy with any of the former.
  • an LP321 or an LP321 variant is administered in concert with a granulocyte colony-stimulating factor (G-CSF), or a G-CSF composition, (such as in the range of about 1.0 to about 10.0 ug/kg weight /day for about 1 to about 10 days in a subject, such as, e.g, an immunocompromised subject, such as, e.g, a sub j ect who has neutropenia or a neutropenic-hke condition) Cryptdins or cryptdin-bke compositions exhibit antimicrobial activity against enteric microorganisms, which can become blood-borne pathogens if an epithebal layer is breached, for example, such as the epithebal layer in the intestines or an epithebum of the airway, such as, e.g, in the lungs (such as, e.g, the alveob or an aveolar sac).
  • G-CSF granulocyte colony-sti
  • Cryptdins or a cryptdin-bke molecule can be secreted from a ceU in which it is produced (Satoh, 1988CeU Tiss Res. 251:87-93; Satoh et al. 1988Acta Histochem 83 185- 188). It should be appreciated that various modifications can be made to an LP321 amino acid sequence without diminishing the antimicrobial activity of an LP peptide of the invention. It is intended that LP peptides exhibiting such modifications (including, e.g, amino acid additions, deletions and/or substitutions) are aU within the scope of the term "LP321" and, therefore, within the scope of the invention.
  • LP321 variants which are devoid of one or more amino acids located N-terminal to the first cysteine residue in the primary structure, are aU within the scope of the present invention.
  • Such an LP321 analog or variant can be synthesized using art-known methods or those described or referenced herein.
  • methods of prognosing, diagnosing, and/or treating a microbial infection, condition, disease, or state such as, e.g, otitis media using an LP321, an LP321 variant, or an LP321 binding agent of the invention.
  • 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.
  • Anti-LP321 binding agents can be used to determine the presence of an LP321 or an LP321 variant in a biological sample such as, e.g, a histological sample, or a lavage product, blood, an exudate or another biological sample.
  • a biological sample such as, e.g, a histological sample, or a lavage product, blood, an exudate or another biological sample.
  • a section of a smaU intestine is fixed by art-known means and incubated with anti-LP321 antibodies such as, e.g, an IgG fraction of LP antiserum.
  • the anti- LP321 antibody is detectably labeled or an appropriate detectable second antibody is used to identify the presence of the primary antibody attached to an LP321 or an LP321 variant.
  • Alternative methods of determining the presence of an LP321, or an LP321 variant in a biological sample obtained, for example, by intestinal lavage or by disrupting ceUs or tissues can be useful to determine the presence of an inflammatory process such as, for example, cobtis, Crohns disease, inflammatory bowel syndrome, pancreatitis, a mabgnancy, an infection, or an ileititic condition, etc.
  • a concentration of an LP321, or an LP321 variant is significantly altered from a concentration found in a normal condition or state.
  • a deviation from a normal level of an LP321 or an LP321 variant by about one to about two standard deviations from an estabbshed basebne control is typicaUy indicative of an inflammatory condition and/or state.
  • Non-limiting examples of such an inflammatory state or condition include, for example, cobtis, Crohns disease, inflammatory bowel syndrome, pancreatitis, a mabgnancy, an infection, or an ileititic condition.
  • an LP321, or an LP321 variant is a therapeutic agent for an infection of, e.g, the intestine, the lung, or a biological surface, wherein said surface is a mucosal surface; wherein said surface an epithebum in an airway; wherein said surface is an epitheba surface of skin, wherein said surface is a surface in an oropharyn geal lumen; wherein said surface is a gingival crevice; or wherein said surface is a urogenital surface.
  • an LP321, or an LP321 variant of the invention is useful where a subject is immunocompromised due, for example, to: mabgnancy, malnutrition, chemotherapy, radiation, immunosuppressive viruses, autoimmune disease, or neonatabty.
  • an LP321, or an LP321 variant of the invention is useful in a surgical prophylaxis, for example, by functioning to help steribze the smaU bowel.
  • an LP of the invention can be useful as a medicament for treating a subject having a pathology characterized, in part, by an inflammatory process and/or condition, e.g, such as an inflammatory process state, state or condition described herein.
  • an LP of the invention is useful in a pharmaceutical composition for a topical appbcation.
  • an LP of the invention is useful in a propeptide form.
  • an LP of the invention is useful as being sequestered in a first form (such as, e.g, a propeptide form) and a second composition (having the capacity to cleave a prosegment of an LP321 or an LP321 variant) is also sequestered (such as, e.g, a second composition bke a matrilysin, or matrilysin-bke composition) from the first form propeptide composition, wherein the second composition and the first propeptide form are brought together at a location in a subject to form an active LP321 or an active LP321 variant, for example, such as in the lung, the alveob, the intestinum carcum, the intestinum crassum, the intestinum ileum, the intestinum jejunum
  • a composition of the invention encompasses an LP of the invention that form multimeric complexes.
  • an LP321 or an LP321 variant forms an multimeric LP321 complex that is capable of forming a pore in a membrane, such as a bpid membrane, such as a bpid bilayer.
  • An LP o the invention (or variant thereof), either purified from natural sources or synthetic can be administered to a subject (in need of treatment) by various means, including oraUy, preferably in a slow-release type formulation that wiU avoid release within the stomach.
  • an LP321 can be administered through nasogastric intubation, trans-abdominal catheter, by injection intravenously, or by aerosol administration.
  • LP321 variants encompassed herein are cycUc LP321 variants that are produced by bgation of two truncated LP321s or LP321 variants by adapting the method of Tang, et al. 1999 Science 286:498-502 (which is incorporated herein by reference for its techniques regarding head-to-tail bgation of truncated defensins).
  • An LP of the invention can be administered alone or in combination with other agents (such as, e.g, a defensin or a cryptdin known in the art).
  • An LP of the invention administered in combination can be administered simultaneously or sequentiaUy and can be repeated as necessary.
  • LP321 Antimicrobial Assays The antimicrobial activity of a purified LP321 or LP321 variant is tested against wUd type and phoP mutant S. typhimurium by means of a modified plate diffusion assay (Lehrer, et al. 1991b J. Immunol. Methods 137:167-173, which is incorporated herein by reference for its assay methods) using wild type S. typhimurium (ATCC 10428) or an isogenic phoP mutant of S. typhimurium (strain CS015 phoP102::Tnl0d-Cam, MiUer et al, supra, 1989).
  • the phoP locus is a two-component regulatory locus essential to S.
  • CeUs are grown to log phase in trypticase soy broth at 37 °C, harvested by centrifugation and resuspended to approximately 10 milbon colony forming units (CFU) per ml in 10 mM sodium phosphate buffer (pH 7.4). A 100 ul abquot of each organism is mixed with 10 ml 1.0% agarose in 0.03% (w/v) trypticase soy medium, 10 mM sodium phosphate (pH 7.4) at 42 °C. Five ul samples of peptide solution are pipetted into 3mm diameter weUs formed in agarose with a sterile punch.
  • the inoculated agarose plate is overlaid with 1.0% agarose containing 6.0% trypticase soy medium.
  • antimicrobial activity is demonstrated by clear zones surrounding weUs loaded with antibacterial samples; the areas of the clear zones are typicaUy concentration-dependent.
  • a cryptdin or cryptdin-bke composition's antimicrobial activity in vitro is substantiaUy enhanced in piperazine-N, N'-bis (2-ethane 5-sulfonic acid) (PIPES) or in N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) as compared to its activity in sodium phosphate.
  • Purified LP peptides of the invention are dissolved at various concentrations (such as, e.g, from about 0.1 to about 300 ug/ml) in 0.01% acetic acid and activity is examined against E. cob ML35 (ATCC).
  • a radial diffusion assay 5 ul of peptide solution is transferred into weUs formed in plates of 1% agarose buffered with 10 mM PIPES (pH 7.4) and containing 1 x 106 log-phase bacteria grown in trypticase soy broth. After 3 hr at 37 °C, the plates are overlaid with 0.8% agarose containing 2x trypticase soy broth and incubated overnight.
  • Antibacterial activities of LP peptides are compared with, e.g, antibacterial activities of rabbit neutrophil defensin NP-1, which is purified from peritoneal exudates as described by Selsted, et al 1985 J. Biol. Chem. 260:4579-4584 (incorporated herein by reference for such assay teachings) or with a known cryptdin sequence.
  • Antibacterial activity is determined by measuring the diameter of clearing around each weU and plotted as a function of peptide concentration. Positive results wiU typicaUy produce a dose-dependent zone of clearing that demonstrates an inhibition of microbial growth. Potencies of an LP321 or an LP321 variant may vary depending on dosage and modification.
  • 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.
  • Bactericidal assays are performed by incubating approximately 1-2 x 106 log-phase bacteria in 10 mM PIPES contaimng from about .1 to about 10 ug peptide/ml. After incubation for 15 or 30 min at 37 °C, ahquots are removed, senaUy diluted, and plated on trypticase soy agar. Bactericidal activity is quantitated by counting colonies after overnight incubation at 37 °C.
  • the bactericidal activity of, e.g, cryptdin 1 can be compared with an LP321 or LP321 variant peptide of the invention to compare microbial activity.
  • E. cob ML35 ceUs, S. aureus 502A ceUs or mutant or wild type S. typhimurium ceUs are incubated with various concentrations of rat cryptdin 1 or rabbit NP-1.
  • Ten ug/ml rat cryptdin 1 has been reported to kiU about 90% of the S. aureus ceUs and greater than 99% of the E. cob and mutant S. typhimurium ceUs, but is relatively ineffective in kilbng wild type S.
  • Giardia lambba which is the most common cause of protozoan disease in the human smaU intestine
  • trophozoites of the C6 clone of Giardia lambba WB are grown to late log phase in TYI-S-33 medium contaimng bovine bile. Free-swimming trophozoites are discarded and tubes with attached trophozoites are refiUed with warm Dulbecco's PBS.
  • Trophozoites are detached by chilbng 10 min on ice, then harvested by centrifugation, resuspended at 2 x 107/ml in 25 mM HEPES (pH 7.5) contaimng 9.0% (isotonic) sucrose and incubated for 2 hr at 37 °C with various concentrations of, e.g, mouse cryptdins 1-3 or 6 and an LP peptide.
  • trophozoite viabibty is determined and compared (depending on treatment) by trypan blue exclusion to determine how an LP321 or LP321 variant peptide or mouse cryptdins 1 -3 or 6 klU Giardia trophozoites in a dose-dependent manner (e.g, it has been reported that 20 ug/ml of cryptdin 2 or cryptdin 3 reduces Giardia growth by greater than 2 orders of magnitude thus, indicating that such cryptdins are active against a variety of microorganisms see, WO 96/16075 for cryptdin assay methods (WO96/16075 is incorporated herein by reference for such assay methods).
  • assays for microbi ⁇ dal activities can also be employed to test an LP321 or an LP321 variant of the invention
  • Such assays are commonly art known, and could be adapted for use to test an LP321, an LP321 variant or an LP321 binding agent without undue experimentation, (see, e.g, Selsted, M. E. (1993) in Investigational Approaches for Studying the Structures and Biological Functions of Myeloid Antimicrobial Peptides, ed. Setiow, J. K. (Plenum, New York), Vol. 15, pp. 131—147; which is incorporated herein for such method techniques).
  • T84 ceUs form confluent monolayers of columnar epitheba that display polarized apical and basolateral membranes, high transepithebal resistances, and a regulated C12 secretory pathway analogous to that found in native crypt epithebum (Dharmsathaphorn & Madara 1990 Methods Enzymol. 192:354—359).
  • C12 secretion is assessed as a short circuit current (Isc) using standard electrophysiologic techniques (Lencer, et al. 1992 J. CeU Biol.
  • cAMP and cGMP are assessed in ethanol extracts of T84 ceU monolayers by radioimmune assay kit (NEN) Hanks' balanced salt solution (HBSS; containing 1.67 mM CaC12 0.8 mM MgSO4 5 mM KC1 0.45 mM KH2P04 137 mM NaCl 0.33 mM Na2HP04 5 mM glucose 10 mM Hepes, pH 7.4) is used for aU assays unless otherwise stated.
  • NNN radioimmune assay kit
  • HBSS Hanks' balanced salt solution
  • HBSS Hanks' balanced salt solution
  • Isc short circuit current
  • BCECF-acid 29,79-b ⁇ s-(2-carboxyethyl)-5-(and-6)- carboxy-fluorescein.
  • Peptide Purification and Synthesis To compare a cryptdin with an LP321 or an LP321 variant of the invention one can use any standard recombinant method employing a pubbshed cryptdin sequence to generate a cryptdin peptide of interest, or one can use a low molecular weight peptide fraction (P-60 cryptdin fraction; from which aU known cryptdins to date have been purified) to purify a cryptdin of interest. Briefly, the peptide fraction is prepared by Biogel P-60 gel chromatography of an acid extract homogenate of adult outbred Swiss Webster mouse smaU intestine using a method of Selsted, et al 1992 J. CeU Biol.
  • mice cryptdins 1—6 are purified to homogeneity by HPLC using the methods of Selsted, supra; or OueUette, et al. 1992 FEBS Lett. 304, 146-148 (which is inco ⁇ orated herein by reference for these assay method teachings).
  • Nonpolarized T84 ceUs grown on glass coversbps
  • polarized monolayers grown on filter supports
  • BCECF-acid 0.1 uM
  • coversbps or monolayers on their filter supports are washed in fresh HBSS contaimng 0.1 uM BCECF at 37°C to remove the peptide.
  • coversbps or monolayers are washed again in fresh HBSS and examined by epifluorescence (490 nM excitation, 520 emission) and bright field microscopy using Nomarski optics for the presence of the fluorophore outside of the ceUs of interest.
  • 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.
  • an LP317, or an LP317 variant can function as a new class of sodium channel blockers.
  • LP317 shares sequence similarity with amylase inhibitors Elevation of serum amylase is associated with lung cancer (Grove, A. 1994 APMIS 102(2):135-44), myeloma (Fuju, et al.
  • compositions comprising LP317 polypeptides, polynucleotides, its agonists/antagonists and/or antibodies are useful for diagnosis, treatment and intervention of cancer, pancreatitis, and tooth decay.
  • LP317 is also expressed in prostate stroma.
  • compositions comprising LP317 polypeptides, polynucleotides, its agonists/antagonists and/or antibodies are also useful for the treatment of defects in or wounds to prostate.
  • Defensins are a family of structuraUy related cysteine- nch peptides active against many Gram-negative and Gram-positive bacteria, fungi, and viruses (see, e.g, Lehrer, et al. CeU 64:229-230; Kagan, et al. 1994 Toxicology 87-131-149; Lehrer, et al. 1993 Annu. Rev. Immunol. 11:105-128, and White, et al. 1995 Curr. Opin. Struct. Biol. 5:521-527).
  • defensins are also caUed corticostatins (CS) because they inhibit corticotropin-stimulated corticosteroid production.
  • CS corticostatins
  • Defensins can k ⁇ l ceUs by forming voltage -regulated multimeric channels in the membrane of the susceptible ceU.
  • Defensins play a significant role in innate immunity to infection and neoplasia.
  • Antimicrobial peptides are a prevalent mechanism of host defense found throughout nature (Kaiser & Diamond 2000 J Leukoc Biol 6:779-84). In mammals, defensins are among the most abundant of these broad-spectrum antibiotics, and are expressed in epithebal and hematopoietic ceUs.
  • defensin peptides are especiaUy abundant in neutrophils; however, gene expression is limited to the promyelocyte stage.
  • defensin genes are found as both constitutively expressed and inducible. Induction has been observed in vitro by stimulation with bacterial bpopolysaccharide as weU as inflammatory mediators. In vivo, up-regulation of several defensin genes occurs in both infectious and inflammatory states. Gene regulation occurs via signal transduction pathways common to other innate immune responses, using transcription factors such as nuclear factor (NF)-kappa beta and NF interleukin-6.
  • NF nuclear factor
  • 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
  • NP-4. AU these peptides range in length from approximately 29 to about 35 amino acids and typicaUy, at the primary amino acid sequence level, they possess invariant cysteine residues that are involved in intrachain disulfide bonding.
  • LP317 nucleic acid sequence (SEQ ID NO 17) is only found in a human brain and prostate stroma of a LIFESEQ GOLDTM database tissue and cDNA bbrary Table 7 Primate, e , human, LP317 polynucleotide sequence (SEQ ID NO 17) and corresponding polypeptide (SEQ ID NO 18)
  • LP317 (start (atg) and stop (tga) codons are indicated in bold typeface and underlined) .
  • a predicted mature LP317 sequence is as follows: GTTTT VAEARVCMGKSQHHSFPCISDRLCSNECVKEDGG TAGYCHLRYCRCQKAC
  • An LP317 Variant Sequence (56aa) : An alternate LP317 mature sequence with the N terminal portion before the first cysteine shortened. TTTTMVAEARVCMGKSQHHSFPCISDRLCSNECVKEDGG TAGYCHLRYCRCQKAC
  • An LP317 Variant Sequence (49aa) : An alternate LP317 mature sequence with the N terminal portion before the first cysteine shortened.
  • LP317 interesting segments of LP317 are the segments from about Arg-36 to about Cys-82 (RVCMGKSQHHSFPCISDRLCSNECVKEDGGWTAGYCHLRYCRCQKAC), and from about Arg-36 to about Cys-59 ( RVCMGKSQHHSFPCISDRLCSNEC ) , which have been discovered to exhibit gamma-thionin-bke domain signatures.
  • Other interesting segments of LP317 are the segments from about Val-37 to about Asp-52 (VC GKSQHHSFPCISD), and from about Gly-69 to about Cys-82 (GYCHLRYCRCQKAC) , which have been discovered to exhibit a purothionin- bke signature.
  • Gamma-purothionin inhibits protein translation in ceU-free systems.
  • a further interesting segment of LP317 is the segment from about Cys-49 to about Cys-78 (CISDRLCSNECVKEDGGWTAGYCHLRYCRC), which has been discovered to exhibit a scorpion- short-toxin-bke signature.
  • LP317 Other interesting segments of LP317 are discovered fragments are the discovered portions of LP317 from about Ser-6 to about Ala-16; from about Leu-13 to about Tyr-23; from about Cys-38 to about Cys-49; from about Ser-56 to about Thr-67; from about Gly-65 to about Tyr-75; from about Cys-38 to about Cys-49, from about Asp-63 to about Cys-71; from about Cys-11 to about Ser-21, from about His-22 to about Val-32; from about Met-31 to about Gly-40; from about Val-37 to about Ser-46, from about Pro-49 to about Val-71, from about Leu-54 to about Thr-67; and from about Val-60 to about Cys-71; whose discoveries were based on an analysis of hydrophobicity, hydropathicity, and hydrophibcity plots.
  • LP317 Additional interesting sections of LP317 are the discovered portions of LP317 from about Ala-8 to Tyr-23; from about Asp-24 to about Glu-34; from about Arg-36 to about Lys-41; from about Pro-48 to about Cys-59; from about Cys-59 to about Gly-69; and from about Ser-70 to about Arg-80. These fragments were discovered based on analysis of antigenicity plots. Further, particularly interesting LP317 structures (e.g, such as a hebx, a strand, or a coil) that have been discovered is the foUowing LP317 hebx structure: from about Thr-9 to about Leu-17.
  • LP317 hebx structure from about Thr-9 to about Leu-17.
  • Particularly interesting discovered coil structures are from about His-22 to about Thr-27; from about Gly-40 to about Ile-50; from about Ser-51 to about Gly-69; and from about Gln-79 to about Cys-82.
  • a particularly interesting discovered strand structure is from about Thr-30 to about Val-32 (TMV). Further encompassed by the invention are contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one coil-strand-coil-coil motif of LP317 combines the coil from about His-22 to about Thr-27, the strand from about Thr-30 to about Val-32, the coil from about Gly-40 to about Ile-50, and the coil from about Ser-51 to about Gly-69 to form an interesting fragment of contiguous amino acid residues from about His-22 to about to about Gly-69.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP peptide encompasses cationic peptide LP variants which, as defined herein, refer to an LP peptide of the invention with a net positive charge within the pH range of from about pH4.0 to about pHlO.O, including pH values of: 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.7, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.9, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, and 10.
  • a cationic LP peptide variant is at least five contiguous amino acids in length of an LP peptide described herein, and has at least one basic amino acid (e.g, arginine, lysine, histidine).
  • a cationic LP peptide variant typicaUy does not have more than about 25, about 27, about 30, about 35, about 40, about 45, about 50, about 55 or about 60 amino acids, and typicaUy has about 12, 13, 14, 15, 16, 17, 18, 19, amino acid residues; more preferably at least about: 20, 21, 22, 24, 26, or 29 amino acid residues, favorably at least about: 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acid residues, more preferably, at least about: 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 amino acid residues; desirably at least about: 50, 51, 52, 53, 54, 55,
  • nucleotides particularly at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69 amino acid residues; more particularly at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79; predominandy at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89; and even more favorably at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues.
  • Similar cationic-bke peptide examples include, for instanced, vertebrate defensins, such as human neutrophil defensins [HNP 1-4], paneth ceU defensins of mouse and human smaU intestine (Oulette and Selsted, FASEB J. 10:1280, 1996; Porter et al. Infect. Immun. 65:2396, 1997), vertebrate defensins, such as HBD-1 of human epithebal ceUs (Zhao et al, FEBS Lett. 368:331, 1995), HBD-2 of inflamed human skin (Harder et al.
  • vertebrate defensins such as human neutrophil defensins [HNP 1-4]
  • paneth ceU defensins of mouse and human smaU intestine Oulette and Selsted, FASEB J. 10:1280, 1996; Porter et al. Infect. Immun. 65:
  • LP cationic peptide variant encompassed by the term LP peptide is a cationic peptide that has been conjugated with a bioactive agent, such as a one described herein.
  • Additional cationic peptide variants encompassed by the term LP peptides are peptides that have one or more amino acids altered to a corresponding D-amino acid.
  • the N-terminal and/or C-terminal amino acid can be a D-amino acid.
  • 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.
  • the antibiotic activity of such LP analogs or variants can be determined using any art known method, such as an assay described herein. As an iUustration, an in vivo assay to measure anti-microbial activity is used as described herein. An in vivo assay can also be used to evaluate the activity of a cationic peptide analog or variant for treatment of tumors.
  • in vitro assays can provide a simple test for anti-nepotistic LP analogs or anti-nepotistic variant LP peptides, such as the methylthiazoltetrazobum (MTT) or the lactate dehydrogenase (LDH) assay.
  • MTT assay is a tetrazobum dye colormetric assay that measures ceU viabibty, while the LDH assay measures ceU cytotoxicity.
  • LP317 Functions Given the teachings suppbed herein, for example, of: LP317 primary amino acid, LP317 higher order structures, the relationship of LP317 amino acid sequence to higher order structural features of thiomns and short scorpion toxins, the comparabihty of LP317 sequence and/or LP317 higher order structure with known thiomns and short scorpion toxins, and the relationship of higher order structural features of such proteins with their known functions, it is bkely that an LP317, an LP317 variant, and/or an LP317 binding agent (e.g, such as an LP317 antibody (or fragment thereof)) plays a similar role/s in a variety of physiological processes.
  • an LP317, an LP317 variant, and/or an LP317 binding agent e.g, such as an LP317 antibody (or fragment thereof)
  • Some non-bmiting examples of functions an LP317, an LP317 variant, an LP317 hetero- or an LP317 homomultimer, or an LP317 binding agent or LP binding compound is bkely to participate in are, for example, those such as: mucosal immunity (such as, e.g, mucosal surfaces in, e.g, epitheba in the airway, skin, oropharynx, gingival crevice, and urogemtal); host defense; anti-microbial activity (such as, e.g, against bacteria; (such as, e.g. Gram-positive, Gram-negative, S. typhimurim, S. typhimurium, S.
  • mucosal immunity such as, e.g, mucosal surfaces in, e.g, epitheba in the airway, skin, oropharynx, gingival crevice, and urogemtal
  • host defense anti-microbial activity (such as,
  • protists such as, e.g, G.
  • ion flow such as, e.g, calcium, sodium, potassium, or chloride ions
  • modulation of an ionic flow through a membrane wherein said mediation of innate immunity
  • modulate chloride ion flow create an ionic pore in a membrane, creating an lomc pore in a membrane, wherein said membrane is in an epithebum, wherein said epithebum is a broncho epithebum; wherein said epithebum is in a lung; creating an ionic pore in a membrane, wherein said membrane is in a microbe, mounting and maintaining defense against
  • LP Antimicrobial Assays The antimicrobial activity of an isolated or recombinant LP317 or LP variant of the invention is tested against wild type and phoP mutant S. typhimurium by means of a modified plate diffusion assay (Lehrer, et al. 1991b J. Immunol. Methods 137:167-173, which is incorporated herein by reference for these assay methods) using wild type S. typhimurium (ATCC 10428) or an lsogenic phoP mutant of S. typhimurium (strain CS015 phoP102::Tnl0d-Cam, MiUer et al, supra, 1989).
  • the phoP locus is a two-component regulatory locus essential to S.
  • 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.
  • Many members of this group play a role in vertebrate development, such as for example in the development, estabbshment, remodebng, and/or maintenance of various organ or organ systems, such as, e.g, the nervous system, the reproductive system, the urogenital system, etc.
  • LP283 exhibits a unique domain architecture having an N-terminal signal peptide sequence, a series of tandem-bke EGF-bke repeats (approximately nine) and a C-terminal CUB-bke domain.
  • the CUB domain (Complement subcomponents Clr/Cls, Uegf, Bmpl; see, Bork & Beckman 1993 J. Mol. Biol. 231:539-45) is a domain spanning approximately 100-110 amino acid residues, which were first reported in the complement subcomponents Clr/Cls, epidermal-growth-factor-related sea urchin protein and bone morphogenetic protein 1.
  • CUB domains are involved in protein-protein and glycosaminoglycan interactions.
  • a number of proteins have been identified that contain both EGF and CUB domains, including Drosophila tolloid, the mammaban toUoid-related genes BMP1 and mTll,f ⁇ ropellin I and III from sea urchin, and the serum glycoprotein attractin. Each of these proteins is impbcated in the regulation of extraceUular processes such as communication, adhesion, and guidance. Based on sequence and domain architecture similarity between LP283 and such proteins it is bkely that LP283 or an LP283 variant (or a fragment thereof) wiU also function in such a role/s.
  • the domain architecture and sequence of LP283 shows similarity at the amino acid sequence level to a mammaban gene family that is defined by two proteins designated SCUBE1 and SCUBE2 (signal peptide-CUB domain-EGF-related 1, and 2; see, Grimmond, et al. 2000 Genomics 70:74-81; Grimmond, et al. 2001 Mech Dev 102:209-211) and to the fibropeUins (Bisgrove, et al. 1995 J. Mol. Evol 41:34-45).
  • 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).
  • fibropeUins function during developmental periods when the organization of the sea urchin embryo is changing rapidly due to mesenchymal ceU ingression, gastrulation, and larval morphogenesis, aU of which are processes that are bebeved to involve the interaction of migrating ceUs and ECM components (Bisgrove and Raff 1993 Dev Biol 157:526-538).
  • fibropeUins function by mediating ceU movements.
  • EGF and CUB domains are impbcated in the physical association of the fibropeUins with ECM proteins.
  • LP283, LP283 variants, or LP283 fragments wiU play a role in the development of vertebrate organs or organ systems, such as, for example, the central nervous system, the reproductive system, the urogenital system, and/or the development of the bmbs.
  • 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, LP283 variants e.g, LP344, LP345, & LP346) or fragments thereof have both specific and general utibty.
  • compositions comprising P283, LP344, LP345, & LP346 polypeptides or polynucleotides, (fragments thereof), P283, LP344, LP345, & LP346 agonists or antagonists, and/or binding compositions (e.g, P283, LP344, LP345, & LP346 antibodies) wiU also be useful for diagnosis, and/or prognosis, of such a disease, condition, syndrome, or state.
  • the underlined portion is a predicted signal sequence (Met-1 to Ala-27)
  • a predicted SP cleavage site is between Ala-20 and Gln-23 indicated as follows 1 MGSGRVPGLCLLVLLVHARA ⁇ AQ 22
  • An alternate predicted SP cleavage site is between Cys-40 and Thr-43 indicated as follows' 1 MGSGRVPGLCLLVLLVHARAAQYSKAAQDVDECVEGTDNC ⁇ ILT 43
  • Each mature LP283 version is encompassed herein
  • An LP encompassed herein includes full-length forms encoded by an ORF disclosed herein, as well as any mature forms therefrom Such a mature LP could be formed, for example, by the removal of a signal peptide and/or by aminopeptidase modification
  • a "mature" LP encompasses, e g , post-translational modifications other than proteolytic cleavages (such as, e g , by way of a non-limiting example, glycosy
  • a predicted mature LP283 sequence is as follows:
  • 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).
  • the alternate predicted mature LP283 sequence (LP344) is as follows: GSGRVPGLCLLVLLVHARAAQYSKAAQDVDECVEGTDNCHIDAICQNTPRSYKCICKSGYTGDGKHCKDVD ECEREDNAGCVHDCVNIPGNYRCTCYDGFHLAHDGHNCLDVDECAEGNGGCQQSCVNMMGSYECHCREGFFL SDNQHTCIQRPEEGM CM KNHGCAHICRETPKGGIACECRPGFELTKNQRDCKLTCNYGNGGCQHTCDDTE QGPRCGCHIKFVLHTDGKTCIETCAVNNGGCDSKCHDAATGVHCTCPVGFMLQPDRKTCKDIDECRLN GGC DHICR TVGSFECSCKKGYKLLINER CQDIDECSFDRTCDHICVNTPGSFQCLCHRGYLLYGITHCGDVDE CSINRGGCRFGCINTPGSYQCTCPAGQGRLHW GKDCTEPLKCQGSPGASKAMLSC
  • 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, .
  • LP283 splice variants are LP283 splice variants. Still another such LP variant is listed below. Applicants discovered that this LP283 variant (also known as LP346 (SEQ ID NO: 23)) is the result of loss of processing
  • Exon 7 normally encodes the LP283 portion from about Gly-238 to about Asn-253 (GERRLEQHIPTQAVSN).
  • Loss of the alternative form of exon 7 and of exon 17 leads to a truncated LP283 variant and an altered sequence C- terminad to the normal exon 7 amino acid sequence so that the EGF-like domain sequence CAVNNGGCDSKCHDAATGVHCTCPVGFMLQPDRKTC is changed to
  • Hs6_7350ch 20560 CACCTTACCCCCCATTTCCTTCTCTCTCCTCCAGATGCCAGTGGTACTCCCTCTCAGCTC
  • Hs6_7350ch 20620 CACCAGCAACCCTGTTTCTTCCTCACCAACTCCAGCCTTCCATCTCTTACCTTGATTTGA A _,*****************************************************
  • Hs6_7350ch 30220 CTGGGTGGTGGGAAATGCGGGGGTGGGTGGCTAGCGCGGCCGACTCTCCCTCAGTCAGCT
  • Hs6_7350ch 30280 GCCCGCAGGGAACGTATTACCACGGCCAGACGGAGCAGTGTGTGCCATGCCCAGCGGGCA
  • SCUBElh4ds 1782 CCTTCCAGGAGAGAGAAGGGCAGCTCTCCTGCGACCTTTGCCCTGGGAGTGATGCCCACG
  • Hs6_7350ch 30340 CCTTCCAGGAGAGAGAAGGGCAGCTCTCCTGCGACCTTTGCCCTGGGAGTGATGCCCACG
  • SCUBElh4ds 1842 GGCCTCTTGGAGCCACCAACGTCACCACGTGTGCAG Hs6_7350ch 30400 GGCCTCTTGGAGCCACCAACGTCACCACGTGTGCAGGTGCCAGGGGAACAAACAATACAG
  • interesting segments of LP283 are the foUowing segments: from about Cys-33 to about Cys-68, from about Cys-74 to about Cys-110, from about Cys-1 6 to about Cys-151, from about Cys-201 to about Cys-236, from about Cys-256 to about Cys-291, from about Cys-297 to about Cys-332, from about Cys-338 to about Cys-371, from about Cys-377 to about Cys-413, from about Cys-55 to about Cys-68, from about Cys-95 to about Cys-110, from about Cys-136 to about Cys-151, from about Cys-182 to about Cys-197, from about Cys-276 to about Cys-291, from about Cys-317 to about Cys-332, from about Cys-357 to about Cys-371, and from about Cys-161 to about Cys-197, which have been discovered to be EGF-Uke domains.
  • AdditionaUy interesting segments of LP283 are the segments: from about Asp-29 to about Cys-55, from about Asp -70 to about Cys-95, from about Asp-112 to about Cys-136, from about Asp-293 to about Cys-317, from about Asp-334 to about Cys- 357, and from about Asp-151 to about Arg-166 which are aU identified as a calcium-binding EGF-Uke domains.
  • Additional interesting segments of LP283 are: from about Cys-46 to about Cys-57, from about Cys-86 to about Cys-97, from about Cys-127 to about Cys-138, from about Cys-308 to about Cys-319, from about Cys-348 to about Cys-359, and from about Cys-388 to about Cys-399), which have been discovered to be aspartic acid and/or asparagine hydroxylation-Uke sites.
  • a further interesting segment of LP283 is from about Cys-95 to about Thr-231, which has been discovered to be a keratin B2-Uke domain.
  • a further interesting segment of LP283 is from about Cys-392 to about Thr-465, which has been discovered to be a metaUothionein-Uke domain. Accordingly, one could test an LP283 or LP283 variant for possible metaUoproteinase activity using any standard method in the art without requiring undue experimentation. For example, commerciaUy available kits can be purchased which test for specific matrix metaUoproteinase activity (see, e.g., Biotrack MMP Bioassays from Amersham Pharmacia Biotech Limited) or the method of Hojima, et al. (1985 J. Biol.
  • Chem 260:15996-16003; incorporated herein for its assay methods can be adapted for use with an LP of the invention to test for enzymatic activity, however, other methods are also known and can be adapted for use here given the teachings suppUed herein of the LP283 sequence.
  • a further interesting segment of LP283 is from about Cys-820 to about Tyr-929, which has been discovered to be a CUB-Uke domain.
  • LP283 Other interesting segments of LP283 are discovered portions of LP283 from about Leu-11 to about Ser-24; from about Lys-55 to about Asn-79; from about Arg-154 to about Pro-176; from about Lys- 192 to about Lys-226; from about His-230 to about Val-251; from about Asn-253 to about Ala-271; from about Thr-272 to about Thr-290; from about Cys-291 to about Cys-308; from about Gly-353 to about Asp-375; from about Cys-377 to about Cys-399; from about Trp-408 to about Gly-421; from about Lys-427 to about Ser-447; from about Ala-449 to about Pro- 466; from about Ala-470 to about Ala-499; from about Phe-501 to about Gly-526; from about Pro-527 to about Thr-538; from about Phe-539 to about Val-563; from about Thr- 564 to about Gly-584; from about Asn-608 to
  • LP283 Additional interesting sections of LP283 are the discovered portions of LP283 from about Leu-12 to about Val-30; from about Asp-31 to about Ile-45; from about Cys-46 to about Tyr-61; from about Thr-62 to about Asp-72; from about Cys-82 to about Tyr-98; from about Asp-99 to about Cys-110; from about Cys-114 to about Val-128; from about Val-129 to about Leu-144; from about Ser-145 to about Gly-167; from about Ile-171 to about Lys-192: from about Lys-198 to about Asp-214; from about Asp-232 to about Glu- 244; from about Thr-248 to about Asp-264; from about Ser-265 to about Val-274; from about His-275 to about Asp-287; from about Asp-295 to about His-306; from about Ile-307 to about Cys-319; from about Leu-326 to about Phe-340 from about Arg-342 to about Cys-
  • particularly interesting LP 283 segments are LP secondary structures (e.g., such as a heUx, a strand, or a coil).
  • Particularly interesting LP283 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 His-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; from
  • Particularly interesting heUx structures are from about His-17 to about Ala-27; from about Leu-242 to about Glu-243; from about Leu-570 to about Glu-571; from about Arg-589 to about Leu-596; from about Leu-600 to about Lys-605; from about Tyr-936 to about Arg-945; from about Gln-956 to about Phe-969; and from about Ser-992 to about Leu-997.
  • Particularly interesting strand structures are the foUowing: from about Lys-54 to about Cys-57; from about Cys-95 to about Cys-97; from about Leu-Ill to about Val-113; from about Ile-180 to about Cys-182; from about Ile-225 to about Leu-229; from about Cys- 256 to about Ala-257; from about His-275 to about Cys-276; from about Cys-297 to about Arg-298; from about His-306 to about Cys-308; from about Leu-325 to about Ile-327; from about His-346 to about Val-349; from about Gln-356 to about Leu-358; from about Leu-364 to about Tyr-366; from about Tyr-395 to about Cys-397; from about Ala-428 to about Leu- 430; from about Thr-460 to about Ser-462; from about Nal-537 to about Leu-542; from about Cys-652 to about Val-653; from about Val-702 to about Thr-704; from
  • one coil-strand-coil-helix-coil motif of LP283 combines the Cys-212 to about Gly-222 coil; with the Ile-225 to about Leu-229 strand; with the Thr-231 to about Lys-234 coil; with the Leu-242 to about Glu-243 heUx; with the His- 245 to about Asn 253 coil to form an interesting fragment of contiguous amino acid residues from Cys-212 to Asn-253.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • interesting segments of LP344 are the foUowing segments: from about Cys-33 to about Cys-68, from about Cys-74 to about Cys-110, from about Cys-116 to about Cys-151, from about Cys-201 to about Cys-236, from about Cys-240 to about Cys-275, from about Cys-281 to about Cys-316, from about Cys-322 to about Cys-355, from about Cys-366 to about Cys-397, from about Cys-55 to about Cys-68, from about Cys-95 to about Cys-110, from about Cys-136 to about Cys-151, from about Cys-182 to about Cys-197, from about Cys-260 to about Cys-275, from about Cys-301 to about Cys-316, from about Cys-341 to about Cys-355, and from about Cys-161 to about Cys-197, which have been discovered to be EGF-Uke domains.
  • AdditionaUy interesting segments of LP344 are the segments: from about Asp-29 to about Cys-55, from about Asp -70 to about Cys-95, from about Asp-112 to about Cys-136, from about Asp-318 to about Cys-341, and from about Asp-151 to about Arg-166 which are aU identified as a calcium-binding EGF-Uke domains.
  • Additional interesting segments of LP344 are: from about Cys-46 to about Cys-57, from about Cys-86 to about Cys-97, from about Cys-127 to about Cys-138, from about Cys-308 to about Cys- 319, from about Cys-348 to about Cys-359, and from about Cys-388 to about Cys-399, which have been discovered to be aspartic acid and/or asparagine hydroxylation-Uke sites.
  • a further interesting segment of LP344 is from about Cys-95 to about Thr-231, which has been discovered to be a keratin B2-Uke domain.
  • a further interesting segment of LP344 is from about Cys-376 to about Thr-449, which has been discovered to be a metaUothionein-Uke domain.
  • a further interesting segment of LP344 is from about Cys-804 to about Tyr-913, which has been discovered to be a CUB-Uke domain.
  • LP344 portions of LP344 from about Leu-11 to about Ser-24; from about Lys-55 to about Asn-79; from about Arg-154 to about Pro-176; from about Lys-192 to about Lys-226; from about Asn-238 to about Ala-255; from about Thr-256 to about Thr-274; from about Cys-275 to about Cys-382; from about Gly-337 to about Asp-359; from about Cys-361 to about Cys-383; from about Trp-492 to about Gly-405; from about Lys-411 to about Ser-431; from about Ala-433 to about Pro-450; from about Ala-454 to about Ala-483; from about Phe-585 to about Gly-510; from about Pro-511 to about Thr-512; from about Phe-522 to about Val-557; from about Thr-558 to about Gly-568; from about Asn-692 to about Glu- 617; from about Ala-619 to about Tyr-644;
  • LP344 Additional interesting sections of LP344 are the discovered portions of LP344 from about Leu-12 to about Val-30; from about Asp-31 to about Ile-45; from about Cys-46 to about Tyr-61; from about Thr-62 to about Asp-72; from about Cys-82 to about Tyr-98; from about Asp-99 to about Cys-110; from about Cys-114 to about Val-128; from about Val-129 to about Leu-144; from about Ser-145 to about Gly-167; from about Ile-171 to about Lys-192: from about Lys-198 to about Asp- 214; from about Thr-232 to about Asp-248; from about Ser-249 to about Nal-258; from about H ⁇ s-259 to about Asp-271; from about Asp-279 to about His-290; from about Ile-291 to about Cys-303; from about Leu-280 to about Phe-324; from about Arg-325 to about Cys- 343; from about Tyr-350 to
  • particularly interesting LP 283 segments are LP secondary structures (e.g., such as a heUx, a strand, or a coil).
  • Particularly interesting LP344 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-920 to about Arg-929; from about Gln-940 to about Phe-953; and from about Ser-986 to about Leu-981.
  • Particularly interesting strand structures are the foUowing: from about Lys-54 to about Cys-57; from about Cys-95 to about Cys-97; from about Leu- 11 to about Val-113; from about Ile-180 to about Cys-182; from about Ile-225 to about Leu-229; from about Cys-240 to about Ala-241; from about His-269 to about Cys-260; from about Cys-281 to about Arg-282; from about His-290 to about Cys- 292; from about Leu-309 to about Ile-301; from about His-330 to about Val-333; from about Gln-340 to about Leu-342; from about Leu-358 to about Tyr-350; from about Tyr-379 to about Cys-371 ; from about Ala-412 to about Leu-414; from about Thr-444 to about Ser-446; from about Val-521 to about Leu-526; from about Cys-636 to about Nal-637; from about Val-676 to about Thr-678; from about
  • one coil-strand-coil-heUx-coil motif of LP344 combines the Cys-212 to about Gly-222 coil; with the Ile-225 to about Leu-229 strand; with the Thr-231 to about Lys-234 coil; to form an interesting fragment of contiguous amino acid residues from Cys-212 to Lys-234.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • interesting segments of LP345, are the foUowing segments: from about Cys-33 to about Cys-68, from about Cys-74 to about Cys-110, from about Cys-116 to about Cys-151, from about Cys-201 to about Cys-236, from about Cys-240 to about Cys-275, from about Cys-281 to about Cys-316, from about Cys-322 to about Cys-355, from about Cys-366 to about Cys-397, from about Cys-55 to about Cys-68, from about Cys-95 to about Cys-110, from about Cys-136 to about Cys-151, from about Cys-182 to about Cys-197, from about Cys-260 to about Cys-275, from about Cys-301 to about Cys-316, from about Cys-341 to about Cys-355, and from about Cys-161 to about Cys-197, which have been discovered to be EGF-Uke domains.
  • AdditionaUy interesting segments of LP345 are the segments: from about Asp-29 to about Cys-55, from about Asp -70 to about Cys-95, from about Asp-112 to about Cys-136, from about Asp-318 to about Cys-341, and from about Asp-151 to about Arg-166 which are aU identified as a calcium-binding EGF-Uke domains.
  • Additional interesting segments of LP345, are: from about Cys-46 to about Cys-57, from about Cys-86 to about Cys-97, from about Cys-127 to about Cys-138, from about Cys-308 to about Cys- 319, from about Cys-348 to about Cys-359, and from about Cys-388 to about Cys-399, which have been discovered to be aspartic acid and/or asparagine hydroxylation-Uke sites.
  • a further interesting segment of LP345, is from about Cys-95 to about Thr-231, which has been discovered to be a keratin B2-like domain.
  • a further interesting segment of LP345, is from about Cys-376 to about Thr-449, which has been discovered to be a metaUothionein- Uke domain.
  • a further interesting segment of LP345, is from about Cys-750 to about Tyr- 859, which has been discovered to be a CUB-Uke domain.
  • LP345 Other interesting segments of LP345, are discovered portions of LP345, from about Leu-11 to about Ser-24; from about Lys-55 to about Asn-79; from about Arg-154 to about Pro-176; from about Lys-192 to about Lys-226; from about Asn-238 to about Ala-255; from about Thr-256 to about Thr-274; from about Cys-275 to about Cys-382; from about Gly-337 to about Asp-359; from about Cys-361 to about Cys-383; from about Trp-492 to about Gly-405; from about Lys-411 to about Ser- 431; from about Ala-433 to about Pro-450; from about Ala-454 to about Ala-483; from about Phe-585 to about Gly-510; from about Pro-511 to about Thr-512; from about Phe-522 to about Val-557; from about Thr-558 to about Gly-568; from about Asn-692 to about Glu- 617; from about Gly-658
  • LP345 Additional interesting sections of LP345, are the discovered portions of LP345, from about Leu-12 to about Nal-30; from about Asp-31 to about Ile-45; from about Cys-46 to about Tyr-61; from about Thr-62 to about Asp-72; from about Cys-82 to about Tyr-98; from about Asp-99 to about Cys-110; from about Cys-114 to about Val-128; from about Val-129 to about Leu-144; from about Ser-145 to about Gly-167; from about Ile-171 to about Lys-192: from about Lys-198 to about Asp-214; from about Thr-232 to about Asp-248; from about Ser-249 to about Val- 258; from about H ⁇ s-259 to about Asp-271; from about Asp-279 to about His-290; from about lle-291 to about Cys-303; from about Leu-280 to about Phe-324; from about Arg-325 to about Cys-343; from about Tyr-350 to
  • 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.
  • Particularly interesting strand structures are the foUowing: from about Lys-54 to about Cys-57; from about Cys-95 to about Cys-97; from about Leu-I ll to about Val-113; from about Ile-180 to about Cys-182; from about Ile-225 to about Leu-229; from about Cys-240 to about Ala-241 ; from about His-269 to about Cys-260; from about Cys-281 to about Arg-282; from about His-290 to about Cys- 292; from about Leu-309 to about Ile-301; from about His-330 to about Nal-333; from about Gln-340 to about Leu-342; from about Leu-358 to about Tyr-350; from about Tyr-379 to about Cys-371; from about Ala-412 to about Leu-414; from about Thr-444 to about Ser-446; from about Nal-521 to about Leu-526; from about Val-622 to about Thr-624; from about Thr-567 to about Phe-
  • contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one coil-strand-coil-heUx-coil motif of LP345 combines the Cys-212 to about Gly-222 coil; with the Ile-225 to about Leu-229 strand; with the Thr-231 to about Lys-234 coil; to form an interesting fragment of contiguous amino acid residues from Cys-212 to Lys-234.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined.
  • LP346 portions of LP346 from about Pro-7 to about His-17; from about Ala-18 to about Glu-32; from about Cys-33 to about Ile- 43; from about Asp-43 to about Ser-52; from about Lys-58 to about Nal-71; from about Asp- 72 to about Asn-88; from about Ala-104 to about Val-113; from about Asp-114 to about Asn-129; from about Asp-146 to about Met-159; from about Asn-160 to about His-170; from about Ile-171 to about Pro-186; from about Cys-201 to about His-210; from about Thr-211 to about Cys-218; from about Thr-231 to about Gln-249; from about Pro-250 to about Leu-259; from about Leu-11 to about Ala-20; from about Ala-21 to about Glu-32; from about Cys-33 to about Ile-42; froma about Asp-43 to about Cys-57; from about Lys-
  • LP346 Additional interesting sections of LP346 are the discovered portions of LP346 from about Leu-11 to about Ala-21; from about Gln-22 to about Asp-31; from about Glu-32 to about Ile-42; from about Asp-43 to about Tyr-53; from about Tyr-53 to about Thr-62; from about Gly-63 to about Cys-74; from about Glu-75 to about Val-87; from about Asn-88 to about Tyr-98; from about Asp-99 to about Asp-112; from about Val-113 to about Gln-124; from about Gln-125 to about Phe-142; from about Leu-144 to about Glu-157; from about Gly-158 to about Cys-168; from about Cys-172 to about Cys-182; from about Glu-183 to about Lys-192; from about Leu-199 to about Cys-208; from about Gln-209 to about His- 224; and from about Gln-245 to about Leu-259.
  • particularly interesting LP346 segments are LP secondary structures (e.g., such as a heUx, a strand, or a coil).
  • Particularly interesting LP346 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 bout 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 His-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 abou Ser-145 to about His-149; from about Gln-153 to about Asn-160; from about Asn-163 to about Cys-168; from about Arg-173 to about Gly-179
  • a particularly interesting heUx structure is from about His-17 to about Ala-27.
  • Particularly interesting strands are from about Lys-54 to about Cys-57; from about Cys-95 to about Cys-97; from about Leu-111 to about Val-113; from about Ile-180 to about Cys-182; and from about Ue-225 to about Leu-229.
  • Further encompassed by the invention are contiguous amino acid residue combinations of any of the predicted secondary structures described above.
  • one coil-strand-coil-coil motif of LP346 combines the His-105 to about Cys-110 coil; with the Leu-111 to about Nal-113 strand; with the Glu-118 to about Cys-123 coil; with the Met-131 to about Gly-141 coil to form an interesting fragment of contiguous amino acid residues from about His-105 to about Gly-141.
  • Other combinations of contiguous amino acids are contemplated as can be easily determined
  • LP283 and variants Functions Given the teachings suppUed herein, for example, of: LP283 (or variants) primary amino acid, the sequence information and knowledge of the secondary structural features of proteins that exhibit sequence similarity to LP283 or variants, such as, for example, SCUBE1, SCUBE2, Drosophila toUoid, the mammaUan toUoid-related genes BMP1 and mTU, fibropeUin I and III from sea urchin, and the serum glycoprotein attractin sequence, and how these features map onto LP283 sequence presented herein (e.g., such as the relationship between the primary amino acid sequence of LP283 active regions and higher order structure of similar CUB-Uke domains such as, the crystal structure of ISPP (Romero, et al.
  • 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.”
  • AA SEQ ID NO: Y The corresponding translated amino acid sequence of a particular NT SEQ ID NO:X, typicaUy beginning with the methionine, is identified as "AA SEQ ID NO: Y," although other reading frames can also be easily translated using techniques known in molecular biology.
  • a polypeptide produced using an alternative open reading frame/s is also specificaUy encompassed by the present invention.
  • the first and last amino acid position of a SEQ ID NO: Y of the predicted signal peptide is identified as "First AA of Signal Pep" and "Last AA of Signal Pep.”
  • the predicted first amino acid position of SEQ ID NO: Y of the secreted portion is identified as "Predicted First AA of Secreted Portion.” FinaUy, the amino acid position of SEQ ID NO: Y of the last amino acid in the open reading frame is identified as "Last AA of ORF.”
  • An LP polypeptide or fragment thereof, identified from SEQ ID NO: Y may be used, e.g., as an immunogen to generate an antibody that specificaUy and/or selectively binds a protein comprising an LP polypeptide sequence (or fragment thereof) of the invention and/or to a mature LP polypeptide or secreted LP protein, e.g., encoded by a polynucleotide sequence described herein
  • An LP polypeptide of the invention can be prepared in any manner suitable to those known in the art.
  • 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 present invention provides an isolated or recombinant LP polynucleotide comprising, or alternatively consisting of, a nucleic acid molecule having a mature polynucleotide sequence of SEQ ID NO: X wherein said polynucleotide sequence or said cDNA encodes at least 12 contiguous amino acids of a mature polypeptide of SEQ ID NO: Y.
  • LP polynucleotide refers to a molecule comprising a nucleic acid sequence contained in a Table herein or in a sequence of SEQ ID NO:X.
  • 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.
  • an LP polynucleotide sequence is at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 contiguous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
  • An LP polynucleotide sequence can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typicaUy, double-stranded or a mixture of single-and double-stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stabiUty or for other reasons.
  • Modified bases can include, e.g., for example, tritylated bases and unusual bases such as inosine.
  • tritylated bases can include, e.g., for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, the term "polynucleotide" embraces chemicaUy, enzymaticaUy, or metaboUcaUy modified forms.
  • 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" in a nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimaUy aUgned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generaUy at least 56%, more generaUy at least 59%, ordinarily at least 62%, more ordinarily at least 65%, often at least 68%, more often at least 71%, typicaUy at least 74%, more typicaUy at least 77%, usuaUy at least 80%, more usuaUy at least about 85%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides.
  • 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.
  • the length of similarity comparison may be over longer stretches, and in certain embodiments wiU be over a stretch of at least about 17 nucleotides, usuaUy at least about 20 nucleotides, more usuaUy at least about 24 nucleotides, typicaUy at least about 28 nucleotides, more typicaUy at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides, e.g., 150, 200, etc.
  • typicaUy one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optical aUgnment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology aUgnment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci.
  • 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.
  • the term "about” includes, e.g., a particularly recited temperature (e.g., 50°C), and/or a temperature that is greater or lesser than that of the stated temperature by, e.g., one, two, three, four, or five degrees Celsius (e.g., 49°C or 51 °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.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is indicative of a similar functional and/or biological role for the nucleotide sequence and its correspondingly encoded polypeptide sequence.
  • 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.
  • a polynucleotide that hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA of the invention), or to a complementary stretch of T (or U) residues, is not included, e.g., in the definition of an "LP polynucleotide” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (i.e., practicaUy any double-stranded cDNA clone generated using oUgo dT as a primer).
  • Still another non-Umiting example of a stringent hybridization condition is one that employs, e.g.: low ionic strength and high temperature for washing (e.g., 15mM sodium chloride/1.5 mM sodium citrate/0.1% sodium dodecyl sulfate at 50°C); a denaturing agent (during hybridization) such as formamide (e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoU/0.1% polyvinylpyrroUdone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride/75 mM sodium citrate at 42°C); or 50% formamide, 5X SSC (750 ⁇ M sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/mL), 0.1% SDS, and 10% dex
  • 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.
  • a "recombinant" nucleic acid or polynucleotide sequence is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of any genetic engineering technique, e.g., products made by transforming ceUs with any non-naturaUy occurring vector are encompassed, as are nucleic acids comprising sequence derived using any synthetic oUgonucleotide process. A similar concept is intended for a recombinant LP polypeptide. SpecificaUy included are synthetic nucleic acid molecules which, due to the redundancy of the genetic code, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
  • an "LP protein” shaU encompass, when used in a protein context, a protein or polypeptide having an amino acid sequence shown in SEQ ID NO: Y or a significant fragment of such a protein or polypeptide, preferably a natural embodiment.
  • the term “protein” or “polypeptide” is meant any chain of contiguous amino acid residues, regardless of length or postranslation modification (e.g., glycosylation, or phosphorylation).
  • an LP protein or an LP polypeptide encompass polypeptide sequences that are pre- or pro-proteins.
  • 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.
  • polypeptides may be branched, for example, as a result of ubiquitination, and they may be cycUc, with or without branching.
  • Cychc, branched, and branched cycUc polypeptides may result from posttransla ⁇ on natural processes or may be made by synthetic methods.
  • Modifications include, e.g., acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a Upid or Upid derivative, covalent attachment of phosphotidyUnositol, cross-Unking, cycUzation, disulfide bond formation, demethylation, formation of covalent cross-Unks, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation,
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues that produce a silent change and result in a functionaUy equivalent LP.
  • DeUberate ammo acid substitutions may be made based on similarity in polarity, charge, solubiUty, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of the LP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophiUcity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophiUcity values may include: leucine, isoleucine, and vaUne; glycine and alanine; and phenylalanine and tyrosine.
  • 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.
  • an LP polypeptide may be desirable to purify an LP polypeptide from recombinant ceU proteins or polypeptides.
  • Various art known methods of protein purification may be employed (see, e.g., Deutscher, (1990) Methods in Enzymology 182: 83-9 and Scopes, (1982) Protein Purification: Principles and Practice. Springer- Verlag, NY.)
  • Solubility of an LP protein or polypeptide is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions (see, Freifelder (1982) Physical Biochemistry (2d ed.) W.FI. Freeman & Co., San Francisco, CA; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3, W.H. Freeman & Co., San Francisco, CA).
  • a soluble particle or polypeptide wiU typicaUy be less than about 30S, more typicaUy less than about 15S, usuaUy less than about 10S, more usuaUy less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S.
  • SolubiUty of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubiUty, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. TypicaUy, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C.
  • 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 usuaUy be a biologicaUy compatible buffer, of a type used for preservation of biological activities, and wiU usuaUy approximate a physiological solvent.
  • 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 present invention encompasses "mature" forms of a polypeptide comprising a polypeptide sequence Usted in a Table herein, or a polypeptide sequence of SEQ ID NO: Y.
  • Methods for predicting whether a protein has a signal sequence, as weU as the cleavage point for that sequence, are known in the art (see, e.g., McGeoch, 1985 Virus Res. 3:271-286 and Henrik Nielsen et al. (1997) Protein Engineering 10: 1-6). Employing such known art methods a signal sequence for an LP polypeptide was made. However, cleavage sites may vary and cannot be predicted with absolute certainty.
  • the present invention provides secreted LP polypeptides having a sequence Usted in a Table herein, or a polypeptide sequence of SEQ ID NO: Y, in which a particular N-terminus variant polypeptide sequence can begin within five, four, three, two, or one amino acid residues (e.g., +5, +4, +3, +2, +1, or -5, -4, -3, -2, -1) from a particular cleavage point designated as such herein.
  • cleavage of a signal sequence of a secreted protein is not uniform, resulting in more than one secreted species for a given protein (e.g., a cleavage variant).
  • Such cleavage variant LP polypeptides, and the polynucleotides encoding them are also encompassed by the present invention.
  • 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 variants of an LP polynucleotide sequence disclosed in a table herein or SEQ ID NO: X and/or the complementary strand thereto.
  • the present invention also encompasses variants of a polypeptide sequence disclosed in a table herein or SEQ ID NO: Y.
  • variant refers to a polynucleotide or polypeptide differing from an LP polynucleotide sequence or an LP polypeptide of the present invention, but retaining essential properties thereof.
  • GeneraUy variants are closely similar overaU in structural and/or sequence identity, and, in many regions, identical to an LP polynucleotide or polypeptide of 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.
  • the present invention is also directed to polypeptides that comprise, or alternatively consist of, an amino acid sequence that is at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, e.g., a polypeptide sequence of SEQ ID NO: Y (or fragments thereof); a polypeptide sequence encoded by a cDNA contained in a deposited clone, and/or a polypeptide fragment of any of these polypeptides (e.g., those fragments as defined herein).
  • 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).
  • test sequence for a test sequence to exhibit at least 95% identity to a referent sequence, up to 5% of the nucleotides in the referent may differ, e.g., be deleted or substituted with another nucleotide, or a number of nucleotides (up to 5% of the total number of nucleotides in the reference sequence) may be inserted into the reference sequence.
  • the test sequence may be: an entire polynucleotide sequence, e.g., as shown in a Table herein, the ORF (open reading frame), or any fragment, segment, or portion thereof (as described herein).
  • 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.
  • a peptide or polypeptide in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence that comprises an amino acid sequence of the present invention, which contains at least: one, but not more than: 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.
  • the number of additions, substitutions, and/or deletions in an polypeptide sequence of the present invention or fragments thereof is at least: 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, 10-50, or 50-150; wherein conservative amino acid substitutions are more preferable than non-conservative substitutions.
  • the present invention is also directed to fragments of an LP polynucleotide.
  • An LP polynucleotide "fragment” encompasses a short polynucleotide of a nucleic acid molecule, or a portion of a polynucleotide sequence of SEQ ID NO: X or a complementary strand thereto, or a portion of a polynucleotide sequence encoding a polypeptide of SEQ ID NO: Y (or fragment thereof).
  • Polynucleotide fragments of the invention encompass a polynucleotide sequence that is preferably at least about 15 nucleotides, more preferably at least about: 20, 21, 22, 24, 26, or 29 nucleotides, favorably at least about: 30, 32, 34, 36, 38, or 39 nucleotides, and even more preferably, at least about: 40, 42, 44, 46, 48, or 49 nucleotides, desirably at least about: 50, 52, 54, 56, 58, or 59 nucleotides, particularly at least about 75 nucleotides, or at least about 150 nucleotides in length.
  • 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.
  • fragments e.g., 50, 150, 500, 600, or 2000 nucleotides
  • Representative examples of various lengths of polynucleotide fragments encompassed by the invention include, e.g., fragments comprising, or alternatively consisting of, a polynucleotide sequence of SEQ ID NO:X from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701
  • the term "about” includes, e.g., a particularly recited polynucleotide fragment range herein, and/or ranges that have lengths that are larger or smaUer by several nucleotides (e.g., 5, 4, 3, 2, or lnt), at either terminus or at both termini.
  • these fragments encode a polypeptide possessing biological activity as defined herein, e.g., lmmunogenicity, or antigenicity.
  • a polynucleotide fragment can be used as a probe or primer as discussed herein.
  • the present invention also encompasses a polynucleotide that stably hybridizes to a polynucleotide fragment described herein under either stringent or lowered stringency hybridization conditions.
  • AdditionaUy inco ⁇ orated are polypeptides encoded by a polynucleotide fragment or a hybridized polynucleotide stably bound to a polynucleotide fragment of the invention.
  • AdditionaUy encompassed by the invention is a polynucleotide encoding a polypeptide, which is specifically or selectively bound by an antibody directed to/or generated against a mature polypeptide of the invention (or fragment thereof), e.g., a mature polypeptide of SEQ ID NO: Y.
  • 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.
  • lengths of polypeptide fragments or segments encompassed by the invention include, e.g., fragments comprising, or alternatively consisting of, from about amino acid residue number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-170, 171- 180, 181-190, 191-200, 201-210, etc., to the end of the mature coding region of a polypeptide of the invention (or fragment thereof).
  • a polypeptide segment of the invention can have a length of contiguous amino acids of a polypeptide of the invention (or fragment thereof) that is at least about: 7, 8, 9, 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 amino acids in length.
  • polypeptide comprising more than one of the above polypeptide fragments is encompassed by the invention; including a polypeptide comprising at least: one, two, three, four, five, six, seven, eight, mne, ten, or more fragments, wherein the fragments (or combinations thereof) may be of any length described herein (e.g., a fragment of 12 contiguous amino acids and another fragment of 30 contiguous amino acids, etc.).
  • the invention also encompasses proteins or polypeptides comprising a pluraUty of distinct, e.g., non-overlapping, segments of specified lengths.
  • 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.
  • Polypeptide fragments of SEQ ID NO: Y falling within conserved domains are specificaUy encompassed by the present invention. Moreover, polynucleotides encoding these domains are also encompassed. Other preferred polypeptide segments are biologicaUy active fragments. BiologicaUy active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of an LP polypeptide (or fragment thereof). The biological activity of the fragments may include, e.g., an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
  • the polynucleotide fragments of the invention encode a polypeptide that demonstrates a functional activity.
  • the phrase "functional activity" encompasses a polypeptide segment that can accompUsh one or more known functional activities associated with a fuU-length (complete) polypeptide of invention protein.
  • Such functional activities include, e.g., without Umitation, biological activity, antigenicity [abiUty to bind (or compete with a polypeptide of the invention for binding) to an antibody to a polypeptide of the invention], lmmunogenicity (abiUty to generate antibody that binds to a polypeptide of the invention), abiUty to form multimers with a polypeptide of the invention, and the ability to bind to a receptor or Ugand of a polypeptide of the invention.
  • a polypeptide of the invention can be assayed by various methods.
  • various immunoassays known in the art can be used, including, e.g., without Umitation, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme Unked immunosorbent assay), "sandwich” immunoassays, lmmunoradiometnc assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using coUoidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays,
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding can be assayed, e g., by using reducing and non- reducing gel chromatography, protein affinity chromatography, and affinity blotting (see generaUy, Phizicky, et al. (1995) Microbial. Rev. 59:94-123).
  • physiological correlates of binding of a polypeptide of the invention to its substrates can be assayed with common techniques.
  • assays described herein see, e.g., the "Examples" section of the appUcation), or otherwise known in the art, can routinely be appUed to measure the abiUty of a polypeptide of the invention (its fragments, variants derivatives and analogs thereof) to eUcit a related biological activity (either in vitro or
  • the present invention encompasses a polypeptide comprising, or alternatively consisting of, an epitope of SEQ ID NO: Y or a table herein, or encoded by a polynucleotide that stably hybridizes to form a hybridization complex, under stringent hybridization conditions (or lower stringency hybridization conditions) as defined herein, to a complement of a sequence of SEQ ID NO: X.
  • 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
  • epitope refers to a portion of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as weU as the polynucleotide encoding this polypeptide.
  • an "immunogenic epitope,” as used herein, is defined as a portion of a protein or a Uneanzed polypeptide (or fragment thereof) that eUcits an antibody response in an animal, as determined by any art known method (e.g , by the methods for generating antibodies described herein or otherwise known, see, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 308 1:3998-4002).
  • an "antigenic epitope,” as used herein, is defined as a portion of a protein or polypeptide to which a binding composition, e.g., an antibody or antibody binding fragment, selectively binds or is specificaUy lmmunoreactive with as determined by any known art method, e g , by an immunoassay described herein. Selective binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody. Antigenic epitopes need not necessarily be immunogenic.
  • a protein or fragment e.g., an LP protein
  • the interaction is dependent upon the presence of a particular structure, e.g., an antigenic determinant (or epitope) recognized by a binding composition.
  • an antibody is specific for epitope "A”
  • the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction contaimng free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • the specified antibodies bind to a particular protein or polypeptide sequence and do not sigmficandy bind other proteins or other polypeptide sequences that are present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity and/or selectivity for a particular protein.
  • antibodies raised to the protein immunogen with an amino acid sequence depicted in SEQ ID NO: Y can be selected to obtain antibodies specificaUy immunoreactive with LP proteins or LP polypeptides and not with other proteins or polypeptides. These antibodies wiU also recogmze proteins or polypeptide sequences that have an above average degree of similarity or identity to an LP protein or LP polypeptide sequence. Fragments that function as epitopes can be produced by any conventional means such as, e.g., (1985) Houghten, Proc. Nad. Acad Sci. USA 82:5131-5135, further described in U.S. Patent No. 4,631,211.
  • an antigenic or immunogenic epitope preferably contains a polypeptide sequence of at least four, at least five, at least six, at least seven, more preferably at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, favorably, between about 15 to about 30 contiguous amino acids of a mature polypeptide of SEQ ID NO: Y or a Table herein.
  • Preferred polypeptide fragments of contiguous amino acid residues of SEQ ID NO: Y comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous amino acid residues in length.
  • Additional non-exclusive preferred antigenic epitopes include, e.g., the antigenic epitopes disclosed herein, as weU as portions thereof. Antigenic epitopes are useful, e.g., to generate antibodies, including monoclonal antibodies that specificaUy bind the epitope. Preferred antigenic epitopes include, e.g., the antigenic epitopes disclosed herein, as weU as any pluraUty thereof, e.g., at least: two, three, four, five or more of these antigenic epitopes in any combination or structural arrangement. Antigenic epitopes can be used as the target molecules in immunoassays (see, e.g., Wilson, et al.
  • immunogenic epitopes can be used, e.g., to induce antibodies according to any known art method (see, for instance, SutcUffe, et al. supra; Wilson, et al. supra; Chow, et al. Proc. Nad. Acad. Sci. USA 82:910-25914; and Bitde, et al. (1985) J. Gen. Virol. 66:2347-2354.
  • immunogenic epitopes include, e.g., an immunogenic epitope disclosed herein, as weU as a pluraUty or any combination thereof, e.g., of at least two, three, four, five or more of these immunogenic epitopes including, e.g., repeats of a particular epitope.
  • a polypeptide comprising a pluraUty of epitopes may be used to eUcit an antibody response with a carrier protein, such as, e.g., an albumin, to an animal system (such as, e.g., a rabbit or a mouse), or, if a polypeptide is of sufficient length (e.g., at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as, e.g., an albumin
  • an animal system such as, e.g., a rabbit or a mouse
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have also been shown to be sufficient to generate antibodies and to be useful since they are capable of binding to, e.g., Unear epitopes in a denatured polypeptide such as in Western blotting.
  • Polypeptides or proteins bearing an epitope of the present invention may be used to generate antibodies according to known methods including, e.g., without Umitation, in vivo immunization, in vitro immunization, and phage display methods (see, e.g., SutcUffe, et al. supra; Wilson, et al. supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. "Binding Composition"
  • binding composition refers to molecules that bind with specificity and/or selectivity to an LP of the invention or fragment thereof (such as, e.g , in an antibody-antigen interaction).
  • other compositions e.g., antibodies, oUgonucleotides, proteins (e.g., receptors), peptides, or smaU molecules
  • specificaUy and/or selectivity associate bind with the LP in contrast to other molecules.
  • the association wiU be in a natural physiologicaUy relevant protein-protein interaction (either covalent or non-covalent) and it may include members of a multi-protein complex (including carrier compounds or dimerization partners).
  • the composition may be a polymer or chemical reagent.
  • a functional analog may be a protein with structural modifications or may be a whoUy unrelated molecule (such as, e.g., one that has a molecular shape that interacts with the appropriate binding determinants).
  • the proteins may serve as agonists or antagonists of the binding partner, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (cur. ed.) Pergamon Press, Tarrytown, N.Y.
  • the LP may be used to screen for binding compositions that specificaUy and/or selectively bind an LP of the invention or fragment thereof (e.g., a binding composition can be a molecule, or part of one, that selectively and/or stoichiomet ⁇ caUy binds, whether covalendy or not, to one or more specific sites of an LP (or fragment thereof) such as, e.g , in an antigen-antibody interaction, a hormone-receptor interaction, a substrate-enzyme interaction, etc.). At least one and up to a pluraUty of test binding compositions can be screened for specific and/or selective binding with the LP.
  • a binding composition can be a molecule, or part of one, that selectively and/or stoichiomet ⁇ caUy binds, whether covalendy or not, to one or more specific sites of an LP (or fragment thereof) such as, e.g , in an antigen-antibody interaction,
  • a binding composition thus identified is closely related to a natural Ugand of an LP (such as, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner; see, e.g., CoUgan, et al. (1991) Current Protocols in Immunology l (2):Chapter 5.)
  • a natural Ugand of an LP such as, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner; see, e.g., CoUgan, et al. (1991) Current Protocols in Immunology l (2):Chapter 5.
  • binding agentLP complex refers to a complex of a binding agent and a LP (or fragment thereof) which is formed by specific and/or selective binding of the binding agent to the respective LP (or fragment thereof).
  • Specific and/or selective binding of the binding agent means that the binding agent has a specific and/or selective binding site that recognizes a site on the LP protein (or fragment thereof).
  • antibodies raised against a LP protein (or fragment thereof) that recogmze an epitope on the LP protein (or fragment thereof) are capable of forming a binding agentLP complex by specific and/or selective binding.
  • typingUy the formation of a binding agentLP complex aUows the measurement of LP protein (or fragment thereof) in a mixture of other proteins and/or biologies.
  • antibodyLP complex refers to an embodiment in which the binding agent, e.g., is an antibody.
  • the antibody may be monoclonal, polyclonal, or a binding fragment of an antibody (including, without Umit, e.g., Fv, Fab, or F(ab)2 fragments; diabodies, Unear antibodies (Zapata, et al., (1995) Protein Engin 8(10): 1057-62); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments).
  • the antibody is a polyclonal antibody.
  • Antibodies can be raised to various LP proteins, including individual, polymorphic, aUeUc, strain, or species variants, and fragments thereof, both in their naturaUy occurring
  • Antibodies of the invention include, e.g , without Umitation, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression Ubrary, anti-idiotypic (anti-Id) antibodies (including, e g , anti-Id antibodies to antibodies of the invention), and an epitope-binding fragment of any of the above.
  • human antibodies includes, e.g., without Umitation, antibodies having an amino acid sequence of a human immunoglobuUn including, e.g., without Umitation, an antibody isolated from a human immunoglobuUn Ubrary or from an ammal transgenic for one or more human immunoglobuUns and that do not express endogenous immunoglobuUns, as described herein or, as taught, e.g., in U.S. Patent No. 5,939,598.
  • An antibody of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of an LP polypeptide (or fragment thereof) or may be specific for both a polypeptide of the present invention as weU as for a heterologous epitope, such as a heterologous polypeptide or sohd support material (see, e.g., WO 2093/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al. (1991) J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; or 5,601,819; or Kostelny, et al. (1992) J. Immunol. 148:1547-1553 .
  • an antibody that selectively binds a polypeptide, which is encoded by a polynucleotide that stably hybridizes, under stringent hybridization conditions (as described herein), to an LP polynucleotide sequence.
  • An antibody of the present invention may also be characterized or specified in terms of its binding affinity to a protein or polypeptide (fragment thereof), or epitope of the invention.
  • a preferred binding affinity of a binding composition includes, e.g., a binding affinity that demonstrates a dissociation constant or Kd of less than about: 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 "3 M, 5 X 10 '4 M, 10 "4 M, 5 X 10 "5 M, 10 "5 M, 5 X 10 "6 M, 10 '6 M, 5 X 10 "7 M, 10 "7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 10 M, 10- ,0 M, 5 X 10 "n M, 10 n M, 5 X 10 "12 M, 10 “12 M, 5 X 10 “13 M, 10 ",3 M, 5 X 10 ⁇ 14 M, 10 "14 M, 5 X 10- 15 M, or 10 " 15 M.
  • the invention also encompasses antibodies that competitively inhibit binding of a binding composition to an epitope of the invention as determined by any known art method for determining competitive binding, e.g., the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of an LP polypeptide (or fragment thereof).
  • neutraUzing antibodies that bind a Ugand and prevent it binding to a receptor.
  • Ugand-binding antibodies that inhibit receptor activation without inhibiting receptor binding.
  • Ugand-binding antibodies that activate a receptor are also included.
  • Antibodies of the invention may act as receptor agonists, e.g., by potentiating or activating either aU or a subset of the biological activities of the Ugand-mediated receptor activation, e.g., by inducing dimerization of a receptor.
  • the antibodies may be specified as agonists, antagonists, or inverse agonists for biological activities comprising the specific biological activities of a peptide of the invention disclosed herein.
  • An antibody agonist can be made using known methods art (see, e.g., WO 96/40281; U.S. Patent No.
  • Antibodies of the present invention may be used, e.g., without Umitation, to purify, detect, or target a polypeptide (or fragment thereof) of the present invention for, e.g., in vitro and/or in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for quaUtatively and/or quantitatively measuring levels of a polypeptide (or fragment thereof) of the present invention in a biological sample (see, e.g., Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, cur. ed.; incorporated by reference).
  • the term "monoclonal antibody” as used herein is not Umited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Methods for producing and screening for specific antibodies using hybridoma technology are routine and known in the art. For an overview of the technology for producing human antibodies, see, e.g., Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). In addition, commercial companies such as, e.g., Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be hired to produce human antibodies. Completely human antibodies that recognize a selected epitope can be generated by
  • antibodies of the invention can, in turn, be used to generate anti-idiotype antibodies that "mimic” a polypeptide (or fragment thereof) of the invention using known techniques (see, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff,J. (1991) Immunol. 147(8):2429-2438).
  • the present invention encompasses antibodies recombinandy fused or chemicaUy conjugated (including both covalent and non-covalent conjugations) to a polypeptide (or portion thereof, preferably comprising at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 contiguous amino acids of a polypeptide of SED ID NO:X) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through Unker sequences.
  • the antibodies may be specific for antigens other than a polypeptide of the invention (or portion thereof, preferably at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 contiguous amino acids) of the present invention.
  • antibodies may be used to target an LP polypeptide (or fragment thereof) to particular ceU types, either in vitro or in vivo, by fusing or conjugating a polypeptide (or fragment thereof) of the present invention to an antibody specific for a particular ceU surface receptor.
  • Antibodies fused or conjugated to a polypeptide of the invention may also be used in in vitro immunoassays and in purification methods using known art methods (see e.g., Harbor, et al., supra, and WO 9312 1232; EP 439,095; Naramura et al. (1994) Immunol. Lett. 39:9 1-99).
  • the present invention further includes compositions comprising a polypeptide of the invention (or fragment thereof) fused or conjugated to an antibody domain other than a variable region.
  • a polypeptide of the invention (or fragment thereof) may be fused or con j ugated to an antibody Fc region, or portion thereof.
  • the antibody portion that is fused to a polypeptide of the invention (or fragment thereof) may comprise a constant region, a hinge region, a CHI domain, a CH2 domain, and/or a CH3 domain or any combination of whole domains or portions thereof.
  • a polypeptide of the invention (or fragment thereof) may also be fused or conjugated to an antibody portion described herein to form multimers.
  • Fc portions fused to a polypeptide of the invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • Methods for fusing or conjugating a polypeptide of the invention (or fragment thereof) to an antibody portion are known (see, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; WO 96/04388).
  • the Fc part of a fusion protein is beneficial in therapy and diagnosis, and thus can result in, e.g., improved pharmacokinetic properties (see, e.g , EP A232, 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified can be favored
  • an antibody of the present invention or fragment thereof can be fused to marker sequences, such as a peptide to facihtate purification.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described U.S. Patent No. 4,676,980.
  • An antibody (or fragment thereof) of the invention may be utiUzed for immunophenotyping of ceU Unes and biological samples.
  • the translation product of an LP polynucleotide sequence (or fragment thereof) may be useful as a ceU specific marker, or more specificaUy, as a ceUular marker (which is differentiaUy expressed at various stages of differentiation and/or maturation of particular ceU types).
  • a particular protein can be measured by a variety of immunoassay methods see, e.g., Stites and Terr (eds.) (1991) Basic and CUnical Immunology (7th ed.); Price and Newman (eds.) (1991) Principles and Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays Plenum Press, NY.; Stites and Terr (eds.) Basic and CUnical Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay. supra; and Harlow and Lane Antibodies. A Laboratory Manual, supra.
  • the abiUty of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., Western blot analysis.
  • One of skiU in the art would be knowledgeable as to the parameters are modifiable to increase binding of an antibody to an antigen and to decrease background (e.g., by pre-clearing the ceU lysate with sepharose beads).
  • Further discussion of immunoprecipitation protocols can be found in, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John WUey & Sons, Inc., New York.
  • the present invention further encompasses antibody-based therapies that involve administering LP antibody to an animal, preferably a mammal, most preferably a primate
  • an antibody of the invention can be used to modulate, treat, inhibit, ameliorate, or prevent diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide (or fragment thereof) of the invention, including, e.g., without Umitation, any one or more of the diseases, disorders, syndromes or conditions described herein.
  • the treatment, ameUoration, and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, e.g., without Umitation, ameUorating symptoms associated with those diseases, disorders or conditions.
  • Antibodies of the invention may be provided in pharmaceuticaUy acceptable compositions as known in the art or as described herein.
  • DNAs which encode a LP protein or fragments thereof can be obtained by chemical synthesis, screening cDNA Ubraries, or by screening genomic Ubraries prepared from a wide variety of ceU Unes or tissue samples. Methods for doing so, or making expression vectors are either art known or are described herein.
  • DNAs can be expressed in a wide variety of host ceUs for the synthesis of a fuU-length protein or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure /function studies.
  • Each LP protein or its fragments can be expressed in host ceUs that are transformed or transfected with appropriate expression vectors.
  • transformed is meant a ceU into which (or into an ancestor of which) a DNA molecule has been introduced, by means of recombinant techniques, which encodes an LP polypeptide or fragment thereof.
  • Expression vectors are typicaUy self-repUcating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably Unked to appropriate genetic control elements that are recognized in a suitable host ceU.
  • the specific type of control elements necessary to effect expression depends on the host ceU used.
  • GeneraUy genetic control elements include a prokaryotic promoter system or a eukaryotic promoter expression control system
  • typicaUy include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • expression vectors also contain an origin of repUcation that aUows the vector to repUcate independendy of the host ceU.
  • An expression vector will preferably include, e.g., at least one selectable marker. Such markers include, e.g., without Umit, dihydrofolate reductase, G418, or neomycin resistance for eukaryotic ceU culture and tetracycUne, kanamycin or ampiciUin resistance genes for culturing in E. coli and other bacteria.
  • the vectors of this invention contain DNAs which encode an LP protein, or a fragment thereof, typicaUy encoding, e.g., a biologicaUy active polypeptide, or protein.
  • the DNA can be under the control of a viral promoter and can encode a selection marker.
  • This invention further contemplates use of expression vectors capable of expressing eukaryotic cDNA coding for a LP (or fragment) in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the protein is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.
  • expression vectors are designed for stable repUcation in their host ceUs or for ampUfication to gready increase the total number of copies of the desirable gene per ceU. It is not always necessary to require that an expression vector repUcate in a host ceU, e.g., it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a repUcation origin that is recogmzed by the host ceU. It is also possible to use vectors that cause integration of an LP protein gene or its fragments into the host DNA by recombination, or to integrate a promoter that controls expression of an endogenous gene. Vectors, as used herein, encompass plasmids, viruses, bacteriophage, integratable
  • Expression vectors are speciaUzed vectors that contain genetic control elements that effect expression of operably Unked genes. Plasmids are the most commonly used form of vector, but many other forms of vectors that perform an equivalent function are also suitable for use (see, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual Elsevier, N.Y.; and Rodnquez, et al. (eds.) (1988) Vectors: A Survey of Molecular Clomng Vectors and Their Uses Buttersworth, Boston, MA).
  • Suitable host ceUs include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e g., E. coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Picbia, and species of the genus Dictyostelum.
  • Higher eukaryotes include estabUshed tissue culture ceU Unes from animal ceUs, both of non-mammaUan origin, e.g., insect ceUs, and birds, and of mammaUan origin, e.g., human, primates, and rodents.
  • Prokaryotic host- vector systems include a variety of vectors for many different species. As used herein, E. coli and its vectors wiU be used genericaUy to include equivalent vectors used in other prokaryotes.
  • a representative vector for ampUfying DNA is pBR322 or its derivatives. Vectors that can be used to express these proteins or protein fragments include, but are not Umited to, such vectors as those containing the lac promoter (pUC- senes); trp promoter (pBR322-trp); Ipp promoter (the pIN-senes); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540).
  • ptrc99a pKK223-3, pKK233-3, pDR540, pRIT5 (available from Pharmacia Biotech, Inc).
  • Higher eukaryotic tissue culture ceUs are typicaUy the preferred host ceUs for expression of the functionaUy active LP protein.
  • suitable expression vectors include pCDNAl; pCD (Okayama, et al. (1985) Mol. CeU Biol. 5:1136-1142); pMClneo Poly-A, (Thomas, et al. (1987) CeU 51:503-512); and a baculovirus vector such as pAC 373 or pAC 610.
  • Additional eukaryotic vectors include, e.g., without Umit, pWLNEO, pSV2CAT, pOG44, pXTl and pSG (available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (available from Pharmacia Biotech, Inc.).
  • a polypeptide (or fragment thereof) of the present invention can also be recovered from natural sources, including, e.g., without Umit, bodily fluids, tissues, and ceUs, (whether directiy isolated or cultured), products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host (including, e.g., bacterial, yeast, higher plant, insect, and mammaUan ceUs).
  • natural sources including, e.g., without Umit, bodily fluids, tissues, and ceUs, (whether directiy isolated or cultured), products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host (including, e.g., bacterial, yeast, higher plant, insect, and mammaUan ceUs).
  • LP proteins need not be glycosylated to eUcit biological responses.
  • it wiU occasionaUy be desirable to express an LP protein or LP polypeptide in a system that provides a specific or defined glycosylation pattern.
  • the usual pattern wiU be that provided naturaUy by the expression system.
  • the pattern wiU be modifiable by exposing the polypeptide, e.g., in unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the LP protein gene may be co-transformed with one or more genes encoding mammaUan or other glycosylating enzymes. It is further understood that over glycosylation may be detrimental to LP protein biological activity, and that one of skiU may perform routine testing to optimize the degree of glycosylation which confers optimal biological activity.
  • an LP polypeptide may also include, e g., an initial modified methiomne residue (in some cases because of host-mediated processes).
  • an initial modified methiomne residue in some cases because of host-mediated processes.
  • TypicaUy the N-terminal methionine encoded by the translation initiation codon removed with high efficiency from any protein after translation in aU eukaryotic ceUs. While the N-terminal methionine on most proteins is also efficiendy removed in most prokaryotes, for some proteins depending on the nature of the amino acid to which the N-terminal methiomne is covalently Unked, the removal process is inefficient.
  • 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)).
  • heterologous coding sequence such as, e.g., an LP polynucleotide sequence, (or fragment thereof) under the transcriptional regulation of aU or part of the AOX1 regulatory sequence is expressed at exceptionaUy high levels in Pichia yeast grown in the presence of methanol.
  • the plasmid vector pPIC9K is used to express polynucleotide sequence encoding a polypeptide of the invention, (or fragment thereof) as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998.
  • This expression vector aUows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter Unked to the Pichia pasto ⁇ s alkaUne phosphatase (PHO) secretory signal peptide located upstream of a multiple cloning site.
  • yeast vectors could be used in place of pPIC9K, such as, e.g., pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, PHIL-D2, PHIL-SI, pPIC3.5K, and, PA08, as a skiUed in the artisan would appreciate, as long as the proposed expression construct provides appropriately located and operably Unked signals for transcription, translation, secretion (if desired), and the Uke, (including an in- frame stop codon as required).
  • heterologously expressed proteins or polypeptides can also be expressed in plant ceUs.
  • viral expression vectors e.g., cauUflower mosaic virus and tobacco mosaic virus
  • plasmid expression vectors e.g., Tl plasmid
  • ceUs are available from a wide range of sources (e.g., the American Tissue Type Culture CoUection, Rockland, MD; also, see for example, Ausubel, et al. (cur. ed. and Supplements; expression vehicles may be chosen from those provided e.g., in Pouwels, et al. (Cur. ed..) Cloning Vectors. A Laboratory Manual).
  • a LP protein, or a fragment thereof may be engineered to be phosphatidyl inositol (PI) Unked to a ceU membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phosphoUpase-C. This releases the antigen in a biologicaUy active form, and aUows purification by standard procedures of protein chemistry (see, e.g., Low (1989) Biochem. Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner, et al.
  • PI phosphatidyl inositol
  • LP proteins have been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) SoUd Phase Peptide Synthesis Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis Springer- Verlag, New York, NY; and Bodanszky (1 84) The Principles of Peptide Synthesis Springer-Nerlag, New York, NY.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the Uke.
  • An LP protein of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accompUshed by use of known protein purification techniques or by the use of the antibodies or binding partners herein described (e.g., in immunoabsorbant affinity chromatography).
  • An LP polypeptide, or fragment thereof, can be used to generate a fusion protein.
  • an LP polypeptide, or fragment thereof when fused to a second polypeptide, can be used as an antigenic tag or an immunogen.
  • Antibodies raised against an LP polypeptide can be used to indirecdy detect a second protein by binding thereto
  • an LP protein has amino acid sequence portion that targets a ceUular location (e.g., based on trafficking signals)
  • that portion of the polypeptide can be used by fusing it to another protein (or fragment) to target a protein.
  • domains that can be fused to an LP polypeptide (or fragment thereof) include, e.g., not only heterologous signal sequences, but also other heterologous functional regions.
  • a fusion does not necessarily need to be direct, but may occur, e.g., through Unker sequences.
  • fusion proteins may also be engineered to improve characteristics of an LP polypeptide.
  • a region of additional ammo acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stabiUty and persistence during purification from a host ceU or during subsequent handling and storage.
  • peptide moieties can be added to the polypeptide to faciUtate purification. Such regions may be removed before final preparation of the polypeptide. Additions of peptide moieties to faciUtate handUng are familiar and routine art techniques.
  • an LP polypeptide (including any fragment thereof, and specificaUy an epitope) can be combined with parts of the constant domain of an immunoglobuUn e.g., (IgA, IgE, IgG, IgM) portions thereof (CH 1, CH2, CH3), and any combination thereof including both entire domains and portions thereof), resulting in a chimeric polypeptide.
  • immunoglobuUn e.g., (IgA, IgE, IgG, IgM) portions thereof (CH 1, CH2, CH3), and any combination thereof including both entire domains and portions thereof.
  • Such fusion proteins can faciUtate purification and often are useful to increase the in vivo half-Ufe of the protein (Fountoulakis, et al. (1995) J. B ⁇ ochem.15 270:3958-3964).
  • IgG fusion proteins that have a disulfide-Unked dime ⁇ c structure due to the IgG portion disulfide bonds have also been found more efficient in binding and neutraUzing other molecules than monomenc polypeptides or fragments thereof alone (Fountoulakis, et al. (1995) J. Biochem. 270:3958-3964).
  • a fusion protein can comprise various portions of the constant region of an immunoglobuUn molecule together with a human protein (or part thereof) EP-A-O 464 533 (Canadian counterpart 2045869).
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus, can result in, e.g., improved pharmacokinetic properties (EP-A 0232 262.).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, may be desired.
  • the Fc portion may hinder therapy and/or diagnosis if the fusion protein is used as an immunogen for immunizations.
  • hIL-5 human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists (Bennett, et al. (1995) I. Molecular Recognition 8:52-58; and Johanson, et al. (1995) J. Biol Chem. 270:9459-9471).
  • new constructs may be made by combining similar functional domains from other proteins.
  • protein-binding or other segments may be "swapped" between different new fusion polypeptides or fragments (see, e.g., Cunningham, et al. (1989) Science 243:1330-1336; and O'Dowd, et al. H988) J. Biol Chem. 263:15985-15992).
  • an LP polypeptide (or fragment thereof) can be fused to a marker sequence, such as a peptide, to faciUtate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as, e.g., the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA, 91311), which provides for convenient purification of the fusion protein (Gentz, et al. (1989) Proc. Nad. Acad. S ⁇ . USA 86:821-824).
  • Another useful peptide-purification tag is the "HA" tag, which corresponds to an epitope derived from an influenza hemagglutinin protein (Wilson, et al. (1984) CeU 37:767).
  • Nucleic acid molecules contaimng LP polynucleotide sequences encoding an LP epitope can also be recombined with a gene of interest as an epitope tag (e.g., the "HA” or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the "HA” or flag tag
  • one system purifies non-denatured fusion proteins expressed in human ceU Unes (Janknecht, et al. (1991) Proc. Nad Acad. Sci. USA 88:8972-897).
  • a gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the sequence of interest is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix-binding domain for the fusion protein. Extracts from ceUs infected with the recombinant vaccima virus are loaded onto N ⁇ 2+ nit ⁇ loacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with lmidazole-contai ng buffers.
  • LP fusion constructions may be generated through the techniques of gene-shuffling, motif-shuffling, exon shuffling, and/or codon shuffling (coUectively referred to as "DNA shuffling").
  • DNA shuffling may be employed to modulate an activity of an LP polypeptide.
  • Such methods can be used to generate LP polypeptides (or fragments thereof) with altered activity, as weU as agonists and antagonists of an LP polypeptide (see, e.g., U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten, et al (1997) Cur.
  • “Derivatives" of LP protein antigens include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by Unkage of functionaUties to groups which are found in LP protein amino acid side chains or at the N- or C- termini, by any art known means. These derivatives can include, without Umitation, aUphatic esters or amides of the carboxyl terminus, or of residues contaimng carboxyl side chains, O-acyl derivatives of hydroxyl group-contaimng residues, and N-acyl derivatives of the amino terminal amino acid or amino-group contaimng residues, e.g., lysine or argimne.
  • Acyl groups are selected from the group of alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species. Covalent attachment to carrier proteins may be important when immunogenic moieties are haptens.
  • chemicaUy modified derivative of a polypeptide of the invention may provide additional advantages such as increased solubiUty, increased stabiUty increased circulating time, or decreased lmmunogenicity or antigenicity (see U.S. Patent no: 4,179,337).
  • a chemical moieties for derivatization may be selected from water soluble polymers such as, e.g., polyethyleneglycol, ethylene glycol, propylene glycol, copolymers, carboxymethylceUulose, dextran, polyvinyl alcohol, etc.
  • 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.
  • Amino acid residues having a free carboxyl group may include, e.g., aspartic acid residues, glutamic acid residues, and C- terminal amino acid residues. Sulfhydryl groups may also be used to attach to a polyethylene glycol molecule. For human, a preferred attachment is at an amino group, such as, e.g., an attachment at the N-terminus or a lysine group.
  • polyethylene glycol as an iUustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to a protein (polypeptide) molecule in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminaUy pegylated, e.g., polypeptide.
  • the method of obtaining an N-terminaUy pegylated preparation may be by purification of the N-terminaUy pegylated material from a population of pegylated protein molecules.
  • Selective protein chemical modification at the N-terminus may be accompUshed by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein.
  • substantiaUy selective derivatization of a protein or polypeptide (or fragment thereof) at the N-terminus with a carbonyl-group- containing-polymer is achieved.
  • This invention also encompasses the use of derivatives of an LP protein other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties.
  • GeneraUy these derivatives faU into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes (e.g., with ceU membranes).
  • Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of proteins or other binding proteins.
  • a LP protein antigen can be immobiUzed by covalent bonding to a soUd support such as cyanogen bromide-activated SEPHAROSE, by methods which are weU known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-Unking, for use in an assay or purification of anti-LP protein antibodies or its respective binding partner.
  • An LP protein can also be labeled for use in diagnostic assays with a detectable group (such as, e.g., radioiodinated by the chloramine T procedure; covalently bound to rare earth chelates; or conjugated to another fluorescent moiety). Purification of an LP protein may be effected by immobiUzed antibodies or a binding partner.
  • a polypeptide of the invention may be as a monomer or a multimer (e.g., a dimer, a trimer, a tetramer, or a higher multimer). Accordingly, the present invention encompasses monomers and multimers of a polypeptide of the invention, (or fragment thereof) including, e.g., their preparation, and compositions (preferably, therapeutic compositions) containing them.
  • the polypeptides and/or fragments of the invention are monomers, dimers, trimers, tetramers or higher multimers.
  • a multimer of the invention is at least a dimer, at least a trimer, or at least a tetramer.
  • Multimers encompassed by the invention may be homomers or heteromers.
  • the term "homomer,” refers to a multimer containing only a specific polypeptide (or fragment thereof) corresponding to an amino acid sequence of SEQ ID NO:Y or in a talbe herein (including fragments, variants, spUce va ⁇ ants, and fusion proteins, corresponding to these polypeptides as described herein).
  • a homomer may contain a polypeptide having identical or different amino acid sequences.
  • a homomer of the invention is a multimer containing only polypeptides (or fragments thereof) having identical amino acid sequences.
  • a homomer of the invention is a multimer contaimng polypeptides having different amino acid sequences.
  • a multimer of the invention is a homodimer (e.g., contaimng polypeptides having identical and/or different amino acid sequences) or a homotnmer (e.g., containing polypeptides having identical and/or different amino acid sequences).
  • the homomeric multimer of the invention is at least a homodimer, at least a homotnmer, or at least a homotetramer.
  • heteromeric refers to a multimer contaimng one or more heterologous polypeptides.
  • a multimer of the invention is a heterodimer, a heterotnmer, or a heterotetramer.
  • the heteromeric multimer of the invention is at least a heterodimer, at least a heterotnmer, or at least a heterotetramer.
  • Multimers of the invention may be the result of hydrophobic, hydrophlUc, ionic and/or covalent associations and/or may be indirectly Unked, by e g., Uposome formation.
  • a multimer of the invention such as, e.g., homodimers or homot ⁇ mers, are formed when polypeptides of the invention (or fragments thereof) contact one another in solution.
  • a heteromultimer of the invention such as, e.g., a heterotnmer or a heterotetramer, is formed when, e.g., a polypeptide of the invention contacts an antibody (generated against a polypeptide, or fragment thereof of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention)) in solution.
  • a multimer of the invention is formed by covalent association with and/or between a polypeptide and a binding partner such as mentioned herein (or fragment thereof).
  • Such covalent associations may involve one or more amino acid residues contained in a polypeptide sequence (e.g., as recited in a sequence Usting herein, or contained in a polypeptide encoded by a deposited clone specified herein).
  • a covalent association is a cross-Unk, e.g., between cysteine residues.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations may involve one or more amino acid residues contained in a heterologous polypeptide sequence such as, e.g., a fusion protein of the invention.
  • covalent associations form with a heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent No. 5,478,925).
  • a covalent association is between a heterologous sequence contained in an Fc fusion protein of the invention (as described herein).
  • a covalent association of a fusion protein of the invention is with a heterologous polypeptide sequence such as, e.g., oseteoprotegerin (see, e.g., WO 98149305, incorporated by reference for these teachings).
  • polypeptides of the invention are joined through peptide Unkers.
  • peptide Unkers examples include, e.g., peptide Unkers described in U.S. Pat. No. 5,073,627 (incorporated by reference for these teachings).
  • a protein comprising multiple polypeptides of the invention that are separated by peptide Unkers may be produced using conventional recombinant DNA technology.
  • Recombinant fusion proteins comprising a polypeptide of the invention (or fragment thereof) fused to a polypeptide sequence that dimerizes or trimerizes in solution can be expressed in a suitable host ceU.
  • the resulting soluble multimeric fusion protein can be recovered from a supernatant using any art known technique or method described herein.
  • Trimeric polypeptides of the invention may offer an advantage of enhanced biological activity (as defined herein).
  • Preferred leucine zipper moieties and isoleucine moieties are those that preferentiaUy form trimers.
  • An example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe, et al.
  • polypeptides or proteins of the invention are associated by interactions with a Flag polypeptide sequence (e.g., contained in a fusion protein of the invention having a Flag sequence).
  • a protein or a polypeptide of the invention is associated by an interaction with a heterologous polypeptide sequence (contained in a Flag fusion protein of the invention) and an anti-Flag antibody.
  • a multimer of the invention may be generated using chemical art known techniques.
  • polypeptides (or fragments thereof) desired to be contained in a multimer of the invention may be chemicaUy cross-Unked using a Unker molecule e.g., Unker molecules and Unker molecule length optimization techniques are known in the art; see, e.g., US Patent No. 5,478,925, which is incorporated by reference for such teachings.
  • AdditionaUy a multimer of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-Unks between the cysteine residues (see, e.g., US Patent No. 5,478,925, inco ⁇ orated by reference for these teachings).
  • polypeptide of the invention modified by the addition of cysteine or biotin to the C or N-terminus of a polypeptide can be generated by art known methods (see, e.g., US Patent No. 5,478,925, incorporated by reference for these teachings).
  • a multimer of the invention can be generated by art known methods (see, e.g., US Patent No. 5,478,925, incorporated by reference for these teachings). Alternatively, a multimer of the invention can be generated using other commonly known genetic engineering techniques. In one embodiment, a polypeptide contained in a multimer of the invention is produced recombinandy with fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent No. 5,478,925, incorporated by reference for these teachings).
  • a polynucleotide encoding a homodimer of the invention can be generated by Ugating a polynucleotide sequence encoding a polypeptide (or fragment thereof) of the invention to another sequence encoding a Unker polypeptide and then subsequendy, further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent No. 5,478,925, incorporated by reference for these teachings).
  • recombinant techniques described herein or otherwise known in the art can be appUed to generate a recombinant polypeptide of the invention (or fragment thereof) that contains a transmembrane domain (or hyrophobic or signal peptide) and that can be incorporated by membrane reconstitution techniques into a Uposome (see, e.g., US Patent No. 5,478,925, incorporated by reference for these teachings).
  • the present invention provides reagents that will find use in diagnostic and/or therapeutic appUcations as described herein, e.g., in the description of kits for diagnosis.
  • 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.
  • the invention encompasses a kit, e.g., for analyzing a sample for the presence of a polynucleotide associated with a proUferative disease, syndrome, disorder, or condition.
  • the kit includes, e.g., at least an LP polynucleotide sequence (or fragment thereof) probe containing a polynucleotide sequence that hybridizes with an LP polynucleotide sequencefor fragment thereof) and directions, e.g., such as for disposal.
  • 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.
  • a probe may be useful as a primer for ampUfication using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the present invention is useful as a prognostic indicator, for a subject exhibiting an enhanced or diminished expression of an LP polynucleotide sequence (or fragment thereof) by comparison to a subject expressing the polynucleotide of the present invention (or fragment thereof) at a level nearer a standard level.
  • measuring level of a composition of the present invention is intended to mean herein measuring or estimating (either quaUtatively and/or quantitatively) a level of, e.g., a polypeptide (or fragment thereof), or a polynucleotide (or fragment thereof) including, e.g., mRNA, DNA, or cDNA, in a first sample (e.g., preferably a biological sample) either directiy (e.g., by determining or estimating an absolute protein or mRNA level) or relatively (e.g., by comparing to a polypeptide or mRNA level in a second sample).
  • a first sample e.g., preferably a biological sample
  • directiy e.g., by determining or estimating an absolute protein or mRNA level
  • relatively e.g., by comparing to a polypeptide or mRNA level in a second sample.
  • 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 biological sample can include, e.g., without Umitation, body fluids (e.g., such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) that contain a polypeptide (or fragment thereof), mRNA (or fragment thereof), a protein (or fragment thereof), or polynucleotide (or fragment thereof) of the present invention, by product, or, waste product; and/or other tissue source found to express a polypeptide (or fragment thereof), mRNA (or fragment thereof), or nucleic acid (or fragment thereof), by product, or, waste product; of the present invention.
  • body fluids e.g., such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid
  • tissue biopsies e.g., tissue biopsies, body fluids, ceUs, or waste products from mammals are known in the art.
  • a tissue biopsy is a preferred source.
  • the present invention further encompasses an LP polynucleotide sequence (or fragment thereof) that is chemicaUy synthesized, or reproduced as a peptide nucleic acid (PNA) using art known methods.
  • PNA peptide nucleic acid
  • the use of a PNA is preferred if a polynucleotide (or a fragment thereof) is incorporated, e.g., onto a soUd support, or genechip.
  • a peptide nucleic acid is a polyamide type of polynucleotide analog in which, generally, e.g., the monomeric units for adenine, guanine, thymine and cytosine are available commerciaUy (see, e.g., Perceptive Biosystems). Certain components of a polynucleotide, such as DNA, Uke phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in a PNA. GeneraUy, PNAs bind specificaUy and tighdy to complementary DNA strands and are not degraded by nucleases (Nielsen, et al. (1993)
  • a PNA binds more strongly to DNA than DNA binds to itself, probably, as there is no electrostatic repulsion between PNA/DNA; furthermore, the PNA polyamide backbone is more flexible than DNA. Because of this, PNA/DNA duplexes can bind under a wider range of stringency conditions than DNA/DNA duplexes thus, making it easier to perform multiplex hybridizations. Moreover, smaUer probes can be used with PNA than with DNA due to the strong binding.
  • 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).
  • Another useful antibody-based method for detecting protein or polypeptide expression includes, e.g., an immunoassay Uke an enzyme Unked immunosorbent assay or a radioimmunoassay (RIA).
  • assaying e.g., the level of a secreted protein in a sample
  • a protein can also be detected by in vivo imaging.
  • the invention provides a means for detecting, marking, locating or diagnosing a disease, syndrome, syndrome, disorder, and/or condition comprising assaying the expression of a polynucleotide (or fragment thereof), or a polypeptide (or fragment thereof), of the present invention that is in a sample, e.g., ceUs or body fluid of an individual by comparing one level of expression with another level of expression, e g , a standard level of expression to indicate, e.g., a disease, syndrome, disorder, and/or condition, (or predilection to the same), or to make a prognosis or prediction.
  • an LP polypeptide (or fragment thereof) can be used to treat, prevent, modulate, ameUorate, and/or diagnose a disease, syndrome, condition, and/or a disorder.
  • a subject can be administered a polypeptide (or fragment thereof) of the invention to replace absent or decreased levels of a polynucleotide or polypeptide (e.g., insuhn); to supplement absent or decreased levels of a different polynucleotide or polypeptide (e.g., hemoglobin S for hemoglobin B; SOD to catalyze DNA repair proteins); to inhibit the activity of a polynucleotide or polypeptide (e.g., an oncogene or tumor suppressor); to activate a polynucleotide or polypeptide (e g , by binding to a receptor), to reduce activity of a membrane bound receptor by competing with the receptor for free Ugand (e.g., soluble TNF receptors can be used to reduce inflammation), or to bring about
  • an antibody directed to a polypeptide (or fragment thereof) of the present invention can also be used to treat, prevent, modulate, ameUorate, and/or diagnose a condition, syndrome, state, disease or disorder.
  • administration of an antibody directed to an LP polypeptide (or fragment thereof) can bind and reduce the level of the targeted polypeptide.
  • administration of an antibody can activate an LP polypeptide (or fragment thereof), such as by binding to the polypeptide that is bound to a membrane (e.g., a receptor).
  • Antibodies of the invention can be used to assay polypeptide levels in a sample, e.g., using classical immunohistological methods known to those of skiU in the art (see e.g., Jalkanen, et al., J. CeU. Biol. 101:976-985 (1985); Jalkanen, et al., J. CeU . Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods typicaUy useful for detecting polypeptide expression include, e.g., immunoassays, such as the enzyme Unked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA Unked immunosorbent assay
  • RIA radioimmunoassay
  • Sequences encoding an LP polypeptide are used for the diagnosis of disorders associated with LP (such as, e.g., LP misexpression, LP overexpression, LP underexpression, etc.).
  • disorders associated with LP such as, e.g., LP misexpression, LP overexpression, LP underexpression, etc.
  • disorders associated with LP include, without Umit, a cell proUferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, Flamartoma, sarcoma, teratocarcinom
  • Sequences encoding an LP polypeptide (or fragment thereof) are used in Southern or northern analysis; dot blot or other membrane-based technologies; PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microarrays utilizing fluids or tissues from a subject; to detect an altered LP polypeptide (or fragment thereof) expression.
  • PCR technologies in dipstick, pin, and multiformat ELISA-Uke assays
  • microarrays utilizing fluids or tissues from a subject; to detect an altered LP polypeptide (or fragment thereof) expression.
  • This invention also provides reagents with significant therapeutic value.
  • An LP protein or polypeptide (naturaUy occurring or recombinant), fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to an LP, are useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proUferation, e.g., cancerous conditions, or degenerative conditions. Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using a composition(s) provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaUng by an LP protein is a target for an agonist or antagonist of the protein.
  • 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.
  • Another therapeutic approach included within the invention involves direct admimstration of reagents, formulations, or compositions by any conventional admimstration techmques (such as, e.g., without Umit, local injection, inhalation, or systemic admimstration) to a subject.
  • the reagents, formulations, or compositions included within the bounds and metes of the invention may also be targeted to a ceU by any of the methods described herein (e.g., polynucleotide deUvery techmques).
  • the actual dosage of reagent, formulation, or composition that modulates a disease, disorder, condition, syndrome, etc. depends on many factors, including the size and health of an organism, however one of one of ordinary skiU in the art can use the foUowing teachings describing methods and techmques for determimng dinical dosages (see, e g., Spilker (1984) Guide to CUnical Studies and Developing Protocols. Raven Press Books, Ltd., New York, pp. 7-13, 54-60, Spilker (1991) Guide to CU cal Trials. Raven Press, Ltd., New York, pp. 93-101, Craig and Stitzel (eds. 1986) Modern Pharmacology.
  • GeneraUy in the range of about between 0.5 fg/ml and 500 ⁇ g/ml inclusive final concentration are admimstered per day to a human adult in any pharmaceuticaUy acceptable carrier. Furthermore, ammal experiments provide reUable guidance for the determination of effective does for human therapy. Interspecies scaUng of effective doses can be performed foUowing art known principles (e.g., see, Mordenti and ChappeU (1989) "The Use of Interspecies ScaUng in Toxicokinetics," in Toxicokinetics and New Drug Development; Yacobi, et al. (eds.) Pergamon Press, NY).
  • Effective doses can also be extrapolated using dose-response curves derived from in vitro or animal-model test systems.
  • a dosage is typicaUy 0.1 mg/kg to 100 mg/kg of a recipients body weight.
  • a dosage is between 0.1 mg/kg and 20 mg/kg of a recipients body weight, more preferably 1 mg/kg to 10 mg/kg of a recipients body weight.
  • GeneraUy homo-specific antibodies have a longer half-Ufe than hetero-specif ⁇ c antibodies, (e.g., human antibodies last longer within a human host than antibodies from another species, e.g., such as a mouse, probably, due to the immune response of the host to the foreign composition).
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers fiUed with one or more of the ingredients of the compositions of the invention and instructions such as, e.g., for disposal (typicaUy, in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products).
  • 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.
  • LP protein, fragments thereof, and antibodies to it or its fragments, antagomsts, and agonists may be admimstered direcdy to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their admimstration.
  • Therapeutic formulations may be admimstered in any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typicaUy comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof.
  • 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.
  • the present invention also provides a pharmaceutical composition.
  • a pharmaceutical composition comprises, e.g., a therapeuticaUy effective amount of a composition of the invention in a pharmaceuticaUy acceptable carrier.
  • pharmaceutically acceptable carrier means a carrier approved by a federal regulatory agency of the Umted States of America, or a regulatory/ administrative agency of a state government of the Umted States or a carrier that is Usted in the U.S. Pharmacopeia or other pharmacopeia; which is generaUy recognized by those in the art for use in an ammal, e.g., a mammal, and, more particularly, in a primate, e.g., a human primate.
  • 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.
  • the etiology of an immune disease, disorder, syndrome, or condition may be genetic and/or somatic, (e.g., such as some forms of cancer or some autoimmune conditions acquired by e.g., chemotherapy or toxins or an infectious agent, e.g., a virus or pnon-Uke entity.
  • an LP can be used to mark or detect a particular immune system disease, syndrome, disorder, state, or condition.
  • 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.
  • an LP can be used to modulate hemostatic or thrombolytic activity.
  • 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 may also be useful in ameUorating, treating, preventing, modulating and/or diagnosing an autoimmune disease, disorder, syndrome, and/or condition such as results, e.g., from the inappropriate recognition by a ceU of the immune system of the self as a foreign material.
  • Such an inappropriate recognition results in an immune response leading to detrimental effect destruction on the host, e.g., on a host ceU, tissue, protein, or moiety, e.g., a carbohydrate side chain.
  • administering may be effective in detecting, diagnosing, ameUorating, or preventing such an autoimmune disease, disorder, syndrome, and/or condition.
  • autoimmune conditions examples include, e.g., without Umit Addison's Disease syndrome hemolytic anemia, anti-phosphoUpid syndrome, rheumatoid arthritis, dermatitis, aUergic encephalomyeUtis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease syndrome, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, BuUous Pemphigoid, Pemphigus, Polyendocnnopathies, Purpura, Reiter's Disease syndrome, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, GuiUain-BarreSyndrome, insulin dependent diabetes meUitis, and autoimmune inflammatory eye disease.
  • Umit Addison's Disease syndrome hemolytic anemia, anti-phosphoUpid syndrome, rheumatoid arthritis, dermatitis, aUergic
  • allergic reactions and conditions such as asthma (e.g., aUergic asthma) or other respiratory problems, may also be ameUorated, treated, modulated or prevented, and/or diagnosed by an LP polynucleotide or polypeptide (or fragment thereof), or an agomst or antagonist thereto.
  • inventive compositions can be used to effect, e.g., anaphylaxis, hypersensitivity to an antigenic molecule, or blood group lncompatibiUty.
  • An LP may also be used to modulate, ameUorate, treat, prevent, and/or diagnose organ re j ection or graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • organ rejection occurs by a host's, immune-ceU destruction of a transplanted tissue or ceU.
  • a similarly destructive immune response is involved in GVHD, however, in this case, transplanted foreign immune ceUs destroy host tissues and/or ceUs.
  • Admimstration of a composition of the invention which ameUorates or modulates such a deleterious immune response (e.g., a deleterious proUferation, differentiation, or chemotaxis of a T ceU), can be effective in modulating, ameUorating, diagnosing, and/or preventing organ re j ection or GVHD.
  • an LP may also be used to detect, treat, modulate, ameUorate, prevent, and/or diagnose an inflammation, e.g., by inhibiting the proUferation and/or differentiation of a cell involved in an inflammatory response, or an inflammatory condition (either chronic or acute), including, e.g., without Umitation, chrome prostatitis, granulomatous prostatitis and malacoplakia, an inflammation associated with an infection (such as, e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), lschemia-reperfusion injury, endotoxin lethaUty, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease syndrome, Crohn's disease syndrome, or a condition resulting from an over production of a cytok ⁇ ne(s) (e.g., TNF or IL-1 .)
  • 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.
  • the desired result may be effected either by, e.g., diminishing or blocking an existing immune response, or by preventing the initiation of a new immune response.
  • hyperproUferative states, diseases, disorders, syndromes, and/or conditions include, e.g., without Umitation, a neoplasm of the colon, abdomen, bone, breast, digestive system, Uver, pancreas, peritoneum, endocrine system (e.g., an adrenal gland, a parathyroid gland, the pituitary, the testicles, the ovary, the thymus, or the thyroid), eye, head, neck, nervous system (central or peripheral), the lymphatic system, pelvis, skin, spleen, thorax, and urogemtal system.
  • endocrine system e.g., an adrenal gland, a parathyroid gland, the pituitary, the testicles, the ovary, the thymus, or the thyroid
  • eye head, neck, nervous system (central or peripheral)
  • the lymphatic system pelvis, skin, spleen, thorax, and urogemtal system.
  • 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).
  • the viral vector is defective and only transforms or transfects a proUferating ceU but does not transform or transfects a non-proUferating ceU.
  • an LP polynucleotide sequence is inserted into a proUferating ceU either alone, (or in combination with, or fused to, another polynucleotide sequence, which can subsequendy be modulated via an external stimulus (e.g., a magnetic signal, a specific smaU molecule, a chemical moiety or a drug administration, etc.) that acts on an upstream promoter to induce expression o the LP polypeptide (or fragment thereof).
  • an external stimulus e.g., a magnetic signal, a specific smaU molecule, a chemical moiety or a drug administration, etc.
  • a desired effect of the present invention may be accompUshed based on using an external stimulus.
  • 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.
  • a retroviral, or adenoviral deUvery system (as known in the art or described herein) is used to specificaUy deUver a recombinant construct or to transfect a ceU that is abnormaUy proUferating.
  • An LP polynucleotide sequence may be deUvered directiy to the site of a ceU proUferation, e.g., in an internal organ, body cavity, and the Uke by use of, e.g., an imaging device used to guide the recombinant construct.
  • administration to an appropriate location may be carried out at a time of surgical intervention.
  • 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.
  • biologically inhibiting is meant a partial or total inhibition of mitotic activity and/or a decrease in the rate of mitotic activity or metastatic activity of a targeted cell.
  • a biologicaUy inhibitory dose can be determined by assessing the effects of an LP on abnormaUy proUferating ceU division in a ceU or tissue culture, tumor growth in an animal or any other art known method.
  • an LP can be useful to inhibit angiogenesis associated with abnormaUy proUferative ceUs or tissues, either alone, or as a protein fusion, or in combination with another LP polynucleotide or polypeptide (or fragment thereof), or an agonist or antagonist, thereto.
  • 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 including a protein fusion, may be useful in inhibiting an abnormaUy proUferative ceU or tissue, via an induction of apoptosis.
  • An LP may act either directly, or indirecdy to induce apoptosis in a proUferative ceU or tissue, e.g., by activating the death- domain FA receptor, such as, e.g., tumor necrosis factor (TNF) receptor-1, CD95 (F&APO- I), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis- inducing Ugand (TRAIL) receptor-1 and -2 (see, e.g., Schulze-Osthoff, et al., Eur J Biochem 254 (3): 439-59 (1998), which is hereby inco ⁇ orated by reference for teachings on apoptotic ceU death).
  • TNF tumor necrosis factor
  • TRAMP T
  • 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 conditions include, e.g., heart disease syndrome, such as, e.g., arrhythmias, carcinoid heart disease syndrome, high cardiac ou ⁇ ut, low cardiac ou ⁇ ut, cardiac tamponade, endocarditis (including bacterial endocarditis), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous pericarditis), pneumopericardium, post-pericardiotomy syndrome, pulmonary heart disease syndrome, rheumatic heart disease syndrome, ventricular dysfunction, hyperemia, cardiovascular pregnancy compUcations, Scimitar Syndrome, cardiovascular syphil
  • 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.
  • Additional cardiovascular disorders include, e.g., heart valve disease such as, e.g., aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
  • heart valve disease such as, e.g., aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenos
  • 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 conditions include, e.g., myocardial ischemias such as, e.g., coronary disease syndrome, such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial stunning.
  • myocardial ischemias such as, e.g., coronary disease syndrome, such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial stunning.
  • coronary disease syndrome such as e.g., angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasispasm, myocardial infarction, and myocardial stunning.
  • 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.
  • Ischemic conditions include, e.g., cerebral ischemia, ischemic coUtis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion in j uries, and peripheral Umb ischemia
  • VascuUtic conditions include, e.g., aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obUterans, hypersensitivity vascuUtis, Schoenlein-Henoch purpura, aUergic cutaneous vascuUtis, and Wegener's granulomatosis.
  • An LP can be beneficial in ameUorating critical Umb ischemia and coronary disease.
  • An LP may be administered using any art known method, described herein An LP may admimstered as part of a therapeutic composition or formulation, as described in detail herein. Methods of deUvenng an LP are also described in detail herein. Anti-Hemopoietic Activity
  • 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.
  • a number of serious diseases are dominated by abnormal neovascularization (including, e.g., soUd tumor growth and metastases, arthritis, some types of eye conditions, and psoriasis; see, e.g., reviews by Moses, et al., Biotech. 9630-634 (1991); Folkman, et al, N. Engl. J. Med , 333: 1757-1763 (1995); Auerbach, et al, J. Microvasc. Res. 29:401-4 11 (1985); Folkman, "Advances in Cancer Research", eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol.
  • 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.
  • MaUgnant and metastatic conditions that can be effected in a desired fashion using an LP include, e.g, without Umitation, a maUgnancy, soUd tumor, and a cancer as described herein or as otherwise known in the art (for a review of such disorders, syndromes, etc. see, e.g, Fishman, et al. Medicine, 2d Ed, J. B. Lippincott Co, Philadelphia (1985)).
  • the present invention provides a method of ameUorating, modulating, treating, preventing, and/or diagnosing an angiogenesis-related disease and/or disorder, comprising administering to a sub j ect in need thereof a beneficiaUy effective amount of an LP.
  • cancers that may be so affected using a composition of the invention includes, e g, without Umit a soUd tumor, including e.g, prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, Uver, parotid, biUary tract, colon, rectum, cervix, uterus, endometnum, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; gUoblastoma; Kaposi's sarcoma; leiomyo sarcoma; non-smaU ceU lung cancer; colorectal cancer; advanced maUgnancies; and blood born tumors such as e.g, leukemia.
  • soUd tumor including e.g, prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, Uver, parotid, biUary tract, colon,
  • an LP may be deUvered topicaUy, to treat or prevent cancers such as, e.g, skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
  • an LP may be utilized to treat superficial forms of bladder cancer by, e.g, lntravesical admimstration into the tumor, or near the tumor site; via injection or a catheter.
  • the appropriate mode of admimstration wiU vary according to the cancer to be treated. Other modes of deUvery are discussed herein.
  • An LP may also be useful in modulating, ameUorating, treating, preventing, and/or diagnosing another disease, disorder, syndrome, and/or condition, besides a ceU proUferative condition (e.g, a cancer) that is assisted by abnormal angiogenic activity.
  • a ceU proUferative condition e.g, a cancer
  • Such close group conditions include, e.g, without Umitation, be gn tumors, e.g, such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogemc granulomas; atherosclerotic plaques; ocular angiogenic diseases, e.g, diabetic retinopathy, retinopathy of prematurity, macular degeneration, cornea graft re j ection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye, rheumatoid arthritis; psoriasis; delayed wound heaUng; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular
  • methods for modulating, ameUorating, treating, preventing, and/or diagnosing hypertrophic scars and keloids comprising administering an LP to a site of hypertrophic scar or keloid formation.
  • the method involves a direct injection into a hypertrophic scar or keloid, to provide a beneficial effect, e.g, by preventing progression of such a lesion.
  • 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.
  • corneal neovascularization e.g, corneal infections (e.g, trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g, graft rejection and Stevens- Johnson's syndrome), alkaU burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a compUcation of using contact lenses.
  • corneal infections e.g, trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis
  • immunological processes e.g, graft rejection and Stevens- Johnson's syndrome
  • alkaU burns trauma, inflammation (of any cause)
  • toxic and nutritional deficiency states e.g, as a compUcation of using contact lenses.
  • an LP composition may be prepared for topical administration in saUne (combined with any of the preservatives and anti-microbial agents commonly used in ocular preparations), and administered in drop form to the eye.
  • the solution or suspension may be prepared in its pure form and administered several times daily.
  • anti-angiogenic compositions prepared as described herein, may also be administered directly to the cornea.
  • an anti-angiogenic composition is prepared with a muco-adhesive polymer, which binds to the cornea.
  • an anti-angiogenic factor or anti-angiogenic LP composition may be utiUzed as an adjunct to conventional steroid therapy.
  • 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).
  • This method may also be utiUzed shortly after a corneal insult to prophylacticaUy prevent corneal neovascularization.
  • the composition could be injected into the periUmbic cornea interspersed between the corneal lesion and its undesired potential Umbic blood supply.
  • Such methods may also be utiUzed in a similar fashion to prevent capiUary invasion of transplanted corneas. In a sustained-release form, injections might only be required 2-3 times per year.
  • a steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
  • 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.
  • proUferative diabetic retinopathy comprising administering to a patient a therapeuticaUy effective amount of an LP to the eyes, such that the formation of blood vessels is inhibited.
  • proUferative diabetic retinopathy may be treated by injection into the aqueous or the vitreous humor, to increase the local concentration of a composition of the invention in the retina. Preferably, this treatment should be initiated before the acquisition of severe disease requiring photocoagulation.
  • methods are provided for treating or preventing retrolental fibroplasia, comprising administering to a patient a beneficiaUy effective amount of an LP to the eye, such that the formation of blood vessels is inhibited.
  • 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.
  • diseases, disorders, states, syndromes, and/or conditions that can be modulated, ameUorated, treated, prevented, and/or diagnosed with an LP include, e.g, without Umitation, soUd tumors, blood born tumors such as leukemias, rumor metastasis, Kaposi's sarcoma, benign tumors (e.g., hemangiomas), acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, e.g, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound heaUng, endometriosis, vasculogenesis,
  • 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.
  • surgical meshes that have been coated with an anti-angiogenic composition of the invention may be utiUzed in a procedure in which a surgical mesh might be utiUzed.
  • a surgical mesh laden with an anti-angiogenic composition may be utiUzed during cancer resection surgery (e.g, abdominal surgery subsequent to colon resection) to provide support to the structure, and to release an amount of the anti-angiogenic factor.
  • methods are provided for treating tumor excision sites, comprising administering an LP to the resection margins of a tumor after excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited.
  • 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
  • ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
  • Uver injury such as, e.g, hepatitis related Uver injury, ischemia reperfusion injury, cholestosis (bile duct in j ury), and Uver cancer
  • toxin-induced Uver disease such as, e.g, that caused by alcohol
  • septic shock cachexia
  • cachexia cachexia
  • anorexia anorexia
  • 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 could be used to promote dermal reestabUshment after dermal loss.
  • An LP could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epitheUaUzation from the wound bed.
  • the foUowing is a non-exhaustive Ust of grafts that an LP could be used to increase adherence to: a wound bed, autografts, artificial skin, allografts, autodermic grafts, autoepidermic grafts, avascular grafts, Blair-Brown grafts, bone grafts, brephoplastic grafts, cutis grafts, delayed grafts, dermic grafts, epidermic grafts, fascia grafts, fuU thickness grafts, heterologous grafts, xenografts, homologous grafts, hype ⁇ lastic grafts, lameUar grafts, mesh grafts, mucosal grafts, OUier-Thiersch grafts, omenpal grafts, patch grafts, pedicle grafts, penetrating grafts, spUt skin graf
  • 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.
  • An LP may: promote proUferation of endotheUal ceUs, keratinocytes, and basal keratinocytes; it could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections, it may have a cytoprotective effect on the smaU intestine mucosa; it may also stimulate heaUng of mucositis (mouth ulcers) that result from chemotherapy and viral infections, it could further be used in fuU regeneration of skin in fuU and partial thickness skin defects, including burns, (i.e., re-population of hair foUicles, sweat glands; and sebaceous glands), treatment of other skin defects such as psoriasis, it also could be used to treat epidermolysis buUosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful bUsters by accelerating re-epitheUaUzation of these lesions; it
  • 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.
  • Nervous system lesions that may be modulated, ameUorated, treated, prevented, and/or diagnosed in a subject using an LP composition of the invention, include, e.g, without Umitation, the foUowing lesions of either the central (including spinal cord and brain) or peripheral nervous system: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal in j ury or death, including e.g, cerebral infarction (or ischemia), or spinal cord infarction (or ischemia); (2) traumatic lesions, including, e.g, lesions caused by physical injury or associated with surgery (e.g, lesions that sever a portion of the nervous system), or compression in j uries; (3) maUgnant lesions, in which a portion of the nervous system is comprised by maUgnant tissue, which is either a nervous system associated maUgnancy or a maUgnancy derived from non-nervous-system tissue; (4) infectious lesions, in which
  • an LP can be used to protect a neuronal ceU from the damaging effects of cerebral hypoxia; cerebral ischemia, cerebral infarction; stroke; or a neural ceU injury associated with a heart attack.
  • An LP which is useful for producing a desired effect in a nervous system condition, may be selected by testing for biological activity in promoting survival and/or differentiation of neural ceU.
  • an LP that eUcits any of the foUowing effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased or decreased sprouting of a neural in culture or in vivo; (3) increased or decreased production of a neuron-associated molecule e.g, such as a neurotransmitter in culture or in vivo, e.g, choUne acetyltransferase or acetylchoUnesterase with respect to a motor neuron; or (4) decreasing a symptom of neuronal dysfunction in vivo or in a model system, e.g, such as a mouse model for Parkinsons Syndrome.
  • a model system e.g, such as a mouse model for Parkinsons Syndrome.
  • 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.
  • a neuron-associated molecule e.g, by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc, depending on the molecule to be measured
  • motor neuron dysfunction by, e.g, assessing the physical manifestation of motor neuron disorder, e.g, weakness, motor neuron conduction velocity, or functional disabiUty in a model system.
  • 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
  • Clostndium Bacteroidaceae, Blastom costs, Bordetella, Borrelia (e.g, Borrelia burgdorferi), Brucellosis, Candtdiasis, Campy lobacter,Cocc ⁇ d ⁇ o ⁇ domycos ⁇ s, Cryptococcosis, Dermatocycoses, E. colt (e.g, EnterotoxigenicE. coli and Enterohemorrhagtc E. coli), Enterobactertaceae (Klebstella, Salmonella (e.g.
  • 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.
  • parasitic agents causing diseases, disorders, conditions, syndromes, or symptoms that can be modulated, ameUorated, treated, prevented, and/or diagnosed by an LP include, e.g, without Umitation, a parasitic agent from any of the foUowing groupings: Amebiasis, Babesiosis, Coccidiosis, Cryptospo ⁇ diosis, Dienta oebiasis, Dou ⁇ ne, Ectoparasitic, Giardiasis, Helminthiasis, eishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, Trichomona, Sporo ⁇ oans
  • An LP composition of the invention can be used to modulate, ameUorate, treat, prevent, and/or diagnose any of these diseases, disorders, conditions, syndromes, or symptoms.
  • an LP can be used to modulate, ameliorate, treat, prevent, and/or diagnose malaria.
  • treatment or prevention using an LP is accompUshed either by administering an effective amount of an LP composition to a subject, or by removing ceUs from a subject, deUvering an LP then returning the resulting engineered ceU to the patient (ex vivo therapy).
  • an LP sequence can be used as an antigen in a vaccine to raise an immune response against an infectious disease.
  • 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.
  • kits and methods for detecting the presence of LP protein or a binding partner TypicaUy the kit wiU have a compartment containing either a defined LP protein peptide or gene segment or a reagent, which recognizes one or the other, e.g, binding partner fragments or antibodies.
  • 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.
  • nucleic acid molecule comprising polynucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of a correspondingly encoded First Amino Acid of a Signal Peptide and ending with the nucleotide at about the position of the 3' nucleotide of a Clone Sequence as defined for SEQ ID NO:X in a Table herein.
  • 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 in at least one polynucleotide sequence fragment of SEQ ID NO:X.
  • 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 product is purified by standard methods of protein purification, in certain cases, e.g, coupled with immunoaffinity methods. Immunoaffinity methods are used either as a purification step, as described above, or as a detection assay to determine the separation properties of the protein.
  • 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).
  • transfections should be performed by spUtting the foUowing tasks between two individuals to reduce the time, and to insure that the ceUs do not spend too much time in PBS.
  • person A aspirates off the media from four 24-weU plates of ceUs, and then person B rinses each weU with 0.5-1 ml of PBS.
  • Person A then aspirates off the PBS rinse, and person B (using a 12-channel pipetter with tips on every other channel) adds 200 ⁇ l of DNA/Lipofectamine/Optimem I complex first to the odd weUs, then to the even weUs (of each row on the 24-weU plates). Incubate at 37 °C for 6 hours.
  • 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.
EP02719036A 2001-03-16 2002-03-01 Proteines de mammiferes lp et reactifs associes Withdrawn EP1434783A4 (fr)

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US28854801P 2001-05-03 2001-05-03
US288548P 2001-05-03
US29035101P 2001-05-11 2001-05-11
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GB2381790A (en) * 2001-09-26 2003-05-14 Glaxo Group Ltd LDL-receptor polypeptides
JP4618736B2 (ja) * 2004-07-22 2011-01-26 エーザイ・アール・アンド・ディー・マネジメント株式会社 Ｌｒｐ４／Ｃｏｒｉｎドーパミン産生ニューロン前駆細胞マーカー
CN101027390B (zh) * 2004-07-22 2012-12-12 卫材R&D管理有限公司 Lrp4/Corin多巴胺能神经元祖细胞标志物
JP4717797B2 (ja) * 2004-07-22 2011-07-06 エーザイ・アール・アンド・ディー・マネジメント株式会社 Ｌｒｐ４／Ｃｏｒｉｎドーパミン産生ニューロン前駆細胞マーカー
EP1950292A4 (fr) 2005-08-18 2009-01-21 Eisai R&D Man Co Ltd Nato3, MARQUEUR DE CELLULE PROGÉNITRICE DE CROISSANCE DE NEURONE PRODUCTEUR DE DOPAMINE
JP6090937B2 (ja) 2011-07-27 2017-03-08 国立大学法人京都大学 新規ドーパミン産生神経前駆細胞マーカー
EP2905622A1 (fr) * 2014-02-07 2015-08-12 Institut D'Investigaciones Biomédiques August Pi i Sunyer Diagnostic d'une maladie neurologique

Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996016075A1 (fr) * 1994-11-18 1996-05-30 The Regents Of The University Of California Peptides antibiotiques du type cryptdines et procedes d'utilisation
US5750504A (en) * 1993-12-24 1998-05-12 Zeneca Agrochemicals Antimicrobial proteins
WO1998046743A1 (fr) * 1997-04-15 1998-10-22 The Wellcome Trust Limited As Trustee To The Wellcome Trust Nouveau recepteur de ldl
WO1999002714A1 (fr) * 1997-07-07 1999-01-21 Abbott Laboratories Reactifs et procedes utilises pour detecter les maladies du sein
WO2000055629A2 (fr) * 1999-03-15 2000-09-21 Eos Biotechnology, Inc. Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein
WO2000061754A2 (fr) * 1999-04-09 2000-10-19 Curagen Corporation Nouvelles proteines humaines et polypeptides codant pour ces proteines
WO2001066689A2 (fr) * 2000-03-07 2001-09-13 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001066690A2 (fr) * 2000-03-06 2001-09-13 Smithkline Beecham Corporation Nouveaux composes
WO2001072961A2 (fr) * 2000-03-24 2001-10-04 Smithkline Beecham Corporation Nouveaux composes
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001079294A2 (fr) * 2000-04-19 2001-10-25 Curagen Corporation Nouvelles proteines humaines, polynucleotides les codant et procedes permettant de les utiliser
WO2002000843A2 (fr) * 2000-06-23 2002-01-03 Millennium Pharmaceuticals, Inc. 56739, nouvelle proteine contenant un domaine cub et utilisations de cette proteine
WO2002004600A2 (fr) * 2000-07-12 2002-01-17 Smithkline Beecham Corporation Nouveaux composes
WO2002014368A2 (fr) * 2000-08-16 2002-02-21 Curagen Corporation Nouvelles proteines et acides nucleiques codant pour ces proteines
WO2002063009A2 (fr) * 2001-02-02 2002-08-15 Eli Lilly And Company Proteines de mammiferes lp et reactifs associes
WO2002064792A2 (fr) * 2001-01-05 2002-08-22 Incyte Genomics, Inc. Molecules utiles pour detecter et traiter des maladies
WO2002066643A2 (fr) * 2000-11-13 2002-08-29 Curagen Corporation Proteines, polynucleotides codant pour ces proteines et procedes d'utilisation
WO2002068579A2 (fr) * 2001-01-10 2002-09-06 Pe Corporation (Ny) Kits tels que des dosages d'acides nucleiques comprenant une majorite d'exons ou de transcrits humains, destines a detecter l'expression et pouvant avoir d'autres applications
WO2002070669A2 (fr) * 2001-03-06 2002-09-12 Incyte Genomics, Inc. Proteines secretees
WO2002072794A2 (fr) * 2001-03-12 2002-09-19 Incyte Genomics, Inc. Proteines de la superfamille des immunoglobulines
WO2002079449A2 (fr) * 2001-03-28 2002-10-10 Incyte Genomics, Inc. Molecules permettant de detecter et de traiter des maladies
WO2002101080A2 (fr) * 2001-05-16 2002-12-19 Pe Corporation (Ny) Proteines secretees humaines isolees, molecules d'acide nucleique codant pour ces proteines secretees humaines et utilisations de ces dernieres
WO2003000863A2 (fr) * 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
WO2003002610A1 (fr) * 2001-06-29 2003-01-09 Incyte Genomics, Inc. Messagers extracellulaires
WO2003002765A2 (fr) * 2001-06-27 2003-01-09 Cancer Research Technology Limited Cancer
EP1308459A2 (fr) * 2001-11-05 2003-05-07 Helix Research Institute Séquences d'ADN complementaires pleine longueur
WO2003064616A2 (fr) * 2002-01-31 2003-08-07 Temple University - Of The Commonwealth System Of Higher Education Composes et methodes pour induire l'arret de croissance et l'apoptose

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077255A1 (fr) * 1999-06-11 2000-12-21 Human Genome Sciences, Inc. 49 proteines humaines secretees
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»
EP1074617A3 (fr) * 1999-07-29 2004-04-21 Research Association for Biotechnology Amorces pour la synthèse de cADN de pleine longueur et leur utilisation
WO2001055355A1 (fr) * 2000-01-31 2001-08-02 Human Genome Sciences, Inc. Acides nucleiques, proteines et anticorps
WO2001062927A2 (fr) * 2000-02-24 2001-08-30 Incyte Genomics Inc Molecules utilisees a des fins diagnostiques et therapeutiques
US20040009907A1 (en) * 2001-02-26 2004-01-15 Alsobrook John P. Proteins and nucleic acids encoding same
WO2001087917A1 (fr) * 2000-05-18 2001-11-22 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
CA2421265A1 (fr) * 2000-09-05 2002-03-14 Incyte Genomics, Inc. Molecules utilisees a des fins diagnostiques et therapeutiques
AU2002366951A1 (en) * 2001-12-10 2003-07-09 Nuvelo,Inc. Novel nucleic acids and polypeptides
AU2002361908A1 (en) * 2001-12-31 2003-07-24 Quark Biotech, Inc. Methods for identifying marker genes for cancer

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5731149A (en) * 1992-05-26 1998-03-24 The Shriner's Hospital For Crippled Children Antibiotic cryptdin peptides and methods of their use
US5750504A (en) * 1993-12-24 1998-05-12 Zeneca Agrochemicals Antimicrobial proteins
WO1996016075A1 (fr) * 1994-11-18 1996-05-30 The Regents Of The University Of California Peptides antibiotiques du type cryptdines et procedes d'utilisation
WO1998046743A1 (fr) * 1997-04-15 1998-10-22 The Wellcome Trust Limited As Trustee To The Wellcome Trust Nouveau recepteur de ldl
WO1999002714A1 (fr) * 1997-07-07 1999-01-21 Abbott Laboratories Reactifs et procedes utilises pour detecter les maladies du sein
WO2000055629A2 (fr) * 1999-03-15 2000-09-21 Eos Biotechnology, Inc. Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein
WO2000061754A2 (fr) * 1999-04-09 2000-10-19 Curagen Corporation Nouvelles proteines humaines et polypeptides codant pour ces proteines
WO2001066690A2 (fr) * 2000-03-06 2001-09-13 Smithkline Beecham Corporation Nouveaux composes
WO2001066689A2 (fr) * 2000-03-07 2001-09-13 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001072961A2 (fr) * 2000-03-24 2001-10-04 Smithkline Beecham Corporation Nouveaux composes
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
WO2001079294A2 (fr) * 2000-04-19 2001-10-25 Curagen Corporation Nouvelles proteines humaines, polynucleotides les codant et procedes permettant de les utiliser
WO2002000843A2 (fr) * 2000-06-23 2002-01-03 Millennium Pharmaceuticals, Inc. 56739, nouvelle proteine contenant un domaine cub et utilisations de cette proteine
WO2002004600A2 (fr) * 2000-07-12 2002-01-17 Smithkline Beecham Corporation Nouveaux composes
WO2002014368A2 (fr) * 2000-08-16 2002-02-21 Curagen Corporation Nouvelles proteines et acides nucleiques codant pour ces proteines
WO2002066643A2 (fr) * 2000-11-13 2002-08-29 Curagen Corporation Proteines, polynucleotides codant pour ces proteines et procedes d'utilisation
WO2002064792A2 (fr) * 2001-01-05 2002-08-22 Incyte Genomics, Inc. Molecules utiles pour detecter et traiter des maladies
WO2002068579A2 (fr) * 2001-01-10 2002-09-06 Pe Corporation (Ny) Kits tels que des dosages d'acides nucleiques comprenant une majorite d'exons ou de transcrits humains, destines a detecter l'expression et pouvant avoir d'autres applications
WO2002063009A2 (fr) * 2001-02-02 2002-08-15 Eli Lilly And Company Proteines de mammiferes lp et reactifs associes
WO2002070669A2 (fr) * 2001-03-06 2002-09-12 Incyte Genomics, Inc. Proteines secretees
WO2002072794A2 (fr) * 2001-03-12 2002-09-19 Incyte Genomics, Inc. Proteines de la superfamille des immunoglobulines
WO2002079449A2 (fr) * 2001-03-28 2002-10-10 Incyte Genomics, Inc. Molecules permettant de detecter et de traiter des maladies
WO2002101080A2 (fr) * 2001-05-16 2002-12-19 Pe Corporation (Ny) Proteines secretees humaines isolees, molecules d'acide nucleique codant pour ces proteines secretees humaines et utilisations de ces dernieres
WO2003000863A2 (fr) * 2001-06-22 2003-01-03 Pioneer Hi-Bred International, Inc. Polynucleotides de defensine et methodes d'utilisation
WO2003002765A2 (fr) * 2001-06-27 2003-01-09 Cancer Research Technology Limited Cancer
WO2003002610A1 (fr) * 2001-06-29 2003-01-09 Incyte Genomics, Inc. Messagers extracellulaires
EP1308459A2 (fr) * 2001-11-05 2003-05-07 Helix Research Institute Séquences d'ADN complementaires pleine longueur
WO2003064616A2 (fr) * 2002-01-31 2003-08-07 Temple University - Of The Commonwealth System Of Higher Education Composes et methodes pour induire l'arret de croissance et l'apoptose

Non-Patent Citations (53)

* Cited by examiner, † Cited by third party
Title
BLOCH C ET AL: "A NEW FAMILY OF SMALL (5 KDA) PROTEIN INHIBITORS OF INSECT ALPHA-AMYLASES FROM SEEDS OR SORGHUM (SORGHUM BICOLOR (L) MOENCH) HAVE SEQUENCE HOMOLOGIES WITH WHEAT GAMMA-PUROTHIONINS" FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 279, no. 1, February 1991 (1991-02), pages 101-104, XP002041563 ISSN: 0014-5793 *
DATABASE EMBL [Online] 11 February 2001 (2001-02-11), "human full-length cDNA 5-PRIME end of clone CS0DK007YE05 of HELA CELLS COT 25-NORMALIZED of Homo sapiens (human)" XP002374367 retrieved from EBI accession no. EM_PRO:CNSLT0W8E Database accession no. AL555324 *
DATABASE EMBL [Online] 12 February 2001 (2001-02-12), "human full-length cDNA 3-PRIME end of clone CS0DF038YL21 of FETAL BRAIN of Homo sapiens (human)" XP002305996 retrieved from EBI accession no. EM_PRO:AL566375 Database accession no. AL566375 *
DATABASE EMBL [Online] 12 February 2001 (2001-02-12), "human full-length cDNA 3-PRIME end of clone CS0DF038YL21 of FETAL BRAIN of Homo sapiens (human)" XP002374364 retrieved from EBI accession no. EM_PRO:CNSLT14RD Database accession no. AL566375 *
DATABASE EMBL [Online] 16 November 2001 (2001-11-16), "MEST347-G04.T3 ISUM5-RN Zea mays cDNA clone MEST347-G04 3', mRNA sequence." XP002374329 retrieved from EBI accession no. EM_PRO:BM074930 Database accession no. BM074930 *
DATABASE EMBL [Online] 17 December 2003 (2003-12-17), "Sequence 17027 from Patent EP1074617." XP002305995 retrieved from EBI accession no. EM_PAT:AX882122 Database accession no. AX882122 & EP 1 074 617 A (HELIX RES INST) 7 February 2001 (2001-02-07) *
DATABASE EMBL [Online] 18 November 1999 (1999-11-18), "687021A03.x1 687 - Early embryo from Delaware Zea mays cDNA, mRNA sequence." XP002374330 retrieved from EBI accession no. EM_PRO:AW181161 Database accession no. AW181161 *
DATABASE EMBL [Online] 2 October 2001 (2001-10-02), "Homo sapiens DiGeorge syndrome-related protein FKSG3 mRNA, complete cds." XP002374306 retrieved from EBI accession no. EM_PRO:AF305069 Database accession no. AF305069 *
DATABASE EMBL [Online] 2 October 2001 (2001-10-02), "Homo sapiens DiGeorge syndrome-related protein FKSG5 mRNA, complete cds." XP002374366 retrieved from EBI accession no. EM_PRO:AF305622 Database accession no. AF305622 *
DATABASE EMBL [Online] 20 January 2000 (2000-01-20), "Homo sapiens chromosome 15 clone RP11-300N24 map 15, LOW-PASS SEQUENCE SAMPLING." XP002374317 retrieved from EBI accession no. EM_PRO:AC021676 Database accession no. AC021676 *
DATABASE EMBL [Online] 24 August 1998 (1998-08-24), "Homo sapiens mRNA for MEGF7, partial cds." XP002374308 retrieved from EBI accession no. EM_PRO:AB011540 Database accession no. AB011540 *
DATABASE EMBL [Online] 29 April 1999 (1999-04-29), "rx00304s Rat mixed-tissue library Rattus norvegicus cDNA clone rx00304 3', mRNA sequence." XP002374318 retrieved from EBI accession no. EM_PRO:AI639089 Database accession no. AI639089 *
DATABASE EMBL [Online] 30 April 1998 (1998-04-30), "Homo sapiens unknown mRNA." XP002374307 retrieved from EBI accession no. EM_PRO:AF060862 Database accession no. AF060862 *
DATABASE EMBL [Online] 30 July 2000 (2000-07-30), "HVSMEh0093P24f Hordeum vulgare 5-45 DAP spike EST library HVcDNA0009 (5 to 45 DAP) Hordeum vulgare cDNA clone HVSMEh0093P24f, mRNA sequence." XP002374362 retrieved from EBI accession no. EM_PRO:BE454491 Database accession no. BE454491 *
DATABASE EMBL [Online] 30 March 2001 (2001-03-30), "Homo sapiens yippee-like 3 (Drosophila), mRNA (cDNA clone MGC:10500 IMAGE:3639517), complete cds." XP002374365 retrieved from EBI accession no. EM_PRO:BC005009 Database accession no. BC005009 *
DATABASE EMBL [Online] EMBL Sequence Version Archive issued 14.03.2001 14 December 1999 (1999-12-14), "Homo sapiens BAC clone RP11-561N12 from 7, complete sequence." XP002374314 retrieved from EBI accession no. EM_PRO:AC016769 Database accession no. AC016769 *
DATABASE EMBL [Online] EMBL Sequence Version Archive Issued 14.11.2001 14 December 1999 (1999-12-14), "Homo sapiens BAC clone RP11-561N12 from 7, complete sequence." XP002374313 retrieved from EBI accession no. EM_PRO:AC016769 Database accession no. AC016769 *
DATABASE EMBL [Online] EMBL Sequence version Archive Issued 24.08.2000 24 August 2000 (2000-08-24), "Gallus gallus CEPU-Se alpha 2 isoform (CEPU-Se) mRNA, complete cds." XP002374315 retrieved from EBI accession no. EM_PRO:AF292935 Database accession no. AF292935 *
DATABASE EMBL [Online] Sequence Version Archive Issued 02.03.2001 12 February 2001 (2001-02-12), "human full-length cDNA 5-PRIME end of clone CS0DB003YO14 of NEUROBLASTOMA COT 10-NORMALIZED of Homo sapiens (human)" XP002374316 retrieved from EBI accession no. EM_PRO:AL583491 Database accession no. AL583491 *
DATABASE EMBL [Online] Sequence version archive issued 03.06.2000 7 January 2000 (2000-01-07), "Rattus norvegicus mRNA for MEGF7, partial cds." XP002374309 retrieved from EBI accession no. EM_PRO:AB011533 Database accession no. AB011533 *
DATABASE EMBL [Online] Sequence version Archive Issued 20.10.2001 29 November 2000 (2000-11-29), "Mus musculus 13 days embryo cDNA, RIKEN full-length enriched library, clone:3930402I15, 5' end partial sequence." XP002374322 retrieved from EBI accession no. EM_PRO:BB569837 Database accession no. BB569837 *
DATABASE Geneseq [Online] 15 January 2004 (2004-01-15), "Mouse tumour suppressor mRNA SEQ ID NO:50." XP002374319 retrieved from EBI accession no. GSN:ADD29597 Database accession no. ADD29597 & WO 03/058201 A (QUARK BIOTECH, INC; THE CLEVELAND CLINIC FOUNDATION; FEINSTEIN, ELENA;) 17 July 2003 (2003-07-17) *
DATABASE Geneseq [Online] 22 April 2004 (2004-04-22), "Protein of the invention SEQ ID NO:710." XP002305994 retrieved from EBI accession no. GSP:ADJ33733 Database accession no. ADJ33733 & WO 01/87917 A (CHEN RUI HONG ; HYSEQ INC (US); WANG JIAN RUI (US); WEHRMAN TOM (US);) 22 November 2001 (2001-11-22) *
DATABASE Geneseq [Online] 23 March 2001 (2001-03-23), "Gene 30 human secreted protein homologous amino acid sequence #157." XP002306004 retrieved from EBI accession no. GSP:AAB64519 Database accession no. AAB64519 & WO 00/77255 A (HUMAN GENOME SCIENCES INC ; ROSEN CRAIG A (US); RUBEN STEVEN M (US); K) 21 December 2000 (2000-12-21) *
DATABASE Geneseq [Online] 25 March 2004 (2004-03-25), "NOV25 coding sequence, SEQ ID 59." XP002374311 retrieved from EBI accession no. GSN:ADH48775 Database accession no. ADH48775 & WO 02/068652 A (CURAGEN CORPORATION; ALSOBROOK, JOHN, P., II; ANDERSON, DAVID, W; BALL) 6 September 2002 (2002-09-06) *
DATABASE Geneseq [Online] 25 March 2004 (2004-03-25), "NOV25 protein sequence, SEQ ID 60." XP002374310 retrieved from EBI accession no. GSP:ADH48776 Database accession no. ADH48776 & WO 02/068652 A (CURAGEN CORPORATION; ALSOBROOK, JOHN, P., II; ANDERSON, DAVID, W; BALL) 6 September 2002 (2002-09-06) *
DATABASE Geneseq [Online] 29 January 2004 (2004-01-29), "Novel protein (useful for identifying genetic disorders) #7." XP002374326 retrieved from EBI accession no. GSP:ADE07852 Database accession no. ADE07852 & WO 03/054152 A (HYSEQ, INC; TANG, Y., TOM; ASUNDI, VINOD; GOODRICH, RYLE, W; REN, FEIY) 3 July 2003 (2003-07-03) *
DATABASE Geneseq [Online] 30 July 2002 (2002-07-30), "Human dithp polynucleotide #256." XP002374320 retrieved from EBI accession no. GSN:ABK71790 Database accession no. ABK71790 & WO 02/20754 A (INCYTE GENOMICS, INC; STUART, JACKSON; LINCOLN, STEPHEN, E; ALTUS, CHR) 14 March 2002 (2002-03-14) *
DATABASE Geneseq [Online] 4 December 2001 (2001-12-04), "Human diagnostic and therapeutic polynucleotide (DITHP) #202." XP002374328 retrieved from EBI accession no. GSN:AAS31187 Database accession no. AAS31187 & WO 01/62927 A (INCYTE GENOMICS INC; PANZER, SCOTT; SPIRO, PETER, A; BANVILLE, STEVEN,) 30 August 2001 (2001-08-30) *
DATABASE Geneseq [Online] 6 November 2001 (2001-11-06), "Human digestive system antigen SEQ ID NO: 1647." XP002374363 retrieved from EBI accession no. GSP:AAM92298 Database accession no. AAM92298 & WO 01/55314 A (HUMAN GENOME SCIENCES, INC; ROSEN, CRAIG, A; BARASH, STEVEN, C; RUBEN,) 2 August 2001 (2001-08-02) *
DATABASE Geneseq [Online] 8 February 2001 (2001-02-08), "Human ORFX ORF2934 polynucleotide sequence SEQ ID NO:5867." XP002374324 retrieved from EBI accession no. GSN:AAC77379 Database accession no. AAC77379 & WO 00/58473 A (CURAGEN CORPORATION; SHIMKETS, RICHARD, A; LEACH, MARTIN) 5 October 2000 (2000-10-05) *
DATABASE Geneseq [Online] 8 February 2001 (2001-02-08), "Human ORFX ORF2934 polypeptide sequence SEQ ID NO:5868." XP002374323 retrieved from EBI accession no. GSP:AAB43170 Database accession no. AAB43170 & WO 00/58473 A (CURAGEN CORPORATION; SHIMKETS, RICHARD, A; LEACH, MARTIN) 5 October 2000 (2000-10-05) *
DATABASE UniProt [Online] 1 June 1998 (1998-06-01), "Neural secreted glycoprotein (CEPU-Se alpha 2 isoform)." XP002374368 retrieved from EBI accession no. UNIPROT:O57596 Database accession no. O57596 *
DATABASE UniProt [Online] 1 May 2000 (2000-05-01), "LDL receptor member LR3." XP002374312 retrieved from EBI accession no. UNIPROT:Q9UES7 Database accession no. Q9UES7 *
DATABASE UniProt [Online] 1 November 1996 (1996-11-01), "Protease inhibitor." XP002374321 retrieved from EBI accession no. UNIPROT:Q39807 Database accession no. Q39807 *
DATABASE UniProt [Online] 1 October 2000 (2000-10-01), "CEGP1 protein." XP002374325 retrieved from EBI accession no. UNIPROT:Q9NQ36 Database accession no. Q9NQ36 *
DATABASE UniProt [Online] 1 October 2000 (2000-10-01), "Cegp1 protein." XP002374327 retrieved from EBI accession no. UNIPROT:Q9JJS0 Database accession no. Q9JJS0 *
DONG Y ET AL: "MOLECULAR CLONING AND CHARACTERIZATION OF LR3, A NOVEL LDL RECEPTOR FAMILY PROTEIN WITH MITOGENIC ACTIVITY" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 251, no. 3, 29 October 1998 (1998-10-29), pages 784-790, XP001076809 ISSN: 0006-291X *
GRIMMOND S ET AL: "Cloning, Mapping, and Expression Analysis of a Gene Encoding a Novel Mammalian EGF-Related Protein (SCUBE1)" GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 70, no. 1, 15 November 2000 (2000-11-15), pages 74-81, XP004437770 ISSN: 0888-7543 *
HUTTNER K M ET AL: "STRUCTURE AND DIVERSITY OF THE MURINE CRYPTDIN GENE FAMILY" GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 19, 1994, pages 448-453, XP002054342 ISSN: 0888-7543 *
J. I. KOURIE AND A. A. SHORTHOUSE: "Properties of cytotoxic peptide-formed ion channels" AM J PHYSIOL CELL PHYSIOL, vol. 278, 2000, pages C1063-C1087, XP002374273 *
KRIEGER M ET AL: "STRUCTURES AND FUNCTIONS OF MULTILIGAND LIPOPROTEIN RECEPTORS: MACROPHAGE SCAVENGER RECEPTORS AND LDL RECEPTOR-RELATED PROTEIN (LRP)" ANNUAL REVIEW OF BIOCHEMISTRY, PALTO ALTO, CA, US, vol. 63, 1994, pages 601-637, XP002937333 ISSN: 0066-4154 *
LIPPMAN D A ET AL: "OPIOID-BINDING CELL ADHESION MOLECULE (OBCAM)-RELATED CLONES FROM ARAT BRAIN CDNA LIBRARY" GENE, ELSEVIER, AMSTERDAM, NL, vol. 117, no. 2, 1992, pages 249-254, XP000602047 ISSN: 0378-1119 *
LODGE A P ET AL: "Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system" MOLECULAR BRAIN RESEARCH, ELSEVIER SCIENCE BV, AMSTERDAM, NL, vol. 82, 2000, pages 84-94, XP002977169 ISSN: 0169-328X *
MENDEZ E ET AL: "PRIMARY STRUCTURE OF OMEGA-HORDOTHIONIN, A MEMBER OF A NOVEL FAMILY OF THIONINS FROM BARLEY ENDOSPERM, AND ITS INHIBITION OF PROTEIN SYNTHESIS IN EUKARYOTIC AND PROLARYOTIC CELL-FREE SYSTEMS" EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN, DE, vol. 239, no. 1, 1996, pages 67-73, XP001182417 ISSN: 0014-2956 *
MORIYAMA TATSUYA ET AL: "Purification and characterization of diacylglycerol lipase from human platelets" JOURNAL OF BIOCHEMISTRY (TOKYO), vol. 125, no. 6, June 1999 (1999-06), pages 1077-1085, XP002305993 ISSN: 0021-924X *
NEELS J G ET AL: "Interaction Between Factor VIII and LDL Receptor-related Protein. MODULATION OF COAGULATION?" TRENDS IN CARDIOVASCULAR MEDICINE, ELSEVIER SCIENCE, NEW YORK, NY, US, vol. 10, no. 1, 2000, pages 8-14, XP002264412 ISSN: 1050-1738 *
OUELLETTE A J ET AL: "A NOVEL MOUSE GENE FAMILY CODING FOR CATIONIC, CYSTEINE-RICH PEPTIDES" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 265, no. 17, 15 June 1990 (1990-06-15), pages 9831-9837,18702, XP000615234 ISSN: 0021-9258 *
SCHOFIELD P R ET AL: "MOLECULAR CHARACTERIZATION OF A NEW IMMUNOGLOBULIN SUPERFAMILY PROTEIN WITH POTENTIAL ROLES IN OPIOID BINDING AND CELL CONTACT" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 8, no. 2, 1 February 1989 (1989-02-01), pages 489-495, XP000602249 ISSN: 0261-4189 *
See also references of WO02074906A2 *
SHARK K B ET AL: "Cloning, sequencing and localization to chromosome 11 of a cDNA encoding a human opioid-binding cell adhesion molecule (OBCAM)" GENE, ELSEVIER, AMSTERDAM, NL, vol. 155, no. 2, 3 April 1995 (1995-04-03), pages 213-217, XP004042416 ISSN: 0378-1119 *
SOUZA CASTRO DE M ET AL: "COMPLETE AMINO ACID SEQUENCES OF TWO GAMMA-THIONINS FROM MAIZE (ZEAMAYS L.) SEEDS" PROTEIN AND PEPTIDE LETTERS, BENTHAM SCIENCE PUBLISHERS, SCHIPHOL, NL, vol. 3, no. 4, 1 August 1996 (1996-08-01), pages 267-274, XP000623654 ISSN: 0929-8665 *
STRUYK A F ET AL: "CLONING OF NEUROTRIMIN DEFINES A NEW SUBFAMILY OF DIFFERENTIALLY EXPRESSED NEURAL CELL ADHESION MOLECULES" JOURNAL OF NEUROSCIENCE, NEW YORK, NY, US, vol. 15, no. 3, March 1995 (1995-03), pages 2141-2156, XP000953464 ISSN: 0270-6474 *

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WO2002074906A3 (fr) 2004-04-22
WO2002074906A2 (fr) 2002-09-26
AU2002250143A1 (en) 2002-10-03

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